Search

Rationale: Platelets are known to play a crucial role in hemostasis. Sphingosine kinases (Sphk) 1 and 2 catalyze the conversion of sphingosine to the bioactive metabolite sphingosine 1-phosphate (S1P). Although platelets are able to secrete S1P on activation, little is known about a potential intrinsic effect of S1P on platelet function., Objective: To investigate the role of Sphk1- and Sphk2-derived S1P in the regulation of platelet function., Methods and Results: We found a 100-fold reduction in intracellular S1P levels in platelets derived from Sphk2(-/-) mutants compared with Sphk1(-/-) or wild-type mice, as analyzed by mass spectrometry. Sphk2(-/-) platelets also failed to secrete S1P on stimulation. Blood from Sphk2-deficient mice showed decreased aggregation after protease-activated receptor 4-peptide and adenosine diphosphate stimulation in vitro, as assessed by whole blood impedance aggregometry. We revealed that S1P controls platelet aggregation via the sphingosine 1-phosphate receptor 1 through modulation of protease-activated receptor 4-peptide and adenosine diphosphate-induced platelet activation. Finally, we show by intravital microscopy that defective platelet aggregation in Sphk2-deficient mice translates into reduced arterial thrombus stability in vivo., Conclusions: We demonstrate that Sphk2 is the major Sphk isoform responsible for the generation of S1P in platelets and plays a pivotal intrinsic role in the control of platelet activation. Correspondingly, Sphk2-deficient mice are protected from arterial thrombosis after vascular injury, but have normal bleeding times. Targeting this pathway could therefore present a new therapeutic strategy to prevent thrombosis., (© 2015 American Heart Association, Inc.)

The aims of this study were to 1) determine whether renal localization of aliskiren and its antihypertensive and renoprotective effects persist after administration of the drug is stopped and 2) define the renal localization of aliskiren by light microscopy autoradiography. Hypertensive double transgenic rats (dTGR) overexpressing genes for human renin and angiotensinogen were treated with aliskiren (3 mg·kg(-1)·day(-1) sc; osmotic minipumps) or enalapril (18 mg/l in drinking water). After a 2-wk treatment, dTGR were assigned to either continued treatment with aliskiren ("continued"), or to cessation of their respective treatment ("stopped") for a 3-wk washout. One week of treatment with aliskiren and enalapril reduced blood pressure and albuminuria vs. baseline. After cessation of either treatment, blood pressure had returned to pretreatment levels and albuminuria remained relatively low for 1 wk, but rose thereafter similarly in both groups. In contrast, renal mRNA for transforming growth factor-β and renal collagen IV was reduced by aliskiren (continued and stopped groups), but not after cessation of enalapril. Similar patterns were found for collagen IV protein expression. Even 3 wk after stopping aliskiren treatment, renal levels of the drug exceeded its IC50, whereas enalaprilat was not detected. To localize aliskiren accumulation, Wistar rats were treated with [(3)H]-aliskiren for 7 days. Autoradiography demonstrated specific labeling in glomeruli, arterioles, and afferent arterioles as well as in the distal nephron. Labeling could still be observed even after 7 days' washout. These results suggest that the renophilic properties of aliskiren are different from enalapril and could have contributed to the renoprotective mechanism of this renin inhibitor.

Brown adipose tissue (BAT) is a key tissue for energy expenditure via fat and glucose oxidation for thermogenesis. In this study, we demonstrate that the myostatin/activin receptor IIB (ActRIIB) pathway, which serves as an important negative regulator of muscle growth, is also a negative regulator of brown adipocyte differentiation. In parallel to the anticipated hypertrophy of skeletal muscle, the pharmacological inhibition of ActRIIB in mice, using a neutralizing antibody, increases the amount of BAT without directly affecting white adipose tissue. Mechanistically, inhibition of ActRIIB inhibits Smad3 signaling and activates the expression of myoglobin and PGC-1 coregulators in brown adipocytes. Consequently, ActRIIB blockade in brown adipose tissue enhances mitochondrial function and uncoupled respiration, translating into beneficial functional consequences, including enhanced cold tolerance and increased energy expenditure. Importantly, ActRIIB inhibition enhanced energy expenditure only at ambient temperature or in the cold and not at thermoneutrality, where nonshivering thermogenesis is minimal, strongly suggesting that brown fat activation plays a prominent role in the metabolic actions of ActRIIB inhibition.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson's disease (PD), but the underlying pathophysiological mechanisms and the normal function of this large multidomain protein remain speculative. To address the role of this protein in vivo, we generated three different LRRK2 mutant mouse lines. Mice completely lacking the LRRK2 protein (knock-out, KO) showed an early-onset (age 6 weeks) marked increase in number and size of secondary lysosomes in kidney proximal tubule cells and lamellar bodies in lung type II cells. Mice expressing a LRRK2 kinase-dead (KD) mutant from the endogenous locus displayed similar early-onset pathophysiological changes in kidney but not lung. KD mutants had dramatically reduced full-length LRRK2 protein levels in the kidney and this genetic effect was mimicked pharmacologically in wild-type mice treated with a LRRK2-selective kinase inhibitor. Knock-in (KI) mice expressing the G2019S PD-associated mutation that increases LRRK2 kinase activity showed none of the LRRK2 protein level and histopathological changes observed in KD and KO mice. The autophagy marker LC3 remained unchanged but kidney mTOR and TCS2 protein levels decreased in KD and increased in KO and KI mice. Unexpectedly, KO and KI mice suffered from diastolic hypertension opposed to normal blood pressure in KD mice. Our findings demonstrate a role for LRRK2 in kidney and lung physiology and further show that LRRK2 kinase function affects LRRK2 protein steady-state levels thereby altering putative scaffold/GTPase activity. These novel aspects of peripheral LRRK2 biology critically impact ongoing attempts to develop LRRK2 selective kinase inhibitors as therapeutics for PD.

