Search

Estrogen ([E.sub.2]) is reported to regulate skeletal muscle mass and contractile function; whether [E.sub.2] exerts its effects through estrogen receptor-[alpha] (ER[alpha]) or -[beta] (ER[beta]) is unclear. We determined the effect of ER[alpha] or ER[beta] elimination on muscle mass and contractile function in multiple muscles of the lower limb, muscles with different locomotor tasks and proportions of fiber types I and II: soleus (Sol), plantaris (Plan), tibialis anterior (TA), and gastrocnemius (Gast) in mature female mice. To determine [E.sub.2] elimination effects on muscle, we also used aromatase (Ar) knockout (KO) and wild-type (WT) mice. ER[alpha] and ArKO body weights were ~10 and 20% higher than WT. Although muscle mass tended to show a commensurate increase in both groups, only the TA was significantly larger in ER[alpha] (P < 0.05). Ratios of muscle mass to body mass revealed significantly lower values for Gast and TA in ArKO mice (P < 0.05). Tetanic tension ([P.sub.o]) per calculated anatomical cross-sectional area (aCSA) in ER[alpha] KO was lower in TA and Gast than in WT. Lower [P.sub.o]/aCSA in ER[alpha] KO Gast and TA was also supported histologically by significantly less [P.sub.o]/fiber areas (P < 0.05). ArKO mice also had lower [P.sub.o]/aCSA in Gast and TA compared with WT. ER[beta] KO and WT mice were comparable in all measures. Our results support the hypothesis that [E.sub.2] effects on skeletal muscle are mediated in part via the ER[alpha] but that [E.sub.2] effects may be mediated via more than one mechanism or receptor. muscle mass; peak tetanic tension; fiber area; myosin protein

GLUT8 is a class III sugar transporter predominantly expressed in testis and brain. In contrast to the class I and class II transporters, hydrophobicity plots predict a short extracellular loop between transmembrane domain (TM)1 and TM2 and a long extracellular loop between TM9 and TM10 that contains the only N-glycosylation site. In vitro translated GLUT8 migrates as a 35-kDa protein that is glycosylated in the presence of microsomal membranes. In heterologous expression systems, glucose transport activity ([K.sub.m] of 2 mM) was inhibited by fructose and galactose. The transporter carries an N[H.sub.2]-terminal endosomal/lysosomal targeting motif ([DE]XXXL[LI]). Accordingly, constitutive GLUT8 has been found to be associated with endosomes and lysosomes but also with membranes of the endoplasmic reticulum. A similar distribution was detected after overexpression of wild-type or tagged GLUT8 in different cell systems. In these cells, none of the conventional signals tested induced a translocation of GLUT8 to the plasma membrane. Therefore, GLUT8 appears to catalyze transport of sugars or sugar derivatives through intracellular membranes. Slc2a8 knockout mice were viable, developed normally, and showed mild alterations in brain (increased proliferation of neuronal cells in dentate gyrus of the hippocampus, hyperactivity), heart (impaired transmission of electrical wave through the atrium), and sperm cells (reduced number of motile sperm cells associated with reduced mitochondrial membrane potential and ATP levels in sperm). The links between molecular function, cellular localization and phenotype of the knockout mouse is unclear and remains to be determined. glucose transporter; knockout models; lysosomes

Growth hormone (GH) signaling influences longevity in mice, with decreased GH signaling associated with longer life span and increased GH signaling with shortened life span. A proposed mechanism through which GH signaling influences life span postulates that decreased GH signaling lowers metabolic rate, thus slowing aging by decreasing production of damaging free radicals. The influence of altered GH signaling on metabolism was tested by monitoring oxygen consumption ([VO.sub.2]), respiratory quotient (RQ), and heat production in long-lived GH receptor knockout (GHRKO) and Ames dwarf mice, and short-lived bovine GH-overexpressing transgenic (bGH TG) mice. Intriguingly, both GHRKO and Ames dwarf mice have increased [VO.sub.2] and heat per gram body weight, and decreased RQ, whereas bGH TG mice have decreased [VO.sub.2] and heat per gram body weight and increased RQ. In conclusion, decreased GH signaling associates with increased metabolism per body weight and may beneficially affect mitochondrial flexibility by increasing the capacity for fat oxidation; generally, GH excess produces opposite metabolic effects. Key Words: Metabolism--Altered growth hormone signaling--Thyroid hormone--oxygen consumption--Respiratory quotient.

Heroin remains a major drug of abuse and is preferred by addicts over morphine. Like morphine, heroin has high affinity and selectivity for [mu]-receptors, but its residual analgesia in exon 1 MOR-1 knockout mice that do not respond to morphine suggests a different mechanism of action. MOR-1 splice variants lacking exon 1 have been observed in mice, humans, and rats, raising the possibility that they might be responsible for the residual heroin and morphine-6[beta]-glucuronide (M6G) analgesia in the exon 1 knockout mice. To test this possibility, we disrupted exon 11 of MOR-1, which eliminates all of the variants that do not contain exon 1. Morphine and methadone analgesia in the exon 11 knockout mouse was normal, but the analgesic actions of heroin, M6G, and fentanyl were markedly diminished in the radiant heat tail-flick and hot-plate assays. Similarly, the ability of M6G to inhibit gastrointestinal transit was greatly diminished in these exon 11 knockout mice, whereas the ability of morphine was unchanged. These findings identify receptors selectively involved with heroin and M6G actions and confirm the relevance of the exon 11-associated variants and raise important issues regarding the importance of atypical truncated G-protein-coupled receptors. knockout | analgesia | opiate receptor

With specific liver X receptor [alpha] and [beta] (LXR[alpha] and LXR[beta]) antibodies, we found that LXR[alpha] is strongly expressed in the luminal and basal cells of prostatic epithelium. The ventral prostates (VP) of LXR[[alpha].sup.-/-] mice are characterized by the presence of smooth-muscle actin-positive stromal overgrowth around the prostatic ducts and by numerous fibrous nodules pushing into the ducts and causing obstruction, so that most of the ducts were extremely dilated. BrdU labeling and Ki67 staining revealed epithelial and stromal proliferation in the fibrous nodules. However, the dense stroma surrounding the ducts was not positive for proliferation markers. There was no detectable difference between WT and LX[[alpha].sup.-/-] mice VP in the expression of the androgen receptor, but there was an increase in nuclear expression of Snail and Smad 2/3, indicating enhanced TGF-[beta] signaling. Upon treatment of WT mice for 3 months with the LXR agonist T2320 or for 3 weeks with [beta]-sitosterol, LXR[alpha] was downregulated, and a VP phenotype similar to that of LXR[[alpha].sup.-/-] mice resulted. We conclude that in rodents, LXR[alpha] seems to control VP stromal growth and that LXR[[alpha].sup.-/-] mice may be a useful model to study prostatic stromal hyperplasia. Because LXR[alpha] is expressed in the epithelium, the excessive stromal growth in LXR[[alpha].sup.-/-] mice indicates that LXR[[alpha] is essential for epithelial stromal communication. Benign prostatic hyperplasia | liver X receptor | TGF-[beta] | mesenchymal transformation | snail

Gene knockout experiments in mice have suggested a hierarchical model of early B cell commitment wherein E2A proteins (E47 and E12) activate early B cell factor (Ebf1), which in turn activates expression of the B cell commitment factor, Pax5. In IL-7 receptor alpha (IL-7R[alpha]) knockout mice, B cell development is blocked before B-lineage commitment at the prepro-B cell stage in adult animals. In IL-7R[[alpha.sup.-/-] prepro-B cells, E47 is expressed and yet is insufficient to transcriptionally activate the putative downstream target gene, Ebf1. In this study, we show that further increases of E47 expression in IL-7R[[alpha].sup.-/-] prepro-B cells fails to activate Ebf1, but rather leads to a dramatic induction of the E2A inhibitory factors, Id2 and Id3. In contrast, enforced expression of Ebf1 in IL-7R[[alpha].sup.-/-] bone marrow potently down-regulates Id2 and Id3 mRNA expression and restores B cell differentiation in vivo. Down-regulation of both Id2 and Id3 during B cell specification is essential in that overexpression of either Id2 or Id3 in wild-type bone marrow blocks B cell specification at the prepro-B cell stage. Collectively, these studies suggest a model where Ebf1 induction specifies the B cell fate by dramatically increasing activity of E47 at the posttranslational level. B cell specification | E2A | Ebf1 | Id proteins | IL-7 receptor

Most transmissible spongiform encephalopathies arise either spontaneously or by infection. Mutations of PRNP, which encodes the prion protein, PrP, segregate with phenotypically similar diseases. Here we report that moderate overexpression in transgenic mice of mPrP(170N, 174T), a mouse PrP with two point mutations that subtly affect the structure of its globular domain, causes a fully penetrant lethal spongiform encephalopathy with cerebral PrP plaques. This genetic disease was reproduced with 100% attack rate by intracerebral inoculation of brain homogenate to tga20 mice overexpressing WT PrP, and from the latter to WT mice, but not to PrP-deficient mice. Upon successive transmissions, the incubation periods decreased and PrP became more protease-resistant, indicating the presence of a strain barrier that was gradually overcome by repeated passaging. This shows that expression of a subtly altered prion protein, with known 3D structure, efficiently generates a prion disease. amyloid | neurodegeneration | prion | species barrier | transgenic mice

