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Oxidative liver injury during steatohepatitis results in aggregation and transglutaminase-2 (TG2)-mediated crosslinking of the keratin cytoplasmic intermediate filament proteins (IFs) to form Mallory-Denk body (MDB) inclusions. The effect of liver injury on lamin nuclear IFs is unknown, though lamin mutations in several human diseases result in lamin disorganization and nuclear shape changes. We tested the hypothesis that lamins undergo aggregation during oxidative liver injury using two MDB mouse models: (i) mice fed the porphyrinogenic drug 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and (ii) mice that harbor a mutation in ferrochelatase (fch), which converts protoporphyrin IX to heme. Dramatic aggregation of lamin A/C and B1 was noted in the livers of both models in association with changes in lamin organization and nuclear shape, as determined by immunostaining and electron microscopy. The lamin aggregates sequester other nuclear proteins including transcription factors and ribosomal and nuclear pore components into high molecular weight complexes, as determined by mass-spectrometry and confirmed biochemically. Lamin aggregate formation is rapid and precedes keratin aggregation in fch livers, and is seen in liver explants of patients with alcoholic cirrhosis. Exposure of cultured cells to DDC, protoporphyrin IX or N-methyl-protoporphyrin, or incubation of purified lamins with protoporphyrin IX, also results in lamin aggregation. In contrast, lamin aggregation is ameliorated by TG2 inhibition. Therefore, lamin aggregation is an early sensor of porphyria-associated liver injury and might serve to buffer oxidative stress. The nuclear shape and lamin defects associated with porphyria phenocopy the changes seen in laminopathies and could result in transcriptional alterations due to sequestration of nuclear proteins.

Purpose: Identifying drug-induced liver injury is a critical task in drug development and postapproval real-world care. Severe liver injury is identified by the liver chemistry threshold of alanine aminotransferase (ALT) >3× upper limit of normal (ULN) and bilirubin >2× ULN, termed Hy's law by the Food and Drug Administration. These thresholds require discontinuation of the causative drug and are seldom exceeded in most patient populations. However, because maintenance of therapy is critical in the treatment of advanced cancer, customized thresholds may be useful in oncology patient populations, particularly for those with baseline liver chemistries elevations., Methods: Liver chemistry data from 31 aggregated oncology clinical trials were modeled through a truncated robust multivariate outlier detection (TRMOD) method to develop the decision boundary or threshold for examining liver injury in oncology clinical trials., Results: The boundary of TRMOD identified outliers with an ALT limit 5.0× ULN and total bilirubin limit 2.7× ULN. In addition, TRMOD was applied to the aggregated oncology data to examine fold-baseline ALT and total bilirubin, revealing limits of ALT 6.9× baseline and bilirubin 6.5× baseline. Similar ALT and bilirubin threshold limits were observed for oncology patients both with and without liver metastases., Conclusions: These higher liver chemistry thresholds examining fold-ULN and fold-baseline data may be valuable in identifying potential severe liver injury and detecting liver safety signals of clinical concern in oncology clinical trials and postapproval settings while helping to avoid premature discontinuation of curative therapy.

Lysine acetylation is an important posttranslational modification that regulates microtubules and microfilaments, but its effects on intermediate filament proteins (IFs) are unknown. We investigated the regulation of keratin 8 (K8), a type II simple epithelial IF, by lysine acetylation. K8 was basally acetylated and the highly conserved Lys-207 was a major acetylation site. K8 acetylation regulated filament organization and decreased keratin solubility. Acetylation of K8 was rapidly responsive to changes in glucose levels and was up-regulated in response to nicotinamide adenine dinucleotide (NAD) depletion and in diabetic mouse and human livers. The NAD-dependent deacetylase sirtuin 2 (SIRT2) associated with and deacetylated K8. Pharmacologic or genetic inhibition of SIRT2 decreased K8 solubility and affected filament organization. Inhibition of K8 Lys-207 acetylation resulted in site-specific phosphorylation changes of K8. Therefore, K8 acetylation at Lys-207, a highly conserved residue among type II keratins and other IFs, is up-regulated upon hyperglycemia and down-regulated by SIRT2. Keratin acetylation provides a new mechanism to regulate keratin filaments, possibly via modulating keratin phosphorylation.