In vitro, concentrations ≥ 10 μM of nilotinib were needed to induce markers of cytotoxicity, apoptosis, and endoplasmic reticulum stress in both neonatal rat ventricular myocytes, a putative target tissue, and non-target heart fibroblasts, indicating a lack of cardiomyocyte-specific nilotinib toxicity in vitro. In rats, oral nilotinib treatment at 80 mg/kg for 4 weeks induced increased heart weight; however, this was not associated with relevant histopathological changes or effects on heart function. Thus, nilotinib at and above clinically relevant concentrations (4.27 μM) did not induce overt cardiovascular pathologies or heart failure in vitro or in vivo under study conditions., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Cytotoxic concentrations of imatinib mesylate (10-50 microM) were required to trigger markers of apoptosis and endoplasmic reticulum stress response in neonatal rat ventricular myocytes and fibroblasts, with no significant differences observed between c-Abl silenced and nonsilenced cells. In mice, oral or intraperitoneal imatinib treatment did not induce cardiovascular pathology or heart failure. In rats, high doses of oral imatinib did result in some cardiac hypertrophy. Multi-organ toxicities may have increased the cardiac workload and contributed to the cardiac hypertrophy observed in rats only. These data suggest that imatinib is not cardiotoxic at clinically relevant concentrations (5 microM)., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The aim of this study was to explore the effects of the renin inhibitor aliskiren in streptozotocin-diabetic TG(mRen-2)27 rats. Furthermore, we investigated in vitro the effect of aliskiren on the interactions between renin and the (pro)renin receptor and between aliskiren and prorenin. Aliskiren distributed extensively to the kidneys of normotensive (non)diabetic rats, localizing in the glomeruli and vessel walls after 2 hours exposure. In diabetic TG(mRen-2)27 rats, aliskiren (10 or 30 mg/kg per day, 10 weeks) lowered blood pressure, prevented albuminuria, and suppressed renal transforming growth factor-beta and collagen I expression versus vehicle. Aliskiren reduced (pro)renin receptor expression in glomeruli, tubules, and cortical vessels compared to vehicle (in situ hybridization). In human mesangial cells, aliskiren (0.1 micromol/L to 10 micromol/L) did not inhibit binding of (125)I-renin to the (pro)renin receptor, nor did it alter the activation of extracellular signal-regulated kinase 1/2 by renin (20 nmol/L) preincubated with aliskiren (100 nmol/L) or affect gene expression of the (pro)renin receptor. Evidence was obtained that aliskiren binds to the active site of prorenin. The above results demonstrate the antihypertensive and renoprotective effects of aliskiren in experimental diabetic nephropathy. The evidence that aliskiren can reduce in vivo gene expression for the (pro)renin receptor and that it may block prorenin-induced angiotensin generation supports the need for additional work to reveal the mechanism of the observed renoprotection by this renin inhibitor.

FTY720 [2-amino-2-[2-(4-octylphenyl) ethyl]propane-1,3-diol hydrochloride] is an oral sphingosine-1-phosphate receptor modulator under development for the treatment of multiple sclerosis (MS). The drug is phosphorylated in vivo by sphingosine kinase 2 to its bioactive form, FTY720-P. Although treatment with FTY720 is accompanied by a reduction of the peripheral lymphocyte count, its efficacy in MS and experimental autoimmune encephalomyelitis (EAE) may be due to additional, direct effects in the central nervous system (CNS). We now show that FTY720 localizes to the CNS white matter, preferentially along myelin sheaths. Brain trough levels of FTY720 and FTY720-P in rat EAE are of the same magnitude and dose dependently increase; they are in the range of 40 to 540 ng/g in the brain tissue at efficacious doses and exceed blood concentrations severalfold. In a rat model of chronic EAE, prolonged treatment with 0.03 mg/kg was efficacious, but limiting the dosing period failed to prevent EAE despite a significant decrease in blood lymphocytes. FTY720 effectiveness is likely due to a culmination of mechanisms involving reduction of autoreactive T cells, neuroprotective influence of FTY720-P in the CNS, and inhibition of inflammatory mediators in the brain.

The purpose of this study was to establish and validate fully automatic measurement of cell proliferation on scanned images of rat cecum and colon. Tissue slides were taken from a 4-week mechanistic study and processed for BrdU immunohistochemistry. Four sections of the cecum and colon per slide were scanned with the Zeiss MIRAX SCAN and transferred to the Definiens eCognition Analyst LS5.0 system for evaluation. Two rule sets for automatic counting of BrdU-positive and negative nuclei from mucosal cells on the image tiles were created by Definiens, one for cecum, one for colon. For validation, manual counting of 16 randomly selected tiles from five different slides of colon and cecum was performed. Negative and positive cell nuclei were counted in each image tile by four different people. Comparison of results from manual counting with the automatic counting showed that the sum as well as single tile data and labeling index (LI) from automatic counting were within the range of manual counting results +/-10%. Automatic counting included only cell nuclei within the mucosa whereas muscularis and lymphoid tissue as well as wrinkles from tissue preparation were excluded. In addition, two data sets from automatic counting of the same image tile were compared: (1) data where image tiles with incorrect detection of mucosa were excluded from further calculation of LI and area, and (2) data where no visual check was performed and all measurements were included. Results were very similar for both data sets. The necessity of the manual correction may therefore be doubted.

ASM 981 has been developed for topical treatment of inflammatory skin diseases. It specifically inhibits the production and release of pro-inflammatory cytokines. We measured the skin penetration of ASM 981 in canine skin and compared penetration in living and frozen skin. To make penetration of ASM 981 visible in dog skin, tritium labelled ASM 981 was applied to a living dog and to defrosted skin of the same dog. Using qualitative autoradiography the radioactive molecules were detected in the lumen of the hair follicles until the infundibulum, around the superficial parts of the hair follicles and into a depth of the dermis of 200 to 500 microm. Activity could not be found in deeper parts of the hair follicles, the dermis or in the sebaceous glands. Penetration of ASM 981 is low in canine skin and is only equally spread in the upper third of the dermis 24 hours after application. Penetration in frozen skin takes even longer than in living canine skin but shows the same distribution.

The experimentally induced neurotoxic effects of paclitaxel and docetaxel have never been compared, since no animal models of docetaxel peripheral neurotoxicity have yet been reported. In this experiment, we examined the effect of the chronic administration of these two taxanes in the Wistar rat using neurophysiological, neuropathological and morphometrical methods. Our results showed that both paclitaxel and docetaxel induced a significant, equally severe and dose-dependent reduction in nerve conduction velocity. On the contrary, the morphometric examination demonstrated that the effect on the nerve fibres was more severe after paclitaxel administration when the same schedule was used. However, the overall severity of the pathological changes was milder than expected on the basis of the neurophysiological results. Our results support the hypothesis that taxanes (and particularly docetaxel) may exert their neurotoxic effect not only on the microtubular system of the peripheral nerves, but also on other less obvious targets.

Purpose: To describe dose-dependent signal intensity (SI) characteristics of experimentally induced soft-tissue abscesses on 1.5-T T1- and T2*-weighted magnetic resonance (MR) images obtained 24 hours after administration of ultrasmall superparamagnetic iron oxide (USPIO) and to describe the relationship between SI and amount of USPIO uptake and macrophage iron content., Materials and Methods: Local institutional review committee on animal care approved the experiments, which were performed according to the guidelines of the National Institutes of Health and the committee on animal research at our institution. Unilateral calf muscle abscesses were induced in 21 rats with an injection of a Staphylococcus aureus suspension. The rats were divided into three groups of seven animals each: low USPIO dose (50 micromol of iron per kilogram of body weight), high USPIO dose (150 micromol Fe/kg), and control (saline solution). All rats were imaged before and 24 hours after USPIO administration at 1.5 T (transverse T1-weighted spin-echo, T2*-weighted fast gradient-echo, and short inversion time inversion-recovery sequences). Images were analyzed quantitatively and qualitatively with regard to SI and signal pattern. Temporal variation of calculated contrast-to-noise ratios was analyzed with the Wilcoxon signed rank test. MR findings were correlated with histopathologic findings, including those of electron microscopy., Results: Twenty-four hours after USPIO administration in the high-dose group, susceptibility effects were present in abscess periphery on postcontrast T2*-weighted images (P=.04), and SI enhancement was noted on postcontrast T1-weighted images within both abscess wall and abscess center (P=.04 for both). In the low-dose group, SI enhancement was noted in entire abscess on T1-weighted postcontrast images (P=.03). Neither significant SI loss (P=.09) nor susceptibility effects were detected in periphery or center of any abscess on postcontrast T2*-weighted images. There was no obvious difference in total amount of macrophages among the groups, but there was a clear difference with regard to individual iron content of iron-positive macrophages between the USPIO dose groups., Conclusion: At 1.5 T, SI characteristics of abscesses on T1- and T2*-weighted images obtained 24 hours after USPIO injection strongly depend on administered dose of the contrast agent. At low doses, T1 effects were stronger than T2* effects.

Spontaneously occurring and chemically induced pheochromocytomas are rare in mice. That the mouse pheochromocytoma is a more appropriate animal model than that of the rat for study of human medullary adrenal tumors has been suggested. The expression of phenylethanolamine-N-methyltransferase (PNMT), the enzyme responsible for production of epinephrine from norepinephrine, is common to both mouse and human pheochromocytomas. This investigation assessed the expression of the immunohistochemical markers PNMT, tyrosine hydroxylase (TH), and chromogranin A (CGA) in spontaneously occurring and chemically induced pheochromocytomas in the B6C3F1 mouse. Spontaneous tumors were derived from control animals from 10 different studies and the pheochromocytomas from treated groups from 4 different studies. All tumors were positive for maximal TH expression. A highly significant difference in PNMT expression (p < 0.01) occurred between spontaneously occurring pheochromocytomas classified as benign or "malignant" by the criteria of toxicologic pathology. Chemically induced tumors showed intermediate PNMT staining. A marked reduction in CGA expression occurred in pheochromocytomas induced by technical grade pentachlorophenol, compared to the other three chemicals and the spontaneously occurring tumors. These findings suggest that immunohistochemistry is a reliable tool in investigating the functional capabilities of pheochromocytomas in mice. PNMT expression is a tightly regulated component of the chromaffin cell phenotype and appears to be readily lost in mouse pheochromocytomas, particularly those with aggressive characteristics.

Preformed and elicited Ab's against the Galalpha1,3Gal terminating carbohydrate chains (alphaGal Ab's) are the primary cause of hyperacute and acute vascular xenograft rejection in pig-to-primate transplantation. alphaGal Ab's are produced by long-lived Ab-producing cells that are not susceptible to pharmacological immunosuppression. We reasoned that antigen-specific elimination of alphaGal Ab's might be achieved in vivo by systemic administration of nonimmunogenic polyvalent alphaGal structures with high avidity for alphaGal Ab's. We devised GAS914, a soluble trisaccharide-polylysine conjugate of approximately 500 kDa that effectively competes for alphaGal binding by alphaGal IgM (IC(50), 43 nM) and IgG (IC(50), 28 nM) in vitro. Injections of GAS914 in cynomolgus monkeys, at the dose of 1 mg/kg, resulted in the immediate decrease of more than 90% of circulating alphaGal Ab's and serum anti-pig cytotoxicity. In baboons, repeated injections of GAS914 effectively reduced both circulating alphaGal Ab's and cytotoxicity over several months. Studies with [(14)C]GAS914 in rhesus monkeys and Gal(-/-) mice indicate that GAS914 binds to circulating alphaGal Ab's and that the complex is quickly metabolized by the liver and excreted by the kidney. Remarkably, posttreatment alphaGal Ab titers never exceeded pretreatment levels and no sensitization to either alphaGal or the polylysine backbone has been observed. Furthermore there was no apparent acute or chronic toxicity associated with GAS914 treatment in primates. We conclude that GAS914 may be used therapeutically for the specific removal of alphaGal Ab's.

Combination of nonhypotensive doses of valsartan and enalapril markedly improved survival (+87%) compared with untreated animals (37%) in spontaneously hypertensive rats (SHRs) with endothelial dysfunction. However, the combination had no effect on kidney function, and proteinuria persisted over the 12 weeks of the study. It was hypothesized that the greater survival was due to improvement in endothelial function or coronary vasculature despite blockade of nitric oxide synthase and high blood pressure. Therefore, endothelial function was evaluated in isolated aortic ring and maximal coronary blood flow was studied in isolated perfused SHR hearts (20-24 weeks) treated with -nitro-l-arginine methyl ester (L-NAME) (50 mg/l) for 4 weeks. The animals received vehicle, valsartan 5 mg/kg/d, enalapril 1 mg/kg/d, valsartan 50 mg/kg/d, or the combination valsartan 5 mg/kg/d with enalapril 1 mg/kg/d in drinking water. Normotensive Wistar-Kyoto (WKY) rats were used as control. Blood pressure was measured by telemetry. Histopathology was performed on heart, kidney (hematoxylin-eosin), and aorta (Masson trichrome). L-NAME elevated blood pressure by 50 mm Hg after vehicle (199 +/- 5 mm Hg). Valsartan 50 mg/kg/d completely abolished this increase (150 +/- 4 mm Hg) whereas the valsartan-enalapril combination synergistically decreased blood pressure (-37 mm Hg at 162 +/- 7 mm Hg) compared with monotherapy (valsartan 5 mg/kg/d -10 mm Hg; enalapril 1 mg/kg/d -15 mm Hg). All treatments improved the histopathology, most markedly in those receiving the valsartan-enalapril combination. The severity mean grades for lesions were 2.1, 1.9, 1.7, 1.1, and 0.9 in vehicle-treated SHRs, enalapril 1 mg/kg/d, valsartan 5 mg/kg/d, valsartan 5 or 50 mg/kg/d, and the valsartan-enalapril combination, respectively, compared with 0.02 in WKY rats. Acetylcholine-induced relaxation was significantly greater in treated SHRs than after vehicle (-40% at 0.1 mmol acetylcholine) but the combination induced the maximal relaxation (-85%). The ratio of maximal tension induced by serotonin in rings with and without endothelium was 1.4 and 1.3 in vehicle and valsartan 5 mg/kg/d-treated rats whereas it did not differ from 1 in WKY rats and all other treated groups. The cardiac hypertrophy (+27%) was prevented by valsartan 50 mg/kg/d and the valsartan-enalapril combination. Coronary reserve was significantly increased by valsartan 50 mg/kg/d (+85% at 7.8 +/- 0.7 ml/min/g) and the valsartan-enalapril combination (+42% at 6.0 +/- 0.4 ml/min/g) compared with 4.2 +/- 0.5 (vehicle). This was not different of 8.8 +/- 0.5 (WKYs). Despite the maintenance of a high blood pressure, low-dose valsartan-enalapril significantly improved endothelial function and histopathology and increased coronary reserve in SHRs chronically receiving L-NAME.