Adenosine has been proposed to promote sleep through [A.sub.1] receptors ([A.sub.1]R's) and/or [A.sub.2A] receptors in the brain. We previously reported that [A.sub.2A] receptors mediate the sleep-promoting effect of prostaglandin [D.sub.2], an endogenous sleep-inducing substance, and that activation of these receptors induces sleep and blockade of them by caffeine results in wakefulness. On the other hand, [A.sub.1]R has been suggested to increase sleep by inhibition of the cholinergic region of the basal forebrain. However, the role and target sites of [A.sub.1]R in sleep-wake regulation remained controversial. In this study, immunohistochemistry revealed that [A.sub.1]R was expressed in histaminergic neurons of the rat tuberomammillary nucleus (TMN). In vivo microdialysis showed that the histamine release in the frontal cortex was decreased by microinjection into the TMN of [N.sup.6]-cyclopentyladenosine (CPA), an [A.sub.1]R agonist, adenosine or coformycin, an inhibitor of adenosine deaminase, which catabolizes adenosine to inosine. Bilateral injection of CPA into the rat TMN significantly increased the amount and the delta power density of non-rapid eye movement (non-REM; NREM) sleep but did not affect REM sleep. CPA-promoted sleep was observed in WT mice but not in KO mice for [A.sub.1]R or histamine [H.sub.1] receptor, indicating that the NREM sleep promoted by [A.sub.1]R-specific agonist depended on the histaminergic system. Furthermore, the bilateral injection of adenosine or coformycin into the rat TMN increased NREM sleep, which was completely abolished by coadministration of 1,3-dimethyl-8-cyclopenthylxanthine, a selective [A.sub.1]R antagonist. These results indicate that endogenous adenosine in the TMN suppresses the histaminergic system via [A.sub.1]R to promote NREM sleep. Adenosine deaminase | histamine | knockout mouse | rat

15,15'-carotenoid monooxygenase (CMO I) is generally recognized as the central carotenoid cleavage enzyme responsible for converting provitamin A carotenoids to vitamin A, while having little affinity for nonprovitamin A carotenoids, such as lycopene. To investigate the role of CMO I in carotenoid metabolism, ~90-d-old C57BL/6 x 129/SvJ [CMO I wild-type (WT)] and B6;129S6-Bcmo1tm1Dnp [CMO I knockout (KO)] mice were fed a high-fat, moderate vitamin A, cholesterol-containing diet supplemented with 150 mg/kg diet of [beta]-carotene, lycopene, or placebo beadlets for 60 d (n = 12-14). CMO I KO mice fed lycopene (Lyc-KO) exhibited significant decreases in hepatic, spleen, and thymus lycopene concentrations and significant increases in prostate, seminal vesicles, testes, and brain lycopene concentrations compared with WT mice fed lycopene (Lyc-WT). Furthermore, in the serum and all tissues analyzed, excluding the testes, there was a significant increase in the percent lycopene cis isomers in Lyc-KO mice compared with Lyc-WT mice. CMO I KO mice fed [beta]-carotene ([beta]C-KO) had significantly lower hepatic vitamin A concentrations (17% of WT mice fed [beta]-carotene [[beta]C-WT]). Concordantly, [beta]C-KO mice had higher serum and tissue [beta]-carotene concentrations than [beta]C-WT mice. In addition, phenotypically CMO I KO mice had significantly higher final body weights and CMO I KO female mice had significantly lower uterus weights than CMO I WT mice. In conclusion, CMO I KO mice fed low levels of vitamin A have altered lycopene biodistribution and isomer patterns and do not cleave [beta]-carotene to vitamin A at appreciable levels.

Frontotemporal dementia (FTD) is characterized by cognitive and behavioral changes and, in a significant subset of patients, Parkinsonism. Histopathologically, FTD frequently presents with tau-containing lesions, which in familial cases result from mutations in the MAPTgene encoding tau. Here we present a novel transgenic mouse strain (K3) that expresses human tau carrying the FTD mutation K3691. K3 mice develop a progressive histopathology that is reminiscent of that in human FTD with the K3691 mutation. In addition, K3 mice show early-onset memory impairment and amyotrophy in the absence of overt neurodegeneration. Different from our previously generated tau transgenic strains, the K3 mice express the transgene in the substantia nigra (SN) and show an early-onset motor phenotype that reproduces Parkinsonism with tremor, bradykinesia, abnormal gait, and postural instability. Interestingly, motor performance of young, but not old, K3 mice improves upon L-dopa treatment, which bears similarities to Parkinsonism in FTD. The early-onset symptoms in the K3 mice are mechanistically related to selectively impaired anterograde axonal transport of distinct cargos, which precedes the loss of dopaminergic SN neurons that occurs in aged mice. The impaired axonal transport in SN neurons affects, among others, vesicles containing the dopamine-synthesizing enzyme tyrosine hydroxylase. Distinct modes of transport are also impaired in sciatic nerves, which may explain amyotrophy. Together, the K3 mice are a unique model of FTD-associated Parkinsonism, with pathomechanistic implications for the human pathologic process. tau | Alzheimer | transgenic | NFT | memory

Pim-1 kinase exerts potent cardioprotective effects in the myocardium downstream of AKT, but the participation of Pim-1 in cardiac hypertrophy requires investigation. Cardiac-specific expression of Pim-1 (Pim-WT) or the dominant-negative mutant of Pim-1 (Pim-DN) in transgenic mice together with adenoviral-mediated overexpression of these Pim-1 constructs was used to delineate the role of Pim-1 in hypertrophy. Transgenic overexpression of Pim-1 protects mice from pressure-overload-induced hypertrophy relative to wild-type controls as evidenced by improved hemodynamic function, decreased apoptosis, increases in antihypertrophic proteins, smaller myocyte size, and inhibition of hypertrophic signaling after challenge. Similarly, Pim-1 overexpression in neonatal rat cardiomyocyte cultures inhibits hypertrophy induced by endothelin-1. On the cellular level, hearts of Pim-WT mice show enhanced incorporation of BrdU into myocytes and a hypercellular phenotype compared to wild-type controls after hypertrophic challenge. In comparison, transgenic overexpression of Pim-DN leads to dilated cardiomyopathy characterized by increased apoptosis, fibrosis, and severely depressed cardiac function. Furthermore, overexpression of Pim-DN leads to reduced contractility as evidenced by reduced [Ca.sup.2+] transient amplitude and decreased percentage of cell shortening in isolated myocytes. These data support a pivotal role for Pim-1 in modulation of hypertrophy by impacting responses on molecular, cellular, and organ levels.

There are surprisingly few experimental models of neural growth and cranial integration. This, and the dearth of information regarding fetal brain development, detracts from a mechanistic understanding of cranial integration and its relevance to the ontogenetic and interspecific patterning of the form of the skull. To address this shortcoming, our research uses transgenic mice expressing a stabilized form of [beta]-catenin to isolate the effects of encephalization on the development of the basi-and neuro-cranium. These mice develop highly enlarged brains due to an increase in neural precursor cells, and differences between transgenic and wild-type mice are predicted to result solely from variation in relative brain size. By focusing on prenatal growth, this project adds to our understanding of a critically important period when major structural and functional interrelationships are established in the skull. Comparisons of wild-type and transgenic mice were performed using microcomputed tomography (microCT) and magnetic resonance imaging (MRI). These analyses show that the larger brains of the transgenic mice are associated with a larger neurocranium and an altered basicranial morphology. However, body size and postcranial ossification do not seem to be affected by the transgene. Comparisons of the rate of postcranial and cranial ossification also point to an unexpected effect of neural growth on skull development: increased fetal encephalization may result in a compensatory decrease in the level of cranial ossification. Therefore, if other life-history factors are held constant, the ontogeny of a metabolically costly structure, such as a brain, may occur at the expense of other cranial structures. These analyses indicate the benefits of a multifactorial approach to cranial integration using a mouse model.

Knockout mice lacking myostatin (Mstn), a negative regulator of the growth of skeletal muscle, develop significant increases in the relative mass of masticator muscles as well as the ability to generate higher maximal muscle forces. Wild-type and Mstn-deficient mice were compared to investigate the postnatal influence of elevated masticatory loads due to increased jaw-adductor and bite forces on the biomineralization of mandibular articular and cortical bone, the internal structure of the jaw joints, and the composition of temporomandibular joint (TMJ) articular cartilage. To provide an inter-specific perspective on the long-term responses of mammalian jaw joints to altered loading conditions, the findings on mice were compared to similar data for growing rabbits subjected to long-term dietary manipulation. Statistically significant differences in joint proportions and bone mineral density between normal and Mstn-deficient mice, which are similar to those observed between rabbit loading cohorts, underscore the need for a comprehensive analysis of masticatory tissue plasticity vis-a-vis altered mechanical loads, one in which variation in external and internal structure are considered. Differences in the expression of proteoglycans and type-II collagen in TMJ articular cartilage between the mouse and rabbit comparisons suggest that the duration and magnitude of the loading stimulus will significantly affect patterns of adaptive and degradative responses. These data on mammals subjected to long-term loading conditions offer novel insights regarding variation in ontogeny, life history, and the ecomorphology of the feeding apparatus.

In addition to its role in the storage of fat, adipose tissue acts as an endocrine organ, and it contains a functional renin-angiotensin system (RAS). Angiotensin-converting enzyme (ACE) plays a key role in the RAS by converting angiotensin I to the bioactive peptide angiotensin II (Ang II). In the present study, the effect of targeting the RAS in body energy homeostasis and glucose tolerance was determined in homozygous mice in which the gene for ACE had been deleted ([ACE.sup.-/-]) and compared with wild-type littermates. Compared with wild-type littermates, [ACE.sup.-/-] mice had lower body weight and a lower proportion of body fat, especially in the abdomen. [ACE.sup.-/-] mice had greater fed-state total energy expenditure (TEE) and resting energy expenditure (REE) than wild-type littermates. There were pronounced increases in gene expression of enzymes related to lipolysis and fatty acid oxidation (lipoprotein lipase, carnitine palmitoyl transferase, long-chain acetyl CoA dehydrogenase) in the liver of [ACE.sup.-/-] mice and also lower plasma leptin. In contrast, no differences were detected in daily food intake, activity, fed-state plasma lipids, or proportion of fat excreted in fecal matter. In conclusion, the reduction in ACE activity is associated with a decreased accumulation of body fat, especially in abdominal fat depots. The decreased body fat in [ACE.sup.-/-] mice is independent of food intake and appears to be due to a high energy expenditure related to increased metabolism of fatty acids in the liver, with the additional effect of increased glucose tolerance. fatty acid metabolism | obesity | ACE knockout mice | glucose tolerance

The most common theories about the pathogenesis of idiopathic kidney stones consider precipitation of calcium phosphate (CAP) within the kidneys critical for the development of the disease. We decided to test the hypothesis that a CaP substrate can promote the deposition of calcium oxalate (CaOx) in the kidneys. Experimental hyperoxaluria was induced by feeding glyoxylate to male mice with knockout (KO) of Na[P.sub.i] IIa (Npt2a), a sodium-phosphate cotransporter. Npt2a KO mice are hypercalciuric and produce CaP deposits in their renal tubules. Experimental hyperoxaluria led to CaOx crystalluria in both the hypercalciuric KO mice and the normocalciuric control B6 mice. Only the KO mice produced CaOx crystal deposits in their kidneys, but the CaOx crystals deposited separately from the Cap deposits. Perhaps CaP deposits were not available for a CaOx overgrowth. These results also validate earlier animal model observations that showed that CaP substrate is not required for renal deposition of CaOx and that other factors, such as local supersaturation, may be involved. The absence of CaOx deposition in the B6 mice despite extreme hyperoxaluria also signifies the importance of both calcium and oxalate in the development of CaOx nephrolithiasis. hypercalciuria; hyperoxaluria; Randall's plaque; Npt2a knockout mice

[[Arg.sup.8]]-vasopressin (Avp), a nonapeptide hormone, is known to regulate blood pressure, water balance, and a variety of behaviors such as anxiety, aggression, and bonding. Although some evidence that Avp modifies ethanol consumption and some of the effects of ethanol on behavior have been reported, the role of Avp in alcohol consumption and preference is poorly understood. The Avp1a receptor (Avpr1a) is ubiquitously expressed in the central nervous system. To determine the role of Avp signaling on the behavioral effects of alcohol, we examined voluntary ethanol consumption in mice with targeted disruptions of the Avpr1a knockout (Avpr1a KO) gene. Avpr1a KO mice displayed both increased ethanol consumption and preference compared with wild-type (WT) mice. Enhanced ethanol consumption was dramatically and reversibly reduced by treatment with N-methyl-D-aspartic acid antagonists. Basal glutamate release was elevated around the striatum in Avpr1a KO mice. Elevation of extracellular glutamate was also produced in WT mice by local application of an Avpr1a antagonist though a dialysis probe, and this elevation was quickly reversed by stopping the perfusion. These results suggest that Avp can inhibit the release of glutamate from the presynaptic terminal via the Avp1a receptor and that elevation of glutamate levels owing to loss of the inhibitory effect via Avp-Avpr1a signaling may play an important role in the preference for ethanol. [[Arg.sup.8]]-vasopressin

Subfamily II of the solute carrier (Slc)39a family contains three highly conserved members (ZIPs 1-3) that share a 12-amino acid signature sequence present in the putative fourth transmembrane domain and function as zinc transporters in transfected cells. The physiological significance of this genetic redundancy is unknown. Here we report that the complete elimination of all three of these Zip genes, by targeted mutagenesis and crossbreeding mice, causes no overt phenotypic effect. When mice were fed a zinc-adequate diet, several indicators of zinc status were indistinguishable between wild-type and triple-knockout mice, including embryonic morphogenesis and growth, alkaline phosphatase activity in the embryo, ZIP4 protein in the visceral yolk sac, and initial rates (30 min) of accumulation/retention of [sup.67]Zn in liver and pancreas. When mice were fed a zinc-deficient diet, embryonic membrane-bound alkaline phosphatase activity was reduced to a much greater extent, and 80% of the embryos of the triple-knockout mice developed abnormally compared with 12% of the embryos of wild-type mice. During zinc deficiency, the accumulation/retention (3 h) of 67Zn in the liver and pancreas of weanlings was significantly impaired in the triple-knockout mice compared with wild-type mice. Thus none of these three mammalian Zip genes apparently plays a critical role in zinc homeostasis when zinc is replete, but they play important, noncompensatory roles when this metal is deficient. pregnancy; stable zinc isotope; triple-knockout mice; zinc homeostasis

Diminished lung function, indicated by a low forced expiratory volume in one second (FE[V.sub.1]), and short physical stature, predict early mortality from all causes, including cardiovascular, among smokers and never smokers. The basis for these associations is unclear, and, it is not known if there is a pulmonary morphological component to the relationship between low FE[V.sub.1] and early death in a general population. Some apolipoprotein E genotypes also predict atherosclerosis and early mortality. These considerations led us to examine the [Apoe.sup.tm1Unc] (Apoe) mouse, in which the apolipoprotein E gene is deleted, and that develops dyslipidemia, atherosclerosis at an early age, and has a shorter life span than the rounder wild-type (wt) strain. We asked if Apoe mice have a morphological or functional pulmonary phenotype. We measured the size, number, and surface area of pulmonary gas-exchange units (alveoli) and mechanical properties of the lung. Compared with wt mice, Apoe mice had: 1) diminished developmental alveologenesis, 2) increased airway resistance in early adulthood, 3) high lung volume and high dynamic and static compliance in later adulthood, 4) more rapid loss of lung recoil with age, and 5) were less long than wt mice. These findings in mice indicate the association of a low FE[V.sub.1] with early death in humans may have developmental, and accelerated ageing, related pulmonary components, and that dietary, genetic, or dietary and genetic influences, on lipid metabolism may be an upstream cause of inflammation and oxidative stress, currently considered to be major risk factors for COPD. FE[V.sub.1]; predict early death; atherosclerosis; short stature; diet; genetics; cholesterol

Restraint stress induces permeability changes in the small intestine, but little is known about the role of tumor necrosis factor (TNF)-[alpha] in the defects of the TJ function. In the present study, we used tumor necrosis factor-R1 knockout mice (TNF-[alpha]-R1KO) to understand the roles of TNF-[alpha] on ileum altered permeability function in models of immobilization stress. The genetic TNF-[alpha] inhibition significantly reduced the degree of 1) TNF-[alpha] production in ileum tissues; 2) the alteration of zonula occludens-I (ZO-1), claudin-2, claudin-4, claudin-5, and [beta]-catenin (immunohistochemistry); and 3) apoptosis (TUNEL staining, Bax, Bc1-2 expression). Taken together, our results demonstrate that inhibition of TNF-[alpha] reduces the tight junction permeability in the ileum tissues associated with immobilization stress, suggesting a possible role of TNF-[alpha] on ileum barrier dysfunction. apoptosis; TNF-[alpha]-deficient mice; zonula occludens-1; claudin-2; [beta]-catenin; tight junction

Epithelial-mesenchymal interactions are essential for growth, differentiation, and regeneration of exocrine and endocrine cells in the pancreas. The keratinocyte growth factor (KGF) is derived from mesenchyme and has been shown to promote epithelial cell differentiation and proliferation in a paracrine fashion. Here, we have examined the effect of ectopic expression of KGF on pancreatic differentiation and proliferation in transgenic mice by using the proximal elastase promoter. KGF transgenic mice were generated following standard procedures and analyzed by histology, morphometry, immunohistochemistry, Western blot analysis, and glucose tolerance testing. In KGF transgenic mice, the number of islets, the average size of islets, and the relation of endocrine to exocrine tissue are increased compared with littermate controls. An expansion of the [beta]-cell population is responsible for the increase in the endocrine compartment. Ectopic expression of KGF results in proliferation of [beta]-cells and pancreatic duct cells most likely through activation of the protein kinase B (PKB)/Akt signaling pathway. Glucose tolerance and insulin secretion are impaired in transgenic animals. These results provide evidence that ectopic expression of KGF in acinar cells promotes the expansion of the [beta]-cell lineage in vivo through activation of the PKB/Akt pathway. Furthermore, the observed phenotype demonstrates that an increase in the [beta]-cell compartment does not necessarily result in an improved glucose tolerance in vivo. [beta]-cell proliferation; protein kinase B; glucose tolerance

Syncoilin is a striated muscle-specific intermediate filament-like protein, which is part of the dystrophin-associated protein complex (DPC) at the sarcolemma and provides a link between the extracellular matrix and the cytoskeleton through its interaction with [alpha]-dystrohrevin and desmin. Its upregulation in various neuromuscular diseases suggests that syncoilin may play a role in human myopathies. To study the functional role of syncoilin in cardiac and skeletal muscle in vivo, we generated syncoilin-deficient ([syncoilin.sup.-/-]) mice. Our detailed analysis of these mice up to 2 yr of age revealed that syncoilin is entirely dispensable for cardiac and skeletal muscle development and maintenance of cellular structure but is required for efficient lateral force transmission during skeletal muscle contraction. Notably, [syncoilin.sup.-/-] skeletal muscle generates less maximal isometric stress than wild-type (WT) muscle but is as equally susceptible to eccentric contraction-induced injury as WT muscle. This suggests that syncoilin may play a supportive role for desmin in the efficient coupling of mechanical stress between the myofibril and fiber exterior. It is possible that the reduction in isometric stress production may predispose the syncoilin skeletal muscle to a dystrophic condition. intermediate filament; sarcomere; mutant mouse

Temporal lobe epilepsy (TLE) is a devastating disease in which aberrant synaptic plasticity plays a major role. We identify matrix metalloproteinase (MMP) 9 as a novel synaptic enzyme and a key pathogenic factor in two animal models of TLE: kainate-evoked epilepsy and pentylenetetrazole (PTZ) kindling-induced epilepsy. Notably, we show that the sensitivity to PTZ epileptogenesis is decreased in MMP-9 knockout mice but is increased in a novel line of transgenic rats overexpressing MMP-9. Immunoelectron microscopy reveals that MMP-9 associates with hippocampal dendritic spines bearing asymmetrical (excitatory) synapses, where both the MMP-9 protein levels and enzymatic activity become strongly increased upon seizures. Further, we find that MMP-9 deficiency diminishes seizure-evoked pruning of dendritic spines and decreases aberrant synaptogenesis after mossy fiber sprouting. The latter observation provides a possible mechanistic basis for the effect of MMP-9 on epileptogenesis. Our work suggests that a synaptic pool of MMP-9 is critical for the sequence of events that underlie the development of seizures in animal models of TLE.

TAZ (transcriptional coactivator with PDZ-binding motif), also called WWTRI (WW domain containing transcription regulator 1). is a 14-3-3-binding molecule homologous to Yes-associated protein. TAZ acts as a coactivator for several transcription factors as well as a modulator of membrane-associated PDZ domain-containing proteins, but its (patho)physiological roles remain unknown. Here we show that gene inactivation of TAZ in mice resulted in pathological changes in the kidney and lung that resemble the common human diseases polycystic kidney disease and pulmonary emphysema. Taz.-null/lacZ knockin mutant homozygotes demonstrated renal cyst formation as early as embryonic day 15.5 with dilatation of Bowman's capsules and proximal tubules, followed by pelvic dilatation and hydronephrosis. After birth, only one-fifth of TAZ-deficient homozygotes grew to adulthood and demonstrated multicystic kidneys with severe urinary concentrating dejects and polyuria. Furthermore, adult TAZ-deficient homozygotes exhibited diffuse emphysematous changes in the lung. Thus TAZ is essential for developmental mechanisms involved in kidney and lung organogenesis, whose disturbance may lead to the pathogenesis of common human diseases. renal disease: knockout mice; transcription factor

Endotoxin-related acute kidney injury has been shown to profoundly induce nitric oxide (NO), which activates sympathetic and renin-angiotensin system, resulting in renal vasoconstriction. While vascular muscle cells are known to upregulate inducible NO synthase (iNOS), less is known about the endothelium as a source of NO during endotoxemia. Studies were, therefore, undertaken both in vitro in mouse microvascular endothelial cells and in vivo in transgenic mice with overexpression of endothelial GTP cyclohydrolase, the rate-limiting enzyme for tetrahydrobiopterin, a cofactor for NO synthase. LPS significantly induced endothelial cell iNOS expression and NO concentration in the culture media, with no change in endothelial NO synthase expression. GTP cyclohydrolase-1 transgenic (Tg) mice demonstrated a significant increase in baseline urine NO-to-creatinine ratio and a more significant increase in renal iNOS expression and serum NO levels with LPS treatment compared with the wild-type (WT) mice. Glomerular filtration rate and renal blood flow decreased significantly in Tg mice with 1.0 mg/kg LPS, while no changes were observed in WT with the same dose of LPS. Serum IL-6 levels were significantly higher in Tg compared with WT mice during endotoxemia. The antioxidant tempol improved the glomerular filtration rate in the Tg mice. Thus endothelium can be an important source of iNOS and serum NO concentration during endotoxemia, thereby increasing the sensitivity to AKI. Reactive oxygen species appear to be involved in this acute renal injury in Tg mice during endotoxemia. nitric oxide; nitric oxide synthase; IL-6; reactive oxygen species

Meprins are membrane-bound and secreted metalloproteinases consisting of [alpha]- and/or [beta]-subunits that are highly expressed in mouse kidney proximal tubules. Previous studies have implied that the meprin [alpha]/[beta]-isoform is deleterious when renal tissue is subjected to ischemia-reperfusion (I/R). To delineate the roles of the meprin isoforms in renal disease, we subjected mice deficient in meprin-[beta] (KO) and their wild-type (WT) counterparts to I/R. WT mice were markedly more susceptible to renal injury after I/R than the meprin-[beta] KO mice as determined by blood urea nitrogen levels. Urinary levels of inflammatory cytokines IL-6 and KC (CXCLI) were significantly higher in WT compared with meprin-[beta] KO mice by 6 h post-I/R. At 96 h postischemia, kidney mRNA expression levels for tumor necrosis factor-[alpha] transforming growth factor-[beta], inducible nitric oxide synthase, and heat shock protein-27 were significantly higher in the WT than meprin-13 KO mice. For WT mice subjected to I/R, there was a rapid (3 h) redistribution of meprin [beta]-subunits in cells in S3 segments of proximal tubules, followed by shedding of apical cell membrane and detachment of cells. These studies indicate that meprin-[beta] is important in the pathogenesis of renal injury following I/R and that the redistribution of active meprin-[alpha]/beta] is a major contributor to renal injury and subsequent inflammation. metalloproteases; kidney; knockout mice; inflammation

In contrast to the other heterotrimeric GTP-binding proteins (G proteins) [G.sub.s] and [G.sub.i], the functional role of Go is still poorly defined. To investigate the role of G[[alpha].sub.o] in the heart, we generated transgenic mice with cardiac-specific expression of a constitutively active form of G[[alpha].sub.o1] * (G[[alpha].sub.o] *), the predominant G[[alpha].sub.o] isoform in the heart. G[[alpha].sub.o] expression was increased 3- to 15-fold in mice from 5 independent lines, all of which had a normal life span and no gross cardiac morphological abnormalities. We demonstrate enhanced contractile function in G[[alpha].sub.o] * transgenic mice in vivo, along with increased L-type [Ca.sup.2+] channel current density, calcium transients, and cell shortening in ventricular G[[alpha].sub.o] *-expressing myocytes compared with wild-type controls. These changes were evident at baseline and maintained after isoproterenol stimulation. Expression levels of all major [Ca.sup.2+] handling proteins were largely unchanged, except for a modest reduction in [Na.sup.+]/[Ca.sup.2+] exchanger in transgenic ventricles. In contrast, phosphorylation of the ryanodine receptor and phospholamban at known PKA sites was increased 1.6- and 1.9-fold, respectively, in G[[alpha].sub.o] * ventricles. Density and affinity of [beta]-adrenoceptors, cAMP levels, and PKA activity were comparable in G[[alpha].sub.o] * and wild-type myocytes, but protein phosphatase 1 activity was reduced upon G[[alpha].sub.o] * expression, particularly in the vicinity of the ryanodine receptor. We conclude that G[[alpha].sub.o] * exerts a positive effect on [Ca.sup.2+] cycling and contractile function. Alterations in protein phosphatase 1 activity rather than PKA-mediated phosphorylation might be involved in hyperphosphorylation of key [Ca.sup.2+] handling proteins in hearts with constitutive [G[alpha].sub.o] activation. G proteins; signal transduction; calcium; contraction; transgenic mice

The metabolic syndrome is a group of disorders including obesity, insulin resistance, atherogenic dyslipidemia, hyperglycemia, and hypertension. To date, few animal models have been described to recapitulate the phenotypes of the syndrome. In this study, we generated and characterized two lines of triple-knockout mice that are deficient in either apolipoprotein E ([Apoe.sup.-/-]) or low-density lipoprotein receptor ([Ldlr.sup.-/-]) and express no leptin ([Lep.sup.ob/ob]) or apolipoprotein B-48 but exclusively apolipoprotein B-100 ([Apob.sup.100/100]). These two lines are referred to as Apoe triple-knockout-Apoe 3KO ([Apoe.sup.-/-][Apob.sup.100/100][Lep.sup.ob/ob]) and Ldlr triple-knockout-Ldlr 3KO ([Ldlr.sup.-/-][Apob.sup.100/100][Lep.sup.ob/ob]) mice. Both lines develop obesity, hyperinsulinemia, hyperlipidemia, hypertension, and atherosclerosis. However, only Apoe 3KO mice are hyperglycemic and glucose intolerant and are more obese than Ldlr 3KO mice. To evaluate the utility of these lines as pharmacological models, we treated both with leptin and found that leptin therapy ameliorated most metabolic derangements. Leptin was more effective in improving glucose tolerance in Ldlr 3KO than Apoe 3KO animals. The reduction of plasma cholesterol by leptin in Ldlr 3KO mice can be accounted for by its suppressive effect on food intake. However, in Apoe 3KO mice, leptin further reduced plasma cholesterol independently of its effect on food intake, and this improvement correlated with a smaller plaque lesion area. These effects suggest a direct role of leptin in modulating VLDL levels and, likewise, the lesion areas in VLDL-enriched animals. These two lines of mice represent new models with features of the metabolic syndrome and will be useful in testing therapies targeted for combating the human condition. atherosclerosis; leptin; hypercholesterolemia; diabetes: obesity

The dystrophin-glycoprotein complex maintains the integrity of skeletal muscle by associating laminin in the extracellular matrix with the actin cytoskeleton. Several human muscular dystrophies arise from defects in the components of this complex. The [[alpha].sub.7][[beta].sub.1]-integrin also binds laminin and links the extracellular matrix with the cytoskeleton. Enhancement of [[alpha].sub.7]-integrin levels alleviates pathology in [mdx/utrn.sup.-/-] mice, a model of Duchenne muscular dystrophy, and thus the integrin may functionally compensate for the absence of dystrophin. To test whether increasing [[alpha].sub.7]-integrin levels affects transcription and cellular functions, we generated [[alpha].sub.7]-integrin-inducible C2C12 cells and transgenic mice that overexpress the integrin in skeletal muscle. C2C12 myoblasts with elevated levels of integrin exhibited increased adhesion to laminin, faster proliferation when serum was limited, resistance to staurospofine-induced apoptosis, and normal differentiation. Transgenic expression of eightfold more integrin in skeletal muscle did not result in notable toxic effects in vivo. Moreover, high levels of [[alpha].sub.7]-integrin in both myoblasts and in skeletal muscle did not disrupt global gene expression profiles. Thus increasing integrin levels can compensate for defects in the extracellular matrix and cytoskeleton linkage caused by compromises in the dystrophin-glycoprotein complex without triggering apparent overt negative side effects. These results support the use of integrin enhancement as a therapy for muscular dystrophy. myoblast proliferation; integrin transgenic mice; microarray

Celiac disease is a gluten intolerance caused by a T-cell response against human leukocyte antigen (HLA)-DQ2 and DQ8-bound gluten peptides. Some subjects experience gastrointestinal symptoms in the absence of villous atrophy. Here we investigate the potential mechanisms of gut dysfunction in gluten-sensitive HLA-DQ8 transgenic mice. HLA-DQ8 mice were sensitized and gavaged with gliadin 3x/wk for 3 wk (G/G). Controls included 1) nonsensitized mice gavaged with rice (C); 2) gliadin-sensitized mice gavaged with rice (G/R); and 3) BSA-sensitized mice gavaged with BSA (BSA/BSA). [CD3.sup.+] intraepithelial lymphocyte, macrophage, and FOX-P3-positive cell counts were determined. Acetylcholine release, small intestinal contractility, and epithelial ion transport were measured. Gut function was investigated after gluten withdrawal and in HLA-DQ6 mice. Intestinal atrophy was not observed in G/G mice. Recruitment of intraepithelial lymphocyte, macrophages, and FOX-P3+ cells were observed in G/G, but not in C, G/R, or BSA/BSA mice. This was paralleled by increased acetylcholine release from the myenteric plexus, muscle hypercontractility, and increased active ion transport in G/G mice. Changes in muscle contractility normalized in DQ8 mice after a gluten withdrawal. HLA-DQ6 controls did not exhibit the abnormalities in gut function observed in DQ8 mice. Gluten sensitivity in HLA-DQ8 mice induces immune activation in the absence of intestinal atrophy. This is associated with cholinergic dysfunction and a prosecretory state that may lead to altered water movements and dysmotility. The results provide a mechanism by which gluten could induce gut dysfunction in patients with a genetic predisposition but without fully evolved celiac disease. muscle contractility; intestinal ion transport; food sensitivity

The X-linked orphan receptor GPR50 shares 45% homology with the melatonin receptors, yet its ligand and physiological function remain unknown. Here we report that mice lacking functional GPR50 through insertion of a lacZ gene into the coding sequence of GPR50 exhibit an altered metabolic phenotype. GPR50 knockout mice maintained on normal chow exhibit lower body weight than age-matched wild-type littermates by 10 wk of age. Furthermore, knockout mice were partially resistant to diet-induced obesity. When placed on a high-energy diet (HED) for 5 wk, knockout mice consumed significantly more food per unit body weight yet exhibited an attenuated weight gain and reduced body fat content compared with wild-type mice. Wheel-running activity records revealed that, although GPR50 knockout mice showed no alteration of circadian period, the overall levels of activity were significantly increased over wild types in both nocturnal and diurnal phases. In line with this, basal metabolic rate ([O.sub.2] consumption, C[O.sub.2] production, and respiratory quotient) was found to be elevated in knockout mice. Using in situ hybridization (wild-type mice) and [beta]-galactosidase activity (from LacZ insertion element in knockout mice), brain expression of GPR50 was found to be restricted to the ependymal layer of the third ventricle and dorsomedial nucleus of the hypothalamus. GPR50 expression was highly responsive to energy status, showing a significantly reduced expression following both fasting and 5 wk of HED. These data implicate GPR50 as an important regulator of energy metabolism. dorsomedial hypothalamus; metabolic rate; obesity; tanycyte

GABA has been proposed to inhibit insulin secretion through [GABA.sub.B] receptors ([GABA.sub.B]Rs) in pancreatic [beta]-cells. We investigated whether [GABA.sub.B]Rs participated in the regulation of glucose homeostasis in vivo. The animals used in this study were adult male and female BALB/C mice, mice deficient in the [GABA.sub.B1] subunit of the [GABA.sub.B]R ([GABA.sub.B.sup.-/-]), and wild types (WT). Blood glucose was measured under fasting/fed conditions and in glucose tolerance tests (GTTs) with a Lifescan Glucose meter, and serum insulin was measured by ELISA. Pancreatic insulin content and islet insulin were released by RIA. Western blots for the [GABA.sub.B1] subunit in islet membranes and immunohistochemistry for insulin and [GABA.sub.B1] were performed in both genotypes. BALB/C mice preinjected with Baclofen ([GABA.sub.B]R agonist, 7.5 mg/kg ip) presented impaired GTTs and decreased insulin secretion compared with saline-preinjected controls. [GABA.sub.B.sup.-/-] mice showed fasting and fed glucose levels similar to WT. [GABA.sub.B.sup.-/-] mice showed improved GTTs at moderate glucose overloads (2 g/kg). Baclofen pretreatment did not modify GTTs in [GABA.sub.B.sup.-/-] mice, whereas it impaired normal glycemia reinstatement in WT. Baclofen inhibited glucose-stimulated insulin secretion in WT isolated islets but was without effect in [GABA.sub.B.sup.-/-] islets. In [GABA.sub.B.sup.-/-] males, pancreatic insulin content was increased, basal and glucose-stimulated insulin secretion were augmented, and impaired insulin tolerance test and increased homeostatic model assessment of insulin resistance index were determined. Immunohistochemistry for insulin demonstrated an increase of very large islets in [GABA.sub.B.sup.-/-] males. Results demonstrate that [GABA.sub.B]Rs are involved in the regulation of glucose homeostasis in vivo and that the constitutive absence of [GABA.sub.B]Rs induces alterations in pancreatic histology, physiology, and insulin resistance. [gamma]-aminobutyric acid; insulin; glycemia; pancreas histology

Cholesterol (CH) homeostasis in the liver is regulated by enzymes of CH synthesis such as 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and catabolic enzymes such as cytochrome P-450, family 7, subfamily A, and polypeptide 1 (CYP7A1). Since a circadian clock controls the gene expression of these enzymes, these genes exhibit circadian rhythm in the liver. In this study, we examined the relationship between a diet containing CH and/or cholic acid (CA) and the circadian regulation of Hmgcr, low-density lipoprotein receptor (Ldlr), and Cyp7a1 gene expression in the mouse liver. A 4-wk CA diet lowered and eventually abolished the circadian expression of these genes. Not only clock genes such as period homolog 2 (Drosophila) (Per2) and brain and muscle arnt-like protein-1 (Bmal1) but also clock-controlled genes such as Hmgcr, Ldlr, and Cyp7a1 showed a reduced and arrhythmic expression pattern in the liver of Clock mutant mice. The reduced gene expression of Cyp7a1 in mice fed a diet containing CA or CH + CA was remarkable in the liver of Clock mutants compared with wild-type mice, and high liver CH accumulation was apparent in Clock mutant mice. In contrast, a CH diet without CA only elevated Cyp7a1 expression in both wild-type and Clock mutant mice. The present findings indicate that normal circadian clock function is important for the regulation of CH homeostasis in the mouse liver, especially in conjunction with a diet containing high CH and CA. circadian; cholesterol-7[alpha]-hydroxylase; Clock mutant mice

In contrast to skeletal muscles that simultaneously express multiple troponin T (TnT) isoforms, normal adult human cardiac muscle contains a single isoform of cardiac TnT. To understand the significance of myocardial TnT homogeneity, we examined the effect of TnT heterogeneity on heart function. Transgenic mouse hearts overexpressing a fast skeletal muscle TnT together with the endogenous cardiac TnT was investigated in vivo and ex vivo as an experimental system of concurrent presence of two classes of TnT in the adult cardiac muscle. This model of myocardial TnT heterogeneity produced pathogenic phenotypes: echocardiograph imaging detected age-progressive reductions of cardiac function; in vivo left ventricular pressure analysis showed decreased myocardial contractility; ex vivo analysis of isolated working heart preparations confirmed an intrinsic decrease of cardiac function in the absence of neurohumoral influence. The transgenic mice also showed chronic myocardial hypertrophy and degeneration. The dominantly negative effects of introducing a fast TnT into the cardiac thin filaments to produce two classes of [Ca.sup.2+] regulatory units in the adult myocardium suggest that TnT heterogeneity decreases contractile function by disrupting the synchronized action during ventricular contraction that is normally activated as an electrophysiological syncytium, myocardial heterogeneity; transgenic mouse; echocardiography; working heart preparation