Inhibition of intestinal brush border DMT1 offers a novel therapeutic approach to the prevention and treatment of disorders of iron overload. Several series of diaryl and tricyclic benzylisothiourea compounds as novel and potent DMT1 inhibitors were discovered from the original hit compound 1. These compounds demonstrated in vitro potency against DMT1, desirable cell permeability properties and a dose-dependent inhibition of iron uptake in an acute rat model of iron hyperabsorption. Tricyclic compounds increased the in vitro potency by up to 16-fold versus the original hit. Diaryl compounds 6b and 14a demonstrated significant iron absorption inhibition in vivo with both 25 and 50 mg/kg doses. The diaryl and tricyclic compounds described in this report represent promising structural templates for further optimization., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Mallory-Denk bodies (MDBs) are hepatocyte inclusions that are associated with poor liver disease prognosis. The intermediate filament protein keratin 8 (K8) and its cross-linking by transglutaminase-2 (TG2) are essential for MDB formation. K8 hyperphosphorylation occurs in association with liver injury and MDB formation, but the link between keratin phosphorylation and MDB formation is unknown. We used a mutational approach to identify K8 Q70 as a residue that is important for K8 cross-linking to itself and other liver proteins. K8 cross-linking is markedly enhanced on treating cells with a phosphatase inhibitor and decreases dramatically on K8 S74A or Q70N mutation in the presence of phosphatase inhibition. K8 Q70 cross-linking, in the context of synthetic peptides or intact proteins transfected into cells, is promoted by phosphorylation at K8 S74 or by an S74D substitution and is inhibited by S74A mutation. Transgenic mice that express K8 S74A or a K8 G62C liver disease variant that inhibits K8 S74 phosphorylation have a markedly reduced ability to form MDBs. Our findings support a model in which the stress-triggered phosphorylation of K8 S74 induces K8 cross-linking by TG2, leading to MDB formation. These findings may extend to neuropathies and myopathies that are characterized by intermediate filament-containing inclusions.

Mallory-Denk bodies (MDBs) are hepatocyte cytoplasmic inclusions found in several liver diseases and consist primarily of the cytoskeletal proteins, keratins 8 and 18 (K8/K18). Recent evidence indicates that the extent of stress-induced protein misfolding, a K8>K18 overexpression state, and transglutaminase-2 activation promote MDB formation. In addition, the genetic background and gender play an important role in mouse MDB formation, but the effect of aging on this process is unknown. Given that oxidative stress increases with aging, the authors hypothesized that aging predisposes to MDB formation. They used an established mouse MDB model-namely, feeding non-transgenic male FVB/N mice (1, 3, and 8 months old) with 3,5 diethoxycarbonyl-1,4-dihydrocollidine for 2 months. MDB formation was assessed using immunofluorescence staining and biochemically by demonstrating keratin and ubiquitin-containing crosslinks generated by transglutaminase-2. Immunofluorescence staining showed that old mice had a significant increase in MDB formation compared with young mice. MDB formation paralleled the generation of high molecular weight ubiquitinated keratin-containing complexes and induction of p62. Old mouse livers had increased oxidative stress. In addition, 20S proteasome activity and autophagy were decreased, and endoplasmic reticulum stress was increased in older livers. Therefore, aging predisposes to experimental MDB formation, possibly by decreased activity of protein degradation machinery.

Three distinct series of substituted pyrazole blockers of divalent metal transporter 1 (DMT1) were elaborated from the high-throughput screening pyrazolone hit 1. Preliminary hit-to-lead efforts revealed a preference for electron-withdrawing substituents in the 4-amido-5-hydroxypyrazole series 6a-l. In turn, this preference was more pronounced in a series of 4-aryl-5-hydroxypyrazoles 8a-j. The representative analogs 6f and 12f were found to be efficacious in a rodent model of acute iron hyperabsorption. These three series represent promising starting points for lead optimization efforts aimed at the discovery of DMT1 blockers as iron overload therapeutics., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Starting from the oxindole 2a identified through a high-throughput screening campaign, a series of Na(V)1.7 blockers were developed. Following the elimination of undesirable structural features, preliminary optimization of the oxindole C-3 and N-1 substituents afforded the simplified analogue 9b, which demonstrated a 10-fold increase in target potency versus the original HTS hit. A scaffold rigidification strategy then led to the discovery of XEN907, a novel spirooxindole Na(V)1.7 blocker. This lead compound, which in turn showed a further 10-fold increase in potency, represents a promising structure for further optimization efforts., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background and Aim: Acute pancreatitis (AP) can result in pancreatic necrosis and inflammation, with subsequent multi-organ failure. AP is associated with increased neutrophil recruitment and a rise in pro-inflammatory cytokines such as TNFα. Pretreatment with haemin, results in recruitment of haem-oxygenase-1 (HO-1)(+) macrophages and protects against experimental pancreatitis. It is not clear whether modulation of HO-1 after onset of disease has a protective role. In this study, we tested the utility of Panhematin, a water-soluble haemin formulation, in activating and inducing pancreatic HO-1, and as a therapeutic agent in treating mouse acute pancreatitis., Methods: We defined the distribution of radiolabelled haemin, then used in vivo HO-1-luciferase bioluminescence imaging and the CO-release assay to test Panhematin-induced upregulation of HO-1 transcription and activity, respectively. Using two well-defined AP murine models, we tested the therapeutic benefit of Panhematin, and quantified cytokine release using a luminex assay., Results: Intravenously administered Panhematin induces rapid recruitment of HO-1(+) cells to the pancreas within 2 h and de novo splenic HO-1 transcription by 12 h. Despite high baseline spleen HO-1 activity, the pancreas is particularly responsive to Panhematin-mediated HO-1 induction. Panhematin-treated mice, at various time points after AP induction had significant reduction in mortality, pancreatic injury, together with upregulation of HO-1 and downregulation of pro-inflammatory cytokines and CXCL1, a potent neutrophil chemoattractant., Conclusions: Despite AP-associated mortality and morbidity, no effective treatment other than supportive care exists. We demonstrate that Panhematin leads to: (i) rapid induction and activation of pancreatic HO-1 with recruitment of HO-1(+) cells to the pancreas, (ii) amelioration of AP even when given late during the course of disease, and (iii) a decrease in leucocyte infiltration and pro-inflammatory cytokines including CXCL1. The utility of Panhematin at modest doses as a therapeutic in experimental pancreatitis, coupled with its current use and safety in humans, raises the potential of its applicability to human pancreatitis.