PTK787/ZK 222584 (1-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, active in the submicromolar range. It also inhibits other class III kinases, such as the platelet-derived growth factor (PDGF) receptor beta tyrosine kinase, c-Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor families, such as epidermal growth factor receptor, fibroblast growth factor receptor-1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and protein kinase C-alpha. PTK787/ZK 222584 inhibits VEGF-induced autophosphorylation of kinase insert domain-containing receptor (KDR), endothelial cell proliferation, migration, and survival in the nanomolar range in cell-based assays. In concentrations up to 1 microM, PTK787/ZK 222584 does not have any cytotoxic or antiproliferative effect on cells that do not express VEGF receptors. After oral dosing (50 mg/kg) to mice, plasma concentrations of PTK787/ZK 222584 remain above 1 microM for more than 8 h. PTK787/ZK 222584 induces dose-dependent inhibition of VEGF and PDGF-induced angiogenesis in a growth factor implant model, as well as a tumor cell-driven angiogenesis model after once-daily oral dosing (25-100 mg/kg). In the same dose range, it also inhibits the growth of several human carcinomas, grown s.c. in nude mice, as well as a murine renal carcinoma and its metastases in a syngeneic, orthotopic model. Histological examination of tumors revealed inhibition of microvessel formation in the interior of the tumor. PTK787/ZK 222584 is very well tolerated and does not impair wound healing. It also does not have any significant effects on circulating blood cells or bone marrow leukocytes as a single agent or impair hematopoetic recovery after concomitant cytotoxic anti-cancer agent challenge. This novel compound has therapeutic potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.

The immunosuppressive cyclosporine A derivative, O-hydroxyethyl-D(Ser)(8)-cyclosporine (SDZ IMM 125), was examined for its ability to induce apoptosis in rat hepatocytes cultured for 4 or 20 h. Four hours after SDZ IMM 125 treatment, chromatin condensation and fragmentation, and the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeled and Annexin V-positive cells increased dose dependently without any observable lactate dehydrogenase leakage. The activity of the cysteine protease, caspase-3, was increased, but not that of caspase-1 and -6. The specific caspase-3 inhibitor, Ac-Asp-Glu-Val-Asp-aldehyde, inhibited caspase-3 activation and attenuated SDZ IMM 125-induced apoptosis and lactate dehydrogenase leakage. After 20 h of SDZ IMM 125 incubation, the parameters of apoptosis were further increased. Decreased mitochondrial membrane potential (measured by rhodamine 123 uptake) and cytochrome c release went in parallel with ultrastructural mitochondrial changes, and might be regarded as early events that trigger the apoptotic cascade. Transmission electron microscopy showed cytoplasmic blebbing after 4 h of SDZ IMM 125 incubation. As observed by transmission electron microscopy, treatment with SDZ IMM 125 resulted in an increase in the number of necrotic cells after 20 h, but not after 4 h. Our findings suggest that in rat hepatocyte cultures, SDZ IMM 125 is a specific inducer of apoptosis after short-term incubation, and this overlaps with necrosis after longer treatment periods. It is very likely that the necrosis occurring later is the result of the early apoptotic events.