Transforming growth factor (TGF)-[beta] plays a critical role in diabetic nephropathy. To isolate the contribution of one of the signaling pathways of TGF-[beta], the Smad3 gene in the mouse was knocked out at exons 2 and 3, and the effect was studied in streptozotocin (STZ)-induced diabetes over a period of 6 wk. TGF-[beta] activity was increased in the diabetic mice but was not able to signal via Smad3 in the knockout (KO) mice. As expected in the wild type, the kidneys of the STZ-diabetic mice showed both structural and functional defects that are characteristic of diabetic renal involvement. In the Smad3-KO mice, however, the defects that were improved were renal hypertrophy, mesangial matrix expansion, fibronectin overproduction, glomerular basement membrane thickening, plasma creatinine, and the blood urea nitrogen. The parameters not significantly altered by the Smad3-KO were albuminuria, reduction in podocyte slit pore density, and the increase in vascular endothelial growth factor abundance and activity. It seems that the absence of Smad3 modifies the natural course of murine diabetic nephropathy, providing renal functional protection and preventing structural lesions relating to kidney hypertrophy and matrix accumulation, even though albuminuria and changes in podocyte morphology persist. In conclusion, the effects of the Smad3-KO mirror the effects of anti-TGF-[beta] therapy in diabetes, suggesting that the chief component of TGF-[beta] signaling that is relevant to kidney disease is the Smad3 pathway. streptozotocin; glomerular basement membrane thickening; mesangial matrix expansion; vascular endothelial growth factor; podocyte slit pore density

The contribution of elevated aldosterone to the pathogenesis of malignant, ANG II-dependent hypertension remains uncertain. Therefore, we examined whether chronic mineralocorticoid receptor blockade attenuates the development of malignant hypertension in transgenic rats (TGRs) with inducible expression of the Ren2 gene [TGR(Cyp1a1Ren2)]. Systolic blood pressure (SBP) was measured by radiotelemetry in male TGRs in three groups: 1) control (n = 9), 2) hypertensives (HT; n = 8), and 3) hypertensives + spironolactone (11 mg x [kg.sup.-1] x day sc; HTS; n = 8). Malignant hypertension was induced with dietary indole-3-carbinol (0.3%) for 10 days. Metabolic measurements were taken at the beginning of the study and at day's 2 and 9. HT exhibited elevated SBP (125 [+ or -] 3 vs. 187 [+ or -] 5 mmHg), plasma renin activity (5 [+ or -] 1 vs. 29 [+ or -] 10 ng ANG I x [ml.sup.-1] x [h.sup.-1]), plasma ANG II (175 [+ or -] 39 vs. 611 [+ or -] 74 fmol/ml), and plasma aldosterone (0.31 [+ or -] 0.04 vs. 5.42 [+ or -] 1.02 nmol/l). Urinary aldosterone excretion increased 5.5-fold by day 2 and an additional 90% by day 9. HT was associated with a 1.8-fold increase in proteinuria by day 9 that was alleviated by treatment with spironolactone (25 [+ or -] 5 vs. 13 [+ or -] 3 mg/day), suggesting that aldosterone contributes to the renal damage observed in malignant hypertension. Urinary Na+ excretion was decreased 76% on day 2, despite a sixfold increase in urinary aldosterone excretion. Decrease in urinary [Na.sup.+] excretion on day 2 in HT suggests that [Na.sup.+] reabsorption was increased in response to the increase in aldosterone; however, the lack of a change in SBP between HT and HTS suggests that mechanisms independent of aldosterone stimulation make a greater contribution to the maintenance of elevated arterial pressure in malignant hypertension in Cyp1a1-Ren2 transgenic rats. angiotensin II; eplerenone; pressure natriuresis; spironolactone

Triadin is involved in the regulation of cardiac excitation-contraction coupling. However, the extent of its contribution to the regulation of sarcoplasmic reticulum (SR) Ca release remains unclear, because overexpression of triadin in single-transgenic mice was associated with the downregulation of its homologous protein, junctin. In the present study, this problem was circumvented by cross-breeding of mice with heart-directed overexpression of triadin and junctin (JxT). This resulted in a stable approximately threefold expression of total triadin but unchanged junctin protein. Transgenic mice exhibited cardiac hypertrophy and structural abnormalities of myofibrils. Measurement of cardiac function by echocardiography and edge detection in myocytes revealed an impaired relaxation in JxT mice. The stimulation of [beta]-adrenergic receptors resulted in a depressed contractility and an impaired relaxation in catheterized hearts and myocytes of JxT mice. The use of a maximum stimulation frequency (5 Hz) was associated with both a lower shortening and relengthening in isolated myocytes of JxT mice. The contractile effects in JxT myocytes were paralleled by similar changes of the intracellular Ca concentration ([[Ca].sub.i]) peak amplitude and Ca transient decay kinetics at basal conditions, under administration of isoproterenol, and with high-frequency stimulation. Finally, we found a higher caffeine-induced [[Ca].sub.i] peak amplitude in JxT myocytes. Our data show that the stable expression of triadin, independent of junctin expression, resulted in cardiac hypertrophy, prolonged basal relaxation, a depressed response to [beta]-adrenergic agonists, and altered Ca transients. Thus the maintenance of triadin expression is essential for normal SR Ca cycling and contractile function. transgenic mice; sarcoplasmic reticulum; hypertrophy; Ca signaling; cardiac function; force-frequency relationship; [beta]-adrenergic receptor stimulation

A key endothelial receptor in leukocyte-endothelial cell (EC) interactions is ICAM-1. ICAM-1 is constitutively expressed at low levels on vascular ECs, and its levels significantly increase following stimulation with many proinflammatory agents. This study provides evidence that in inflamed arterioles of anesthetized mice (65 mg/kg ip Nembutal), ICAM-1 mediates leukocyte rolling, in contrast to its expected role of mediating firm adhesion in venules. The number of leukocytes rolling on arteriolar ECs is decreased in ICAM-1 knockout (KO) compared with wild-type (WT) mice (KO, 6.0 [+ or -] 0.9; WT, 12.0 [+ or -] 1.0 leukocytes/40 s; P < 0.05), whereas the leukocyte-rolling number in venules remains unaffected (KO, 5.6 [+ or -] 0.9; WT, 7.0 [+ or -] 0.7 leukocytes/40 s; n = 13-15 sites). We also show that the fraction of leukocytes that is rolling on arteriolar ECs does so with a higher characteristic velocity (>70 [micro]m/s), and, furthermore, that the distance over which rolling contacts with the arteriolar wall are maintained is ICAM-1 dependent. In ICAM-1 KO animals or in WT mice in the presence of ICAM-l-blocking antibody, leukocytes rolled significantly shorter distances over the sampled 200-[micro]m vessel length compared with WT (68 [+ or -] 6.7 and 55 [+ or -] 9.4 vs. 85 [+ or -] 12.9% total, respectively, n = 4 sites, P < 0.05). We also found evidence that in ICAM-1 KO mice, a significant fraction of leukocyte rolling and adhesive interactions with arteriolar ECs could be accounted for by upregulation of another adhesion molecule, VCAM-1, providing an important illustration of how expression of related proteins can be altered following genetic ablatement of a target molecule (in this case ICAM-1). intracellular adhesion molecule-1 knockout mice; intracellular adhesion molecule-1; vascular cell adhesion molecule-1; leukocyte adhesion; leukocyte rolling; tumor necrosis factor-[alpha]; in vivo; intravital confocal microscopy

Reexamination of the [Akp3.sup.-/-] mouse intestine showed that, despite the lack of intestinal alkaline phosphatase (IAP), the [Akp3.sup.-/-] gut still had considerable alkaline phosphatase (AP) activity in the duodenum and ileum. This activity is due to the expression of a novel routine Akp6 gene that encodes an IAP isozyme expressed in the gut in a global manner (gIAP) as opposed to duodenum-specific IAP (dIAP) isozyme encoded by the Akp3 gene. Phylogenetically, gIAP is similar to the rat IAP I isozyme. Kinetically, gIAP displays a 5.7-fold reduction in catalytic rate constant ([k.sub.cat]) and a 30% drop in Kin, leading to a 4-fold reduction [k.sub.cat]/[K.sub.m] compared with dIAP, and these changes in enzymatic properties can all be attributed to a crucial R317Q substitution. Western and Northern blot analyses document the expression of Akp6 in the gut, from the duodenum to the ileum, and it is upregulated in the jejunum and ileum of [Akp3.sup.-/-] mice. Developmentally, Akp3 expression is turned on during postnatal days 13-15 and exclusively in the duodenum, whereas Akp6 and Akp5 are expressed from birth throughout the gut with enhanced expression at weaning. Posttranslational modifications of gIAP have a pronounced effect on its catalytic properties. Given the low catalytic efficiency of gIAP, its upregulation during fat feeding, its sequence similarity with rat IAP I, and the fact that rat IAP I has been implicated in the upregulation of surfactant-like particles during fat intake, it appears likely that gIAP may have a role in mediating the accelerated fatty acid intake observed in [Akp3.sup.-/-] mice fed a high-fat diet. alkaline phosphatases; site-directed mutagenesis; computer modeling; fat absorption; intestinal physiology