MEKK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the MAPK JNK and is required for microtubule inhibitor-induced apoptosis in B cells. Here, we find that apoptosis induced by actin disruption via cytochalasin D and by the protein phosphatase 1/2A inhibitor okadaic acid also requires MEKK1 activation. To elucidate the functional requirements for activation of the MEKK1-dependent apoptotic pathway, we created mutations within MEKK1. MEKK1-deficient cells were complemented with MEKK1 containing mutations in either the ubiquitin interacting motif (UIM), plant homeodomain (PHD), caspase cleavage site or the kinase domain at near endogenous levels of expression and tested for their sensitivity to each drug. We found that both the kinase activity and the PHD domain of MEKK1 are required for JNK activation and efficient induction of apoptosis by drugs causing cytoskeletal disruption. Furthermore, we discovered that modification of MEKK1 and its localization depends on the integrity of the PHD.

Background/objectives: Although the prevalence of complementary and alternative medicine (CAM) use has been studied among general and specific disease populations, little is known on the use of CAM among Asian dermatology patients. This study assesses prevalence, demographics, disease determinants, expectations and reasons for CAM use among patients visiting a major referral dermatology centre in Singapore., Methods: A descriptive cross-sectional study of 855 dermatology outpatients was done. Consecutive sampling using interviewer-administered questionnaires collected information on patient demographics, dermatological condition, prevalence, reasons and expectations of CAM use. Patient-perceived disease severity was measured via the Dermatological Life Quality Index (DLQI). Dermatologists completed Patient Data Forms, detailing diagnosis, diagnosis date and CAM use., Results: The prevalence of CAM use was 25.7%. Patients who were higher educated, held white collar occupations, had longer disease duration, higher DLQI scores or were suffering from psoriasis or eczema were more likely to have used CAM. More than 60% of patients expected dermatologists to provide at least basic CAM advice and 75% were willing to declare their CAM use. Forty percent of dermatologists accurately knew their patients' current CAM use., Conclusions: Prevalence of CAM use in dermatology patients was high. Many doctors were unaware of patients' CAM use despite most patients being willing to declare it. Patients generally expected dermatologists to provide CAM advice. Dermatologists should make a concerted effort to identify likely CAM users and consider openly discussing CAM use with them., (© 2010 The Authors. Australasian Journal of Dermatology © 2010 The Australasian College of Dermatologists.)

Heme oxygenase-1 (HO-1) induction by hemin or Panhematin protects against experimental pancreatitis. As a preclinical first step toward determining whether HO-1 upregulation is a viable target in acute pancreatitis (AP) patients, we tested the hypothesis that HO-1 expression in peripheral blood mononuclear cell (PBMC) subsets of hospitalized patients with mild AP is upregulated then normalizes upon recovery and that cells from AP patients have the potential to upregulate their HO-1 ex vivo if exposed to Panhematin. PBMCs were isolated on days 1 and 3 of hospitalization from the blood of 18 AP patients, and PMBC HO-1 levels were compared with PMBCs of 15 hospitalized controls (HC) and 7 volunteer healthy controls (VC). On day 1 of hospitalization, AP patients compared with VCs had higher HO-1 expression in monocytes and neutrophils. Notably, AP monocyte HO-1 levels decreased significantly upon recovery. Panhematin induced HO-1 in ex vivo cultured AP PBMCs more readily than in HC or VC PBMCs. Furthermore, PBMCs from acutely ill AP patients on day 1 were more responsive to HO-1 induction compared with day 3 upon recovery. Similarly, mouse splenocytes had enhanced HO-1 inducibility as their pancreatitis progressed from mild to severe. In conclusion, AP leads to reversible PBMC HO-1 upregulation that is associated with clinical improvement and involves primarily monocytes. Leukocytes from AP patients or mice with AP are primed for HO-1 induction by Panhematin, which suggests that Panhematin could offer a therapeutic benefit.

Biomarkers could be useful in evaluating immune reconstitution inflammatory syndrome (IRIS). A cohort of 45 HIV-1-infected, antiretroviral treatment (ART)-naive patients with baseline CD4 T cell counts

We report a 46-year-old male with metastatic functioning paraganglioma of the thoracic spine. Thoracic spine functioning paragangliomas are rare and we describe the benefits of embolisation of the lesion prior to surgical decompression., (Crown Copyright 2009. Published by Elsevier Ltd. All rights reserved.)

Female animals tolerate trauma and hemorrhage better than male animals AND Estrogen has rapid nongenomic effects that protect organs from damage and attenuate insult-induced inflammation MOREOVER The survival deficit from trauma and hemorrhage produced in ovariectomized female animals is repaired with administration of exogenous estrogen AND Women survive injury, sepsis, and trauma-hemorrhage-induced hypoxemia/reperfusion better than men THEREFORE Women rule ... in survival after trauma, thus, men would benefit from being more like women., (Copyright (c) 2010 Mosby, Inc. All rights reserved.)

Identifying the functions of human immunodeficiency virus (HIV)-specific CD8+ T cells that are not merely modulated by the level of virus but clearly distinguish patients with immune control from those without such control is of paramount importance. Features of the HIV-specific CD8+ T-cell response in antiretroviral-treated patients (designated Rx <50) and untreated patients (long-term nonprogressors [LTNP]) matched for very low HIV RNA levels were comprehensively examined. The proliferative capacity of HIV-specific CD8+ T cells was not restored in Rx <50 to the level observed in LTNP, even though HIV-specific CD4+ T-cell proliferation in the two patient groups was comparable. This diminished HIV-specific CD8+ T-cell proliferation in Rx <50 was primarily due to a smaller fraction of antigen-specific cells recruited to divide and not to the numbers of divisions that proliferating cells had undergone. Exogenous interleukin-2 (IL-2) induced proliferating cells to divide further but did not rescue the majority of antigen-specific cells with defective proliferation. In addition, differences in HIV-specific CD8+ T-cell proliferation could not be attributed to differences in cellular subsets bearing a memory phenotype, IL-2 production, or PD-1 expression. Although polyfunctionality of HIV-specific CD8+ T cells in Rx <50 was not restored to the levels observed in LTNP despite prolonged suppression of HIV RNA levels, per-cell cytotoxic capacity was the functional feature that most clearly distinguished the cells of LTNP from those of Rx <50. Taken together, these data suggest that there are selective qualitative abnormalities within the HIV-specific CD8+ T-cell compartment that persist under conditions of low levels of antigen.

Virus-specific CD8+ T cells probably mediate control over HIV replication in rare individuals, termed long-term nonprogressors (LTNPs) or elite controllers. Despite extensive investigation, the mechanisms responsible for this control remain incompletely understood. We observed that HIV-specific CD8+ T cells of LTNPs persisted at higher frequencies than those of treated progressors with equally low amounts of HIV. Measured on a per-cell basis, HIV-specific CD8+ T cells of LTNPs efficiently eliminated primary autologous HIV-infected CD4+ T cells. This function required lytic granule loading of effectors and delivery of granzyme B to target cells. Defective cytotoxicity of progressor effectors could be restored after treatment with phorbol ester and calcium ionophore. These results establish an effector function and mechanism that clearly segregate with immunologic control of HIV. They also demonstrate that lytic granule contents of memory cells are a critical determinant of cytotoxicity that must be induced for maximal per-cell killing capacity.

We report the first case of a human immunodeficiency virus type 1 (HIV-1)-infected individual receiving combination antiretroviral therapy, which included ritonavir, who developed Cushing syndrome with profound complications after epidural triamcinolone injections. This case highlights the potential of ritonavir interactions even with local injections of a corticosteroid.