In rat hepatocytes and isolated liver mitochondrial fractions, Cyclosporine A (CsA) is often used as a specific inhibitor of mitochondrial Ca(2+) release and as a specific blocker of mitochondrial membrane potential and permeability transition (MPT), which are all processes involved in the inhibition of apoptosis. However, neither inhibition nor induction of apoptosis by CsA has yet been described in the rat hepatocyte primary culture during incubation for 4 and 20 h. It was the purpose of the present study to examine by means of morphological and biochemical criteria the effects of CsA on apoptosis and to characterize the underlying mechanisms. Rat hepatocytes were cultured for 4 or 20 h with CsA at concentrations of 0, 10, 25, and 50 microM. Chromatin condensation and fragmentation, DNA fragmentation (TUNEL), membrane phosphatidylserine distribution (Annexin V), caspase-1, -3, and -6 activity, mitochondrial membrane potential (Rhodamine 123), and cytochrome c release into the cytosol were investigated. Four hours after CsA treatment, chromatin condensation and fragmentation and the number of TUNEL- and Annexin V-positive cells increased dose-dependently without any observable enzyme leakage, which indicated the integrity of the outer cell membrane. After 20 h of CsA incubation apoptosis parameters were further increased and were accompanied by the increased activity of the cysteine protease, caspase-3 (CPP 32), and slightly increased caspase-6 (Mch 2), but not caspase-1 (ICE). The caspase-3 inhibitor, Ac-DEVD-CHO, inhibited caspase-3 activation and attenuated CsA-induced apoptosis and LDH leakage. The caspase-6 inhibitor, Ac-VEID-CHO, only marginally inhibited CsA-induced apoptosis. Decreased mitochondrial membrane potential and cytochrome c release went in parallel with ultrastructural mitochondrial changes and might be regarded as early events that trigger the apoptosis cascade. Transmission electron microscopy confirmed an increase in the number of necrotic cells after 20 h, but not after 4 h, compared with controls., (Copyright 2000 Academic Press.)

Intestinal fatty acid-binding protein (I-FABP) is a cytosolic protein expressed at high levels (up to 2% of cytosolic proteins) in the small intestine epithelium. Despite cell transfection studies, its function is still unclear. Indeed, different effects on fatty acid metabolism depending on the cell type and the amount of I-FABP expressed have been reported. Furthermore, a decrease in fatty acid incorporation has been unexpectedly obtained when I-FABP reached 0. 72% of cytosolic proteins in fibroblasts (Prows et al. 1997. Arch. Biochem. Biophys. 340: 135). In the present study, the effect of a high level of I-FABP similar to amounts present in the small intestine was investigated in the human colon adenocarcinoma cell line, Caco-2. After transfection with human I-FABP cDNA, a clone expressing 1.5% I-FABP and unchanged level of liver FABP was selected. These cells, which had a lower rate of proliferation as compared with mock-transfected cells, developed the typical morphological characteristics of differentiated enterocytes. Incubation of differentiated cells with [(14)C]palmitate showed a 34% reduction (P < 0.01) of fatty acid incorporation, whereas the relative distribution of radiolabel into triglycerides was not affected. A nonsignificant 21% reduction of fatty acid incorporation was observed with another clone expressing 10-fold less I-FABP. In conclusion, a high level of I-FABP expressed in a differentiated enterocyte model inhibited fatty acid incorporation, by a mechanism which remains to be defined.

Craniopharyngioma is a rare neoplasm in the rat, and few cases have been described. These lesions are thought to originate from squamous cell remnants of Rathke's pouch, an evagination of primitive stomatodeum. This neoplasm is usually locally invasive, and neither cranial nor extracranial metastases have been described. A spontaneously occurring malignant, metastasizing craniopharyngioma arising from the neurohypophysis was detected in a 2-year-old male albino rat. The infiltrative growth was observed in the wall of the vessels of the circle of Willis, in the perivascular space of Virchow and Robin, in the submeningeal space near the hypothalamus, through the fissura chorioidea, in the medulla oblongata, and along the optic nerve into the periocular region. Metastases were detected in the thalamus and hippocampus. The diagnosis was made on the basis of microscopic, immunocytochemical and ultrastructural findings.

A method based on image analysis and applicable in pharmacology and toxicology is described that has been designed to meet the statistical requirements for the determination of liver hypertrophy or hyperplasia. An algorithm has been developed to detect and count fluorescent nuclei in Feulgen-stained liver sections (12,500 to 25,000 nuclei or 170 to 350 fields of observation per section within 2-4 hr) by means of fully automatic image analysis with the Leitz Texture Analysis System (TAS) (up to 23 sections in series on the microscope stage). The applicability of this method to conventionally derived, formaldehyde-fixed, tissue sections has been tested on 3-microns sections cut from archived paraplast blocks of already diagnosed materials. Alterations in the liver induced by eight different compounds administered perorally for 28 days to rats have been studied. The morphometric results were found to be specific for a given compound and revealed reproducible and statistically significant dose dependencies of hypertrophy or hyperplasia, or both. Increased frequencies of mitotic figures observed by eye correlated reasonably well with the morphometrically determined hyperplasia, but many hyperplastic livers revealed no mitotic figures. By contrast, the histological diagnoses of an increased degree of hypertrophy were only poorly correlated with the morphometric determinations and, in many instances, were made in livers showing no decrease in the frequency of nuclei. On the other hand, the results from electron microscopy agreed well with the corresponding morphometric analyses: in cases with predominant hypertrophy the proliferation of the smooth endoplasmic reticulum was distinct or striking, or the peroxisomes were altered and increased in number, whereas no obvious ultrastructural changes were seen in cases of morphometrically exclusive hyperplasia.

A monoclonal antibody raised against and specific for cytochrome P-450 isoenzyme CYP4A1 was used to investigate the subcellular distribution of this enzyme in the liver, kidney and ileum of nafenopin treated rats by means of immunoelectron microscopy. In the liver and kidney, labelling was restricted to peroxisomes and mitochondria of hepatocytes and proximal tubular epithelial cells whereas in ileum, immunolabelling was exclusively detected in mitochondria of absorptive cells.

The expression of the neural cell adhesion molecules L1 and N-CAM was investigated in developing postnatal mouse hippocampus by immunocytochemical techniques at the light and electron microscopic levels. In the 1, 8 and 21-day-old hippocampus, L1 was only observed on fasciculating axons. L1 was not detectable on dendrites and cell bodies of pyramidal cells, granule cells and interneurons in any of the hippocampal regions studied. Also, synapses were never found to be L1-immunoreactive either pre- or postsynaptically. L1 was not detectable at contacts between astrocytes and axons. Polyclonal N-CAM antibodies reacting with the three components of N-CAM (N-CAM total) stained all neuronal and glial cell types in the 1, 8 and 21-day-old hippocampus. In contrast, the 180 kDa component of N-CAM (N-CAM 180) was only detectable on neuronal cell bodies and dendrites of the 1 and 8-day-old hippocampus. In the 21-day-old hippocampus N-CAM 180 was not recognized on neuronal cell bodies. N-CAM 180 was strongly expressed in postsynaptic sites at all ages studies. We infer from these observations that L1 is predominantly involved in axon fasciculation, whereas N-CAM 180 appears to be more characteristic of stabilizing cell contacts, particularly at the synapse.

GABAA receptors, the major synaptic targets for the neurotransmitter GABA, constitute gated chloride channels. By their allosteric, drug-induced modulation, they serve as control elements for the regulation of anxiety, vigilance, and epileptiform activity. The structural requirements of fully functional GABAA receptors in the mammalian brain have remained elusive so far. We report here on the cloning of the gamma 2-subunit cDNA of rat brain and its functional analysis by coexpression with the alpha 1- and beta 1-subunits in Xenopus oocytes, and on the sites of gene expression of the 3 subunits in the rat brain. The recombinant receptor displayed GABA-inducible currents (Imax = 6 microA; Ka = 75 microM) which were allosterically modulated by benzodiazepine receptor ligands (enhancement and inhibition by diazepam and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, respectively). In the absence of GABA, pentobarbital elicited a maximal current amplitude similar to that of GABA. A minor population of channels is expressed which is open in the absence of GABA or pentobarbital. Mapping subunit gene expression by in situ hybridization histochemistry suggests that the alpha 1-, beta 1-, and gamma 2-subunits are likely receptor constituents in some neuronal populations, e.g., mitral cells of the olfactory bulb, pyramidal cells of the hippocampus, and granule cells of the dentate gyrus and cerebellum.

To investigate the cellular and molecular signals underlying regulation of cell adhesion molecule expression, the influence of interactions between dorsal root ganglion neurons and Schwann cells on their expression of L1 and N-CAM was quantitated by immunogold electronmicroscopy. The numbers of antibody binding sites on cell surfaces of neurons and glia were compared between pure populations and co-cultures. After 3 d of co-culture, expression of L1 was reduced by 91% on Schwann cells and 36% on neurons, with expression in pure cultures being taken as 100%. N-CAM expression was unchanged on neurons and reduced by 43% on Schwann cells. Within 3 d after removal of neurons from Schwann cell-neuron co-cultures by immunocytolysis, expression of L1 and N-CAM on Schwann cell surfaces increased by 69 and 84%, respectively. Cell surface antigens recognized by an antibody to mouse liver membranes were unchanged in co-cultures. Furthermore, in co-cultures of neurons and sciatic nerve fibroblasts neither of the three antibodies detected any changes in expression of antigens when pure and co-cultures were compared. These observations suggest that adhesion molecules are not only involved in neuron-Schwann cell recognition and neurite outgrowth on Schwann cells (Seilheimer, B., and M. Schachner. 1988. J. Cell Biol. 107: 341-351), but that cell interactions, in turn, modulate the extent of adhesion molecule expression.