We determined whether extrahepatic biliary epithelial cells can differentiate into cells with phenotypic features of hepatocytes. Gallbladders were removed from transgenic mice expressing hepatocyte-specific [beta]-galactosidase ([beta]-Gal) and cultured under standard conditions and under experimental conditions designed to induce differentiation into a hepatocyte-like phenotype. Gallbladder epithelial cells (GBEC) cultured under standard conditions exhibited no [beta]-Gal activity. [beta]-Gal expression was prominent in 50% of cells cultured under experimental conditions. Similar morphological changes were observed in GBEC from green fluorescent protein transgenic mice cultured under experimental conditions. These cells showed higher levels of mRNA for genes expressed in hepatocytes, but not in GBEC, including aldolase B, albumin, hepatocyte nuclear factor-4[alpha], aldehyde dehydrogenase 1, and glutamine synthetase, and they synthesized bile acids. Additional functional evidence of a hepatocyte-like phenotype included LDL uptake and enhanced benzodiazepine metabolism. Connexin-32 expression was evident in murine hepatocytes and in cells cultured under experimental conditions, but not in cells cultured under standard conditions. Notch 1, 2, and 3 and Notch ligand Jagged 1 mRNAs were downregulated in these cells compared with cells cultured under standard conditions. CD34, [alpha]-fetoprotein, and Sca-1 mRNA were not expressed in cells cultured under standard conditions, suggesting that the hepatocyte-like cells did not arise from hematopoietic stem cells or oval cells. These results point to future avenues for investigation into the potential use of GBEC in the treatment of liver disease. stern cells; cell culture; transgenic mice; transdifferentiation; biliary epithelium

Dopamine D2 receptor (D2R) knockout (KO) female mice develop chronic hyperprolactinemia and pituitary hyperplasia. Our objective was to study the expression of the mitogen fibroblast growth factor (FGF2) and its receptor, FGFR1, comparatively in pituitaries from KO and wild-type (WT) female mice. We also evaluated FGF2 subcellular localization and FGF2 effects on pituitary function. FGF2-induced prolactin release showed a similar response pattern in both genotypes, even though basal and FGF2-stimulated release was higher in KO. FGF2 stimulated pituitary cellular proliferation (MTS assay and [[sup.3]H]thymidine incorporation), with no differences between genotypes. FGF2 concentration (measured by ELISA) in whole pituitaries or cultured cells was lower in KO (P < 0.00001 and 0.00014). Immunofluorescence histochemistry showed less FGF2 in pituitaries from KO females and revealed a distinct FGF2 localization pattern between genotypes, being predominantly nuclear in KO and cytosolic in WT pituitaries. Finally, FGF2 could not be detected in the conditioned media from pituitary cultures of both genotypes. FGFR1 levels (Western blot and immunohistochemistry) were higher in pituitaries of KO. Basal concentration of phosphorylated ERKs was lower in KO cells (P = 0.018). However, when stimulated with FGF2, a significantly higher increment of ERK phosphorylation was evidenced in KO cells (P [less than or equal to] 0.02). We conclude that disruption of the D2R caused an overall decrease in pituitary FGF2 levels, with an increased distribution in the nucleus, and increased FGFR1 levels. These results are important in the search for reliable prognostic indicators for patients with pituitary dopamine-resistant prolactinomas, which will make tumor-specific therapy possible. dopaminergic D2 receptor; fibroblast growth factor receptor-1; extracellular signal-regulated kinase phosphorylation; prolactin; immunohistochemistry

Calcitonin gene-related peptide (CGRP) is thought to be a prominent neuropeptide in cardiovascular regulation and neuroimmune modulation. There are two isoforms of CGRP ([alpha]CGRP and [beta]CGRP), and the main CGRP receptors are probably composed of a calcitonin receptor-like receptor (CLR) and a receptor activity-modifying protein (RAMP)1. However, the physiological functions of CGRP that are mediated through the CLR/RAMP1 receptors remain to be clarified. For an improved understanding of the functions, we generated mice deficient in RAMP1, a specific subunit of CGRP receptors, by a conditional gene-targeting technique. The RAMP1deficient mice ([RAMP1.sup.-/-]) exhibited high blood pressure, with no changes in heart rate. [alpha]CGRP was found to have a potent vascular relaxant activity compared with [beta]CGRP in the artery of the WT ([RAMP1.sup.+/+]) mice. The activities of both CGRP isoforms were remarkably suppressed in the arteries of the [RAMP1.sup.-/-] mice. The LPS-induced inflammatory responses of the [RAMP1.sup.-/-] mice revealed a transient and significant increase in the serum CGRP levels and high serum levels of proinflammatory cytokines compared with the [RAMP1.sup.+/+] mice. [alpha]CGRP and [beta]CGRP equally suppressed the production of TNF-[alpha] and IL.-12 in bone marrow-derived dendritic cells stimulated with lipopolysaccharide. Their inhibitory effects were not observed in the bone marrow-derived dendritic cells of the [RAMP1.sup.-/-] mice. These results indicate that CGRP signaling through CLR/RAMP1 receptors plays a crucial role in the regulation of both blood pressure by vascular relaxation and proinflammatory cytokine production from dendritic cells. gene-disrupted mice | calcitonin gene-related peptide | adrenomedullin | neuropeptide | dendritic cells

The interaction of renal basolateral organic anion transporter 3 (Oat3) with commonly used pharmacotherapeutics (e.g., NSAIDs, [beta]-lactams, and methotrexate) has been studied extensively in vitro. However, the in vivo role of Oat3 in drug disposition, in the context of other transporters, glomerular filtration, and metabolism, has not been established. Moreover, recent investigations have identified inactive human OAT3 polymorphisms. Therefore, this investigation was designed to elucidate the in vivo role of Oat3 in the disposition of penicillin G and prototypical substrates using an Oat3 knockout mouse model. Oat3 deletion resulted in a doubling of penicillin's half-life (P < 0.05) and a reduced volume of distribution (P < 0.01), together yielding a plasma clearance that was one-half (P < 0.05, males) to one-third (P < 0.001, females) of that in wild-type mice. Inhibition of Oat3 abolished the differences in penicillin G elimination between genotypes. Hepatic accumulation of penicillin was 2.3 times higher in male knockouts (P < 0.05) and 3.7 times higher in female knockouts (P < 0.001). Female knockouts also exhibited impaired estrone-3-sulfate clearance. Oat3 deletion did not impact p-aminohippurate elimination, providing correlative evidence to studies in Oat1 knockout mice that suggest Oat1 governs tubular uptake of p-aminohippurate. Collectively, these findings are the first to indicate that functional Oat3 is necessary for proper elimination of xenobiotic and endogenous compounds in vivo. Thus Oat3 plays a distinct role in determining the efficacy and toxicity of drugs. Dysfunctional human OAT3 polymorphisms or instances of polypharmacy involving OAT3 substrates may result in altered systemic accumulation of [beta]-lactams and other clinically relevant compounds. kidney; liver; transport; Oat1; drug disposition

Serotonin [5-hydroxytryptamine (5-HT)] biosynthesis depends on two rate-limiting tryptophan hydroxylases (Tph): Tph1, which is expressed in peripheral organs, and Tph2, which is expressed in neurons. Because 5-HT is involved in pulmonary hypertension (PH), we investigated whether genetic variations in Tph1 and/or Tph2 affected PH development in mice. To examine the functional impact of peripheral Tph1 deficiency on hypoxic PH, we used [Tph1.sup.-/-] mice characterized by very low 5-HT synthesis rates and contents in the gut and lung and increased 5-HT synthesis in the forebrain. With chronic hypoxia, 5-HT synthesis in the forebrain increased further. Hypoxic PH, right ventricular hypertrophy, and distal pulmonary artery muscularization were less severe (P < 0.001) than in wild-type controls. The Tph inhibitor p-chlorophenylalanine (100 mg x [kg.sup.-1] x [day.sup.-1]) further improved these parameters. We then investigated whether mouse strains harboring the C1473G polymorphism of the Tph2 gene showed different PH phenotypes during hypoxia. Forebrain Tph activity was greater and hypoxic PH was more severe in C57B1/6 and 129X1/SvJ mice homozygous for the 1473C allele than in DBA/2 and BALB/cJ mice homozygous for the 1473G allele, p-Chlorophenylalanine reduced PH in all groups and abolished the difference in PH severity across mouse strains. Hypoxia increased 5-hydroxytryptophan accumulation but decreased 5-HT contents in the forebrain and lung, suggesting accelerated 5-HT turnover during hypoxia. These results provide evidence that dysregulation of 5-HT synthesis is closely linked to the hypoxic PH phenotype in mice and that Tph1 and Tph2 may contribute to PH development. vascular smooth muscle; transgenic mice