The differential expression of chemokines and chemokine receptors, by tissues and leukocytes, respectively, contributes to the specific accumulation of leukocyte subsets to different tissues. CCR10/CCL28 interactions are thought to contribute to the accumulation of IgA Ab-secreting cells (ASC) to mucosal surfaces, such as the gastrointestinal tract and the lactating mammary gland. Although the role of CCL28 in lymphocyte homing is well established, direct in vivo evidence for CCR10 involvement in this process has not been previously shown. In this study, we describe the generation of a CCR10-deficient mouse model. Using this model, we demonstrate that CCR10 is critical for efficient localization and accumulation of IgA ASC to the lactating mammary gland. Surprisingly, IgA ASC accumulation to the gastrointestinal tract is minimally impacted in CCR10-deficient mice. These results provide the first direct evidence of CCR10 involvement in lymphocyte homing and accumulation in vivo, and demonstrate that reliance on CCR10-mediated recruitment of IgA ASC varies dramatically within mucosal tissues.

Objective: To evaluate the efficacy and safety of the selective dopamine D1/D5 antagonist ecopipam for the treatment of obesity., Research Methods and Procedures: Four randomized, double-blind, multicenter trials compared ecopipam (n=1667) and placebo (n=1118) in obese subjects including type 2 diabetic subjects. Subjects received oral ecopipam 10, 30, or 100 mg daily for 12 weeks (Phase 2) or 50 or 100 mg daily for 52 weeks (Phase 3) combined with a weight loss program. Primary efficacy variables were the proportion of subjects with>or=5% weight loss from baseline at 12 weeks (Phase 2) or the distribution of percentage weight loss from baseline at 52 weeks (Phase 3)., Results: In the Phase 2 study, 26% of subjects administered ecopipam 100 mg vs. 6% of placebo subjects achieved>or=5% weight loss after 12 weeks (p<0.01). In the Phase 3 studies, ecopipam 100 mg produced a 3.1% to 4.3% greater weight loss than placebo at 52 weeks. More subjects administered ecopipam vs. placebo achieved a 5% to 10% or >10% weight loss in two non-diabetic phase 3 trials. Ecopipam-treated subjects also maintained more weight loss compared with placebo subjects at 52 weeks. Phase 3 studies were discontinued because of unexpected psychiatric adverse events (ecopipam 31% vs. placebo 15%), including depression, anxiety, and suicidal ideation., Discussion: Ecopipam was effective for achieving and maintaining weight loss in obese subjects, including type 2 diabetic subjects; however, the adverse effects on mood observed in the Phase 3 studies exclude its projected use in weight management.

Perturbations of B cells in HIV-infected individuals are associated with the overrepresentation of distinct B cell populations. Here we describe high extrinsic CD95 ligand (CD95L)-mediated apoptosis in CD10-/CD21lo mature/activated B cells that likely arise from HIV-induced immune activation. In addition, high intrinsic apoptosis was observed in CD10+ immature/transitional B cells that likely arise as a result of HIV-induced lymphopenia. CD10+ B cells expressed low levels of Bcl-2 and Bcl-xL, consistent with their high susceptibility to intrinsic apoptosis. Higher levels of activated Bax and Bak were induced in CD10+ B cells compared with CD95L-treated CD10- B cells, consistent with the greater involvement of mitochondria in intrinsic vs. extrinsic apoptosis. Of interest, both extrinsic apoptosis in CD95L-treated CD10- B cells and intrinsic apoptosis in CD10+ B cells were associated with caspase-8 activation. Our data suggest that two distinct mechanisms of apoptosis are associated with B cells of HIV-infected individuals, and both may contribute to the depletion and dysfunction of B cells in these individuals.

During recycling of synaptic vesicles (SVs), the retrieval machinery faces the challenge of recapturing SV proteins in a timely and precise manner. The significant dilution factor that would result from equilibration of vesicle proteins with the much larger cell surface would make recapture by diffusional encounter with the endocytic retrieval machinery unlikely. If SV proteins exchanged with counterparts residing at steady state on the cell surface, the dilution problem would be largely avoided. In this scenario, during electrical activity, endocytosis would be driven by the concentration of a pre-existing pool of SVs residing on the axonal or synaptic surface rather than the heavily diluted postfusion vesicular pool. Using both live cell imaging of endogenous synaptotagmin Ia (sytIa) as well as pHluorin-tagged sytIa and VAMP-2, we show here that synaptic vesicle proteins interchange with a large pool on the cell axonal surface whose concentration is approximately 10-fold lower than that in SVs.