The cellular and subcellular localization of the neural cell adhesion molecules L1 and N-CAM was studied by pre- and postembedding immunoelectron microscopic labeling procedures in the developing mouse cerebellar cortex. The salient features of the study are: L1 displays a previously unrecognized restricted expression by particular neuronal cell types (i.e., it is expressed by granule cells but not by stellate and basket cells) and by particular subcellular compartments (i.e., it is expressed on axons but not on dendrites or cell bodies of Purkinje cells). L1 is always expressed on fasciculating axons and on postmitotic, premigratory, and migrating granule cells at sites of neuron-neuron contact, but never at contact sites between neuron and glia, thus strengthening the view that L1 is not involved in granule cell migration as a neuron-glia adhesion molecule. While N-CAM antibodies reacting with the three major components of N-CAM (180, 140, and 120 kD) show a rather uniform labeling of all cell types, antibodies to the 180-kD component (N-CAM180) stain only the postmigratory granule cell bodies supporting the notion that N-CAM180, the N-CAM component with the longest cytoplasmic domain, is not expressed before stable cell contacts are formed. Furthermore, N-CAM180 is only transiently expressed on Purkinje cell dendrites. N-CAM is present in synapses on both pre- and post-synaptic membranes. L1 is expressed only preterminally and not in the subsynaptic membranes. These observations indicate an exquisite degree of fine tuning in adhesion molecule expression during neural development and suggest a rich combinatorial repertoire in the specification of cell surface contacts.

To investigate whether neural adhesion molecules are involved in neuron-induced Schwann cell differentiation, cocultures of pure dorsal root ganglion neurons, and Schwann cells were maintained in the presence of antibodies to evaluate possible perturbing effects. Several parameters characteristic of differentiating Schwann cells were studied, such as transition of spindle-shaped to flattened, i.e., more epithelioid morphology, association with neuronal cell bodies, ensheathment of neurites, production of basal lamina and collagen fibrils, and expression of the myelin associated glycoprotein (MAG). A complete ablation of Schwann cell differentiation in all features studied was seen with antibodies to the neural adhesion molecule L1. Antibodies to N-CAM did not reduce the association of Schwann cells with neurites but abolished the interdigitation of Schwann cell processes into neurite bundles, while leaving the other parameters studied unaffected. Fab fragments of antibodies to J1, MAG, and mouse liver membranes did not interfere with the manifestation of any of these parameters. None of the antibodies changed incorporation of [3H]thymidine into Schwann cells.

In order to investigate the expression of cell adhesion molecules in synapses, we have studied the localization of the neural cell adhesion molecule N-CAM in the cerebellum and hippocampus of adult mice by immunocytological and immunochemical methods. Of the three molecular components of N-CAM with relative molecular masses (Mr) of 120, 140, and 180 kD, N-CAM 120 is not detectable in synaptosomal membranes, whereas N-CAM 140 is expressed on both pre- and postsynaptic membranes and N-CAM 180 is restricted to postsynaptic sites, with localization of the N-CAM 180-specific epitope in postsynaptic densities. Specificity of immunoreactivity is indicated by the observation that antibodies to the neural cell adhesion molecule L1 do not label synaptic membranes, whereas antibodies to two major components of postsynaptic densities, actin and erythrocyte spectrin, react with synaptic structures. Interestingly, N-CAM 180 is only detectable in subpopulations of synapses in the intact tissue. Isolated synaptosomes, opened for unimpeded accessibility of antibody by hypoosmotic treatment, also reveal a partial expression of N-CAM 180 in that 67% are labeled by antibodies to N-CAM 180, while antibodies to actin and erythrocyte spectrin react with 95% and 88% of all synaptosomes, respectively. N-CAM 180 does not appear to be differentially expressed in synapses of a particular morphological type, but is detectable in all types of synapses in the cerebellum and hippocampus, except for mossy fiber synapses and synapses between basket and Purkinje cells, which are generally N-CAM 180-negative. Since N-CAM 180 has been shown to be characteristic of stabilized or stabilizing cell contacts, possibly by its association with the cytoskeleton-membrane linker protein spectrin (Pollerberg et al.: J. Cell Biol. 101:1921-1929, '85; Nature 324:462-465, '86; Cell Tissue Res. 250:227-236, '87), we would like to suggest N-CAM 180 plays an important role in determining the stability of contacts between pre- and postsynaptic membranes and state of synaptic activity.

Adhesion molecule on glia (AMOG) is a novel neural cell adhesion molecule that mediates neuron-astrocyte interaction in vitro. In situ AMOG is expressed in the cerebellum by glial cells at the critical developmental stages of granule neuron migration. Granule neuron migration that is guided by surface contacts between migrating neurons and astroglial processes is inhibited by monoclonal AMOG antibody, probably by disturbing neuron-glia adhesion. AMOG is an integral cell surface glycoprotein of 45-50-kD molecular weight with a carbohydrate content of at least 30%. It does not belong to the L2/HNK-1 family of neural cell adhesion molecules but expresses another carbohydrate epitope that is shared with the adhesion molecules L1 and myelin-associated glycoprotein, but is not present on N-CAM or J1.