Smads 2 and 3 are differentially activated by transforming growth factor-beta (TGF-beta ) in quiescent and activated hepatic stellate cells. Constitutive nuclear localization of Smads in activated cells is TGF-beta-independent

Hepatic stellate cells are the primary cell type responsible for matrix deposition in liver fibrosis, undergoing a process of transdifferentiation into fibrogenic myofibroblasts. These cells, which undergo a similar transdifferentiation process when cultured in vitro, are a major target of the profibrogenic agent transforming growth factor-beta (TGF-beta). We have studied activation of the TGF-beta downstream signaling molecules Smads 2, 3, and 4 in hepatic stellate cells (HSC) cultured in vitro for 1, 4, and 7 days, with quiescent, intermediate, and fully transdifferentiated phenotypes, respectively. Total levels of Smad4, common to multiple TGF-beta superfamily signaling pathways, do not change as HSC transdifferentiate, and the protein is found in both nucleus and cytoplasm, independent of treatment with TGF-beta or the nuclear export inhibitor leptomycin B. TGF-beta mediates activation of Smad2 primarily in early cultured cells and that of Smad3 primarily in transdifferentiated cells. The linker protein SARA, which is required for Smad2 signaling, disappears with transdifferentiation. Additionally, day 7 cells demonstrate constitutive phosphorylation and nuclear localization of Smad 2, which is not affected by pretreatment with TGF-beta-neutralizing antibodies, a type I TGF-beta receptor kinase inhibitor, or activin-neutralizing antibodies. These results demonstrate essential differences between TGF-beta-mediated signaling pathways in quiescent and in vitro transdifferentiated hepatic stellate cells.     

AZ 242, a novel PPARalpha/gamma agonist with beneficial effects on insulin resistance and carbohydrate and lipid metabolism in ob/ob mice and obese Zucker rats

Abnormalities in fatty acid (FA) metabolism underlie the development of insulin resistance and alterations in glucose metabolism, features characteristic of the metabolic syndrome and type 2 diabetes that can result in an increased risk of cardiovascular disease. We present pharmacodynamic effects of AZ 242, a novel peroxisome proliferator activated receptor (PPAR)alpha/gamma agonist. AZ 242 dose-dependently reduced the hypertriglyceridemia, hyperinsulinemia, and hyperglycemia of ob/ob diabetic mice. Euglycemic hyperinsulinemic clamp studies showed that treatment with AZ 242 (1 micromol/kg/d) restored insulin sensitivity of obese Zucker rats and decreased insulin secretion. In vitro, in reporter gene assays, AZ 242 activated human PPARalpha and PPARgamma with EC(50) in the micro molar range. It also induced differentiation in 3T3-L1 cells, an established PPARgamma effect, and caused up-regulation of liver fatty acid binding protein in HepG-2 cells, a PPARalpha-mediated effect. PPARalpha-mediated effects of AZ 242 in vivo were documented by induction of hepatic cytochrome P 450-4A in mice. The results indicate that the dual PPARalpha/gamma agonism of AZ 242 reduces insulin resistance and has beneficial effects on FA and glucose metabolism. This effect profile could provide a suitable therapeutic approach to the treatment of type 2 diabetes, metabolic syndrome, and associated vascular risk factors.     

Selective excitation of GABAergic neurons in the substantia nigra of the rat by orexin/hypocretin in vitro

Dysfunction of the orexin/hypocretin neurotransmitter system leads to the sleep disorder narcolepsy. Narcolepsy is characterized by excessive daytime sleepiness and the occurrence of cataplexy--a sudden loss of muscle tone triggered by emotionally arousing events. Both symptoms can be treated with drugs that act on dopaminergic systems. Here we have investigated the effect of orexins on the firing of dopaminergic and GABAergic neurons of the substantia nigra (SN) in brain slices. Surprisingly, dopaminergic neurons in pars compacta were unaffected by orexins. In contrast, bath application of orexin A (100 nM) or orexin B (5-300 nM) greatly increased the firing rate of GABAergic neurons in pars reticulata. The orexin B-mediated excitation was unaffected by blocking synaptic transmission (using low-Ca2+/high-Mg2+ solution). However, the effect of orexin B was reduced significantly by thapsigargin (1 microM) and inhibitors of protein kinase A. The presence of orexinergic fibres in the SN pars reticulata was demonstrated by immunohistochemical methods with the fibre density increasing in the rostrocaudal direction. The orexin excitation of SN reticulata cells may help to maintain their high firing rate during waking. Furthermore, the absence of orexin effects in narcolepsy may predispose affected individuals to attacks of cataplexy.     