The role of ornithine decarboxylase (ODC) in polyamine metabolism has long been established, but the exact source of ornithine has always been unclear. The arginase enzymes are capable of producing ornithine for the production of polyamines and may hold important regulatory functions in the maintenance of this pathway. Utilizing our unique set of arginase single and double knockout mice, we analyzed polyamine levels in the livers, brains, kidneys, and small intestines of the mice at 2 wk of age, the latest timepoint at which all of them are still alive, to determine whether tissue polyamine levels were altered in response to a disruption of arginase I (AI) and II (AII) enzymatic activity. Whereas putrescine was minimally increased in the liver and kidneys from the AII knockout mice, spermidine and spermine were maintained. ODC activity was not greatly altered in the knockout animals and did not correlate with the fluctuations in putrescine. mRNA levels of ornithine aminotransferase (OAT), antizyme 1 (AZ1), and spermidine/spermine-[N.sup.1]-acetyltransferase (SSAT) were also measured and only minor alterations were seen, most notably an increase in OAT expression seen in the liver of AI knockout and double knockout mice. It appears that putrescine catabolism may be affected in the liver when AI is disrupted and ornithine levels are highly reduced. These results suggest that endogenous arginase-derived ornithine may not directly contribute to polyamine homeostasis in mice. Alternate sources such as diet may provide sufficient polyamines for maintenance in mammalian tissues. ornithine; putrescine; spermidine; spermine; decarboxylase

Cystinuria is a hereditary disorder caused by a defect in the apical membrane transport system for cystine and dibasic amino acids in renal proximal tubules and intestine, resulting in recurrent urolithiasis. Mutations in SLC3A1 and SLC7A9 genes, that codify for rBAT/[b.sup.0,+]AT transporter subunits, cause type A and B cystinuria, respectively. In humans, cystinuria treatment is based on the prevention of calculi formation and its dissolution or breakage. Persistent calculi are treated with thiols [i.e., D-penicillamine (DP) and mercaptopropionylglycine (MPG)] for cystine solubilization. We have developed a new protocol with DP to validate our Slc7a9 knockout mouse model for the study of the therapeutic effect of drugs in the treatment of cystine lithiasis. We performed a 5-wk treatment of individually caged lithiasic mutant mice with a previously tested DP dose. To appraise the evolution of lithiasis throughout the treatment a noninvasive indirect method of calculi quantification was developed: calculi mass was quantified by densitometry of X-ray images from cystinuric mice before and after treatment. Urine was collected in metabolic cage experiments to quantify amino acids in DP-treated and nontreated, nonlithiasic mutant mice. We found significant differences between DP-treated and nontreated knockout mice in calculi size and in urinary cystine excretion. Histopathological analysis showed that globally nontreated mutant mice had more severe and diffuse urinary system damage than DP-treated mice. Our results validate the use of this mouse model for testing the efficacy of potential new drugs against cystinuria. D-Penicillamine treatment; cystinuria model; calculi; noninvasive imaging system

Rats harboring the human renin and angiotensinogen genes (dTGR) feature angiotensin (ANG) II/ hypertension-induced cardiac damage and die suddenly between wk 7 and 8. We observed by electrocardiogram (ECG) telemetry that ventricular tachycardia (VT) is a common terminal event in these animals. Our aim was to investigate electrical remodeling. We used ECG telemetry, noninvasive cardiac magnetic field mapping (CMFM) at wk 5 and 7, and performed in vivo programmed electrical stimulation at wk 7. We also investigated whether or not losartan (Los; 30 mg*[kg.sup.-1]*[day.sup.-1]) would prevent electrical remodeling. Cardiac hypertrophy and systolic blood pressure progressively increased in dTGR compared with Sprague-Dawley (SD) controls. Already by wk 5, untreated dTGR showed increased perivascular and interstitial fibrosis, connective tissue growth factor expression, and monocyte infiltration compared with SD rats, differences that progressed through time. Left-ventricular mRNA expression of potassium channel subunit Kv4.3 and gap-junction protein connexin 43 were significantly reduced in dTGR compared with Los-treated dTGR and SD. CMFM showed that depolarization and repolarization were prolonged and inhomogeneous. Los ameliorated all disturbances. VT could be induced in 88% of dTGR but only in 33% of Los-treated dTGR and could not be induced in SD. Untreated dTGR show electrical remodeling and probably die from VT. Los treatment reduces myocardial remodeling and predisposition to arrhythmias. ANG II target organ damage induces VT. magnetocardiography; noninvasive mapping; double-transgenic rat model; in vivo electrophysiological study

The [Na.sup.+]-[K.sup.+]ATPase (NKA) is a transmembrane protein that sets and maintains the electrochemical gradient by extruding three [Na.sup.+] in exchange for two [K.sup.+]. An important physiological role proposed for vascular smooth muscle NKA is the regulation of blood pressure via modulation of vascular smooth muscle contractility (5). To investigate the relations between the level of NKA in smooth muscle and blood pressure, we developed mice carrying a transgene for either the NKA [[alpha].sub.1]- or [[alpha].sub.2]-isoform ([[alpha].sub.1sm+] or [[alpha].sub.2sm+] mice) driven by the smooth muscle-specific [alpha]-actin promoter SMP8. Interestingly, both [alpha]-isoforms, the one contained in the transgene and the one not contained, were increased to a similar degree at both protein and mRNA levels. The total [alpha]-isoform protein was increased from 1.5-fold ([[alpha].sub.1sm+] mice) to 7-fold ([[alpha].sub.2sm+] mice). The increase in total NKA [alpha]-isoform protein was accompanied by a 2.5-fold increase in NKA activity in [[alpha].sub.2sm+] gastric antrum. Immunocytochemistry of the [[alpha].sub.1]- and [[alpha].sub.2]-isoforms in [[alpha].sub.2sm+] aortic smooth muscle cells indicated that [alpha]-isoform distributions were similar to those shown in wild-type cells, [[alpha].sub.2sm+] Mice (high expression) were hypotensive (109.9 [+ or -] 1.6 vs. 121.3 [+ or -] 1.4 mmHg; n = 13 and 11, respectively), whereas [[alpha].sub.1sm+]+ mice (low expression) were normotensive (122.7 [+ or -] 2.5 vs. 117.4 [+ or -] 2.3; n = 11 or 12). [[alpha].sub.2sm+] Aorta, but not [[alpha].sub.1sm+] aorta, relaxed faster from a KCl-induced contraction than wild-type aorta. Our results show that smooth muscle displays unique coordinate expression of the [alpha]-isoforrns. Increasing smooth muscle NKA decreases blood pressure and is dependent on the degree of increased [alpha]-isoform expression. vascular contractility; hypertension; [Na.sup.+]-[K.sup.+]-ATPase [alpha]-isoforms

While several transgenic mouse models exhibit improved contractile characteristics in the heart, less is known about how these changes influence energy metabolism, specifically the balance between carbohydrate and fatty acid oxidation. In the present study we examine glucose and fatty acid oxidation in transgenic mice, generated to overexpress sarco(endo) plasmic reticulum calcium-ATPase (SERCA), which have an enhanced contractile phenotype. Energy substrate metabolism was measured in isolated working hearts using radiolabeled glucose and palmitate. We also examined oxygen consumption to see whether SERCA overexpression is associated with increased oxygen utilization. Since SERCA is important in calcium handling within the cardiac myocyte, we examined cytosolic calcium transients in isolated myocytes using indo-1, and mitochondrial calcium levels using pericam, an adenovirally expressed, mitochondrially targeted ratiometric calcium indicator. Oxygen consumption did not differ between wildtype and SERCA groups; however, we were able to show an increased utilization of glucose for oxidative metabolism and a corresponding decreased utilization of fatty acids in the SERCA group. Cytosolic calcium transients were increased in myocytes isolated from SERCA mice, and they show a faster rate of decay of the calcium transient. With these observations we noted increased levels of mitochondrial calcium in the SERCA group, which was associated with an increase in the active form of the pyruvate dehydrogenase complex. Since an increase in mitochondrial calcium levels leads to activation of the pyruvate dehydrogenase complex (the rate-limiting step for carbohydrate oxidation), the increased glucose utilization observed in isolated perfused hearts in the SERCA group may reflect a higher level of mitochondrial calcium. mitochondrial calcium; pyruvate dehydrogenase; cardiac energetics; sarco(endo)plasmic reticulum calcium-adenosine 5'-triphosphatase

Constitutive myostatin gene knockout in mice causes excessive muscle growth during development. To examine the effect of knocking out the myostatin gene after muscle has matured, we generated mice in which myostatin exon 3 was flanked by loxP sequences (Mstn[f/f]) and crossed them with mice bearing a tamoxifen-inducible, ubiquitously expressed Cre recombinase transgene. At 4 mo of age, Mstn[f/f]/Cre+ mice that had not received tamoxifen had a 50-90% reduction in myostatin expression due to basal Cre activity but were not hypermuscular relative to Mstn[w/w]/Cre+ mice (homozygous for wild-type myostatin gene). Three months after tamoxifen treatment (initiated at 4 mo of age), muscle mass had not changed from the pretreatment level in Mstn[w/w]/Cre+ control mice. Tamoxifen administration to 4-mo-old Mstn[f/f]/Cre+ mice reduced myostatin mRNA expression to less than 1% of normal, which increased muscle mass ~25% over the following 3 mo in both male and female mice (P < 0.005 vs. control). Fiber hypertrophy appeared to be sufficient to explain the increase in muscle mass. The pattern of expression of genes encoding the various myosin heavy-chain isoforms was unaffected by postdevelopmental myostatin knockout. We conclude that, even after developmental muscle growth has ceased, knockout of the myostatin gene induces a significant increase in muscle mass. Cre recombinase; tamoxifen; muscle fiber hypertrophy; myosin heavy chains; conditional knockout