There are many data mining techniques for processing and general learning of multivariate data. However, we believe the wavelet transformation and latent variable projection method are particularly useful for spectroscopic and chromatographic data. Projection based methods are designed to handle hugely multivariate nature of such data effectively. For the actual analysis of the data we have used latent variable projection methods such as principal component analysis (PCA) and partial least squares projection to latent structures based discriminant analysis (PLS-DA) to analyze the raw data presented to the participants of the First Duke Proteomics Data Mining Conference. PCA was used to solve problem #1 (clustering problem) and the PLS-DA was used to solve problem #2 (classification problem). The idea of internal and external cross-validation was used to validate the model obtained from the classification analysis. The simple two-component PLS-DA model obtained from the analysis performed well. The model has completely separated the two groups from all the data. The same model applied on two-thirds of the data showed good performance by external validation with independent test set of remaining 13 specimens obtained by setting aside the spectra of every third specimen (accuracy of 85%).

Public transport driver scheduling problems are well known to be NP-hard. Although some mathematically based methods are being used in the transport industry, there is room for improvement. A hybrid approach incorporating a genetic algorithm (GA) is presented. The role of the GA is to derive a small selection of good shifts to seed a greedy schedule construction heuristic. A group of shifts called a relief chain is identified and recorded. The relief chain is then inherited by the offspring and used by the GA for schedule construction. The new approach has been tested using real-life data sets, some of which represent very large problem instances. The results are generally better than those compiled by experienced schedulers and are comparable to solutions found by integer linear programming (ILP). In some cases, solutions were obtained when the ILP failed within practical computational limits.

Vinblastine and other microtubule-damaging agents, such as nocodazole and paclitaxel, cause cell cycle arrest at the G2/M transition and promote apoptosis in eukaryotic cells. The roles of these drugs in disrupting microtubule dynamics and causing cell cycle arrest are well characterized. However, the mechanisms by which these agents promote apoptosis are poorly understood. We disrupted the MEKK1 kinase domain in chicken bursal B-cell line DT40 by homologous recombination and have shown that it is essential for both vinblastine-mediated apoptosis and vinblastine-mediated c-Jun N-terminal protein kinase activation. In addition, our data indicate that vinblastine-mediated apoptosis in DT40 cells requires new protein synthesis but does not require G2/M arrest, suggesting that vinblastine-mediated cell cycle arrest and apoptosis are two independent processes.

The in vivo role of epidermal growth factor (EGF) is not well defined even though its effects on culture cells were well studied. To understand the developmental, physiological, and pathological roles of EGF, we have generated transgenic mice widely expressing human EGF with the use of the beta-actin promoter. EGF and transforming growth factor alpha (TGFalpha) bind with equal affinity to the EGF receptor, a transmembrane tyrosine kinase, to trigger various biological responses. EGF and TGFalpha signaling are implicated in the development of the reproductive system. EGF also plays a physiological role in reproduction. Removal of the salivary gland in rodents, which reduces circulating EGF, reduces spermatogenesis, which can be corrected by EGF replacement. Here we show that in our transgenic males, only few post-meiosis II gametes were found, and the mice were sterile. This resembles a common cause of infertility in humans. Furthermore, the transgenic males had reduced serum testosterone. Our findings contrast the previous report on transgenic mice overexpressing TGFalpha in testis, which showed normal spermatogenesis. These data suggest that EGF is the active ligand for EGF receptor reported in germ cells, and proper EGF expression is important for completion of spermatogenesis.

With the increased interest in computer-aided image analysis methods, there is a greater need for objective methods of algorithm evaluation. Validation of in vivo MRI studies is complicated by a lack of reference data and the difficulty of constructing anatomically realistic physical phantoms. We present here an extensible MRI simulator that efficiently generates realistic three-dimensional (3-D) brain images using a hybrid Bloch equation and tissue template simulation that accounts for image contrast, partial volume, and noise. This allows image analysis methods to be evaluated with controlled degradations of image data.

Epidermal growth factor (EGF) is derived from a large precursor (EGFP) of unusual structure. As EGFP remains unprocessed in certain tissues, it is of biological relevance to study its activity. Activation of the EGF receptor by EGF is involved in transformation of NIH3T3 fibroblasts. We isolated clones of NIH3T3 expressing full length, cytoplasmic region deleted or EGF-repeats deleted EGFP. All clones formed colonies in soft agarose and tumors in nude mice. Two clones expressing EGF-repeats deleted EGFP formed more and larger colonies. To conclude, EGFP is biologically active. Deletion of the 8 EGF repeats may enhance anchorage independent growth in NIH3T3.

Interleukin (IL)-13 and IL-4 are pleiotropic immunoregulatory cytokines that share many overlapping biological properties reflecting the fact that both can utilize a receptor complex composed of the IL-4 receptor-alpha (IL-4Ralpha) chain and the IL-13Ralpha chain. The cytoplasmic domain of the IL-13Ralpha is 60 amino acids long and is essential for IL-13-dependent growth. It contains a Pro-rich domain in the membrane-proximal region and two Tyr residues. Here we show that a truncated IL-13Ralpha, lacking the 38 carboxyl-terminal residues but retaining the Pro-rich region, can support IL-13-dependent proliferation, although with reduced efficiency. A Y402F mutant of the cytoplasmic domain of IL-13Ralpha supported normal IL-13-induced growth. However, tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3), which we show is induced by IL-13 and IL-4 in cells that express the IL-13Ralpha, was significantly reduced. The cytoplasmic domain of IL-13Ralpha was constitutively associated with STAT3, Tyk2, and Janus kinase 1 (JAK1). IL-13-induced tyrosine phosphorylation of IL-13Ralpha in vivo could not be detected using anti-Tyr(P) antibodies. A glutathione S-transferase fusion protein of the cytoplasmic domain of IL-13Ralpha was phosphorylated on tyrosine in vitro by JAK1, JAK3, and Tyk2, although the tyrosine phosphorylation events mediated by Tyk2 and JAK3 were not detectable using anti-phosphotyrosine antibodies. These data, together with the demonstration that IL-13Ralpha associates constitutively with Tyk2 and that Tyr-402 is involved in IL-13-induced phosphorylation of STAT3, suggest that the latter is mediated by Tyk2. Tyrosine phosphorylation of STAT3, which was not necessary for IL-13-induced proliferation, may account for some of the effects of IL-4 and IL-13 on the function of their targets.

In interleaved spiral MRI, an object's Fourier transform is sampled along a set of curved trajectories in the spatial frequency domain (k-space). An image of the object is then reconstructed, usually by interpolating the sampled Fourier data onto a Cartesian grid and applying the fast Fourier transform (FFT) algorithm. To obtain accurate results, it is necessary to account for the nonuniform density with which k-space is sampled. An analytic density compensation function (DCF) for spiral MRI, based on the Jacobian determinant for the transformation between Cartesian coordinates and the spiral sampling parameters of time and interleaf rotation angle, is derived in this paper, and the reconstruction accuracy achieved using this function is compared with that obtained using several previously published expressions. Various nonideal conditions, including intersecting trajectories, are considered. The new DCF eliminated intensity cupping that was encountered in images reconstructed with other functions, and significantly reduced the level of artifact observed when unevenly spaced sampling trajectories, such as those achieved with trapezoidal gradient waveforms, were employed. Modified forms of this function were found to provide similar improvements when intersecting trajectories made the spiral-Cartesian transformation noninvertible, and when the shape of the spiral trajectory varied between interleaves.

The keratins are a component of the cytoskeleton that is present in fetal and neonatal rat granulosa cells (ROG), but disappears as the cells undergo postnatal steroidogenic differentiation. Steroidogenesis is initiated in the fetus as a low level constitutive function which is cAMP responsive, but becomes responsive to gonadotrophic hormones only after birth. ROG from PMSG-primed immature rats, like mature ROG, are keratin negative, highly steroidogenic and gonadotrophin-responsive, but rapidly lose their steroidogenic capacity in culture. In such cultured cells, transformation with the Kirsten-ras oncogene (v-Ki-ras) maintains low levels of constitutive steroidogenesis and responsiveness to cAMP, and induces the expression of keratin. To determine whether similar changes would occur in cells expressing both the SV40 and v-Ki-ras, cultured ROG were transformed with SV40 early genes, with Kirsten murine sarcoma virus (KiMSV), or with both agents concurrently. Keratin was demonstrated by fluorescence microscopy and Western blots, and progesterone production by RIA. ROG transformed with SV40 alone became immortalized but secreted little steroid and lacked keratin. In contrast, three cell lines, co-transformed with SV40 plus KiMSV, acquired keratin as well as the capacity to secrete progesterone in response to cAMP, closely resembling cells transformed with Ki-ras alone. KiMSV-transformed muscle fascia fibroblasts lacked both steroidogenic potential and keratin. The results show that the complex, v-Ki-ras-induced changes in steroidogenesis and keratin expression are reproducible and tissue specific. The phenotypic resemblance between singly and doubly transformed ROG indicates that the v-Ki-ras oncogene does not act by overcoming SV40-mediated inhibition of differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid and sensitive high-pressure liquid chromatographic (HPLC) assays for ticarcillin in serum and urine have been developed. Sample pretreatment and optimized chromatographic conditions are presented for a C-18-bonded reversed-phase column used in an internal standard assay method. Ticarcillin has a retention time of or approximately 5.3 min at a flow rate of 1.5 ml/min for a mobile phase of acetonitrile-aqueous 0.06 M sodium biphosphate, pH 2.05, (50.5:100). In a two-step extraction procedure, the ticarcillin extraction efficiencies from serum and urine were 76.1 +/- 4.7 and 80.9 +/- 3.2% respectively. The assay sensitivity limit for ticarcillin in these fluids is approximately 1.0 microgram/ml. A comparison is made of the HPLC and microbiological assay results for ticarcillin in 20 different but equally divided serum samples obtained from two volunteers.

To study the effects of a low carbohydrate, isoenergetic diet on pulmonary physiology and sleep behavior, we measured pulmonary functions and respiratory gas exchange and carried out ambulatory electroencephalographic studies after a week's intake of isoenergetic diet containing only 50 g carbohydrate per day in 6 healthy female adult humans in a free-living condition. Compared with their normal intake, during the week of low carbohydrate intake there was a rise in the level of fasting plasma 3-hydroxybutyrate from 0.12 +/- 0.07 (mean +/- SD) to 1.01 +/- 0.40 mmol/L(P less than 0.01, paired t-test); a fall in serum bicarbonate from 26.2 +/- 0.75 to 25.0 +/- 1.41 mmol/L (P less than 0.05) and in serum chloride from 107 +/- 1.3 to 105 +/- 1.8 mmol/L (P less than 0.05). Serum urea rose from 4.3 +/- 0.71 to 5.7 +/- 0.70 mmol/L (P less than 0.01), and serum uric acid from 0.34 +/- 0.08 to 0.39 +/- 0.10 mmol/L (P less than 0.05). Functional residual capacity was increased from 2.07 +/- 0.35 to 2.26 +/- 0.34 L (P less than 0.01). Respiratory gas exchange ratio fell from 0.81 +/- 0.05 to 0.75 +/- 0.04 (P less than 0.05) and partial pressure of expired carbon dioxide reduced from 22 +/- 3.3 to 21 +/- 3.1 mmHg (P less than 0.05). There was a reduction in endogenous carbon dioxide production and arterial carbon dioxide tension. An analysis of ambulatory electroencephalogram showed that REM latency increased from 66 +/- 8 to 111 +/- 38 min (P less than 0.05), with no significant changes in sleep time and stages. These studies show that a low carbohydrate isoenergetic diet is tolerable, influences sleep behavior, reduces carbon dioxide production and respiratory gas exchange ratio, and may be therapeutically useful in patients with hypercapnic respiratory failure.

Two rapid, specific, and sensitive high-pressure liquid chromatographic (HPLC) assays were developed for cloxacillin in serum and urine. A reversed-phase column (RP-8) was selected for use with two different sets of HPLC conditions and sample pretreatment procedures. Cloxacillin extraction efficiencies are reported from serum and urine. Equations are presented for linear relationships between peak height or peak area ratios of cloxacillin to nafcillin (internal standard) and the cloxacillin concentration over a range of 0-80 micrograms/ml. The sensitivity limit of these assays was approximately 0.3 microgram/ml of a standard solution for one method and 0.05 microgram /ml for the other HPLC assay.

To explore the effects of moderate and severe reductions in carbohydrate intake on abnormal pulmonary physiology in chronic hypercapneic respiratory failure, spirometric, metabolic, arterial blood gas tension, and oximetric studies were carried out in eight patients who took, in random order daily for a week, either 50 g or 200 g of carbohydrate in an isocaloric diet. At the end of a week's daily intake of an isocaloric diet containing 200 g of carbohydrate, all patients experienced a subjective improvement; the mean body weight was 55.5 +/- 15.4 kg (1 SD) compared with 56.0 +/- 16.0 kg during the control dietary period, the arterial carbon dioxide tension decreased from a mean of 56.9 +/- 6.7 to 50.9 +/- 6.2 mm Hg (p less than 0.005), and the arterial oxygen tension increased from a mean of 50.6 +/- 7.3 to 62.0 +/- 14.5 mm Hg (p less than 0.02). After a week's intake of 50 g of carbohydrate in an isocaloric diet, the body weight and arterial oxygen tension did not change significantly, but the arterial carbon dioxide tension decreased still further to 48.0 +/- 7.8 mm Hg (p less than 0.05). Mouth pressure at 100 msec after the start of inspiration, as a measure of respiratory center output, was significantly higher during both the low carbohydrate intakes compared with the control dietary period. The spirometric data, ventilation-perfusion distribution measurements, oxygen consumption, and carbon dioxide production did not change significantly during various dietary periods. It is concluded that, under these short-term, hospital-controlled conditions, a reduction in the carbohydrate intake to 200 g a day improves the general well-being of patients with chronic hypercapneic respiratory failure, increases arterial oxygen tension, and decreases arterial carbon dioxide tension. A further reduction in the carbohydrate intake to 50 g a day provides further beneficial effects, and such a diet may be used in patients with intractable respiratory failure.