A new identity for MLK3 as an NIMA-related,cell cycle-regulated kinase that is localized near centrosomes and influences microtubule organization

Although conserved counterparts for most proteins involved in the G(2)/M transition of the cell cycle have been found in all eukaryotes, a notable exception is the essential but functionally enigmatic fungal kinase NIMA. While a number of vertebrate kinases have been identified with catalytic domain homology to NIMA, none of these resemble NIMA within its extensive noncatalytic region, a region critical for NIMA function in Aspergillus nidulans. We used a bioinformatics approach to search for proteins with homology to the noncatalytic region of NIMA and identified mixed lineage kinase 3 (MLK3). MLK3 has been proposed to serve as a component in MAP kinase cascades, particularly those resulting in the activation of the c-Jun N-terminal kinase (JNK). Here we describe the first in-depth study of endogenous MLK3 and report that, like NIMA, MLK3 phosphorylation and activity are enhanced during G(2)/M, whereas JNK remains inactive. Coincident with the G(2)/M transition, a period marked by dramatic reorganization of the cytoplasmic microtubule network, endogenous MLK3 transiently disperses away from the centrosome and centrosomal-proximal sites where it is localized during interphase. Furthermore, when overexpressed, MLK3, like NIMA, localizes to the centrosomal region, induces profound disruption of cytoplasmic microtubules and a nuclear distortion phenotype that differs from mitotic chromosome condensation. Cellular depletion of MLK3 protein using siRNA technology results in an increased sensitivity to the microtubule-stabilizing agent taxol. Our studies suggest a new role for MLK3, separable from its function in the JNK pathway, that may contribute to promoting microtubule instability, a hallmark of M phase entry.     

Hepatitis B markers in southern Altaĭ inhabitants of the village of Mendur-Sokkon (the Republic of Altaĭ). HBsAg subtyping in hepatitis B virus isolates with monoclonal antibodies

Blood samples taken from 231 native inhabitants of the village of Mendur-Sokkon located in the Republic of Altai (South-Western Siberia, Russia) were tested for the presence of virus hepatitis B (HBV) markers. 31 samples (13.4%) were found to contain HBsAg, 111 samples (48.05%) were found to contain total anti-HBc antibodies, 123 samples (53.24%) were found to contain anti-HBs antibodies and 15 blood samples (6.49%), anti-HBc antibodies without anti-HBs antibodies and HBsAg. The age-dependent distribution of the occurrence of HBV markers among the aboriginal population of the South Altal remained unchanged (69.9 +/- 7.9%) for the last 50 years. The vertical and horizontal routes of HBV transmissions were noted. The data obtained in this study are indicative of a highly endemic character of HBV of the territory of Mendur-Sokkon. HBsAg-positive blood samples were taken for HBsAg subtyping with the use of a panel of monoclonal antibodies. Two subtypes of HBsAg were detected: ayw1-2 and ayw3varB with the occurrence of 92.6% and 7.4%, i.e. distributed in the ratio 25/2.     

Mac-1-negative B-1b phenotype of natural antibody-producing cells, including those responding to Gal alpha 1,3Gal epitopes in alpha 1,3-galactosyltransferase-deficient mice

Human natural Abs against Galalpha1-3Galbeta1-4GlcNAc (Gal) epitopes are a major barrier to xenotransplantation. Studies in this report, which use combined multiparameter flow cytometric sorting and enzyme-linked immunospot assay, demonstrate that anti-Gal IgM-producing cells are found exclusively in a small B cell subpopulation (i.e., CD21(-/low) IgM(high) B220(low) CD5(-) Mac-1(-) 493(-) cells) in the spleens of alpha1, 3-galactosyltransferase-deficient mice. All IgM-producing cells were detected in a similar splenic subpopulation of alpha1, 3-galactosyltransferase-deficient and wild-type mice. A higher frequency of B cells with anti-Gal surface IgM receptors was observed in the peritoneal cavity than in the spleen, but these did not actively secrete Abs, and showed phenotypic properties of B-1b cells (CD21(-/low) IgM(high) CD5(-) CD43(+) Mac-1(+)). However, these became Mac-1(-) and developed anti-Gal Ab-producing activity after in vitro culture with LPS. The splenic B cells with anti-Gal receptors consisted of both Mac-1(+) B-1b cells and Mac-1(-) B-1b-like cells. The latter comprised most anti-Gal IgM-producing cells. Our studies indicate that anti-Gal natural IgM Abs are produced by a B1b-like, Mac-1(-) splenic B cell population and not by plasma cells or B-1a cells. They are consistent with a model whereby B-1b cells lose Mac-1 expression upon Ag exposure and that these, rather than plasma cells, become the major IgM Ab-producing cell population.     

Interferon-γ receptors are expressed at synapses in the rat superficial dorsal horn and lateral spinal nucleus

Summary Interferon-γ can facilitate the spinal nociceptive flexor reflex and elicit neuropathic pain-related behavior in rats and mice. Immunoreactivity for the interferon-γ receptor (IFN-γR) occurs in the superficial layers of the dorsal horn and the lateral spinal nucleus in the rat and mouse spinal cord, as well as in subsets of neurons in the dorsal root ganglia. The aim of the present study was to examine the cellular localization and origin of the IFN-γR in the spinal cord. As viewed by confocal microscopy, the immunopositivity for the IFN-γR was co-localized with that of the presynaptic marker synaptophysin and with neuronal nitric oxide synthase in the lateral spinal nucleus, whereas only a minor overlap with these molecules was observed in laminae I and II of the dorsal horn. There was no co-localization of the IFN-γR with markers for astrocytes and microglial cells. Ultrastructurally, the IFN-γR was found predominantly in axon terminals in the lateral spinal nucleus but also at postsynaptic sites in dendrites in laminae I and II. The IFN-γR expressed in neurons in dorsal root ganglia was transported in axons both centrally and peripherally. Hemisection of the spinal cord caused no reduction in immunolabelling of the IFN-γR in the dorsal horn or the lateral spinal nucleus. Since rhizotomy does not effect the immunolabelling in the lateral spinal nucleus, our observation indicates that the presynaptic receptors in this nucleus are derived from intrinsic neurons. The localization of the IFN-γR in the spinal cord differed from that of the AMPA glutamate receptor subunits 2 and 3 and the substance P receptor (NK1). Our results, showing localization of IFN-γR to pre- and postsynaptic sites in the dorsal horn and lateral spinal nucleus indicate that IFN-γ can modulate nociception at the spinal cord level.     

Determination of malondialdehyde in rat brain by capillary zone electrophoresis

A method for determination of malondialdehyde with capillary electrophoresis using UV detection at 267 nm has been developed. The buffer system consisted of 10 mM borax and 0.5 mM CTAB at pH 9.3. Malondialdehyde migrated as the first peak in the electropherogram at 2.6 min. Limit of detection was 1.2 μM corresponding to 7.8 pg. Malondialdehyde was determined before and after stimulating lipid peroxidation with the addition of ferrous ammonium sulphate to homogenates of rat brain tissue. Proteins were precipitated by boiling and removed from the brain homogenates with centrifugation. No further pretreatment was made before injecting the homogenates on the CE system. Non-precipitated homogenates could also be analyzed, but this required washing of the capillary with 0.1 M NaOH before introduction of the next sample.     

The American Alligator Detects Food Chemicals in Aquatic and Terrestrial Environments

American alligators (Alligator mississippiensis) were offered raw meat or animal carcasses under water or on land in experiments where only chemoreception could be used to discriminate between these and control materials. Alligators also were presented with aqueous extracts and airborne chemicals from meat. Juvenile alligators, tested in indoor tanks, opened more cheesecloth packets containing meat than control packets when these materials were placed under water and on platforms above the water surface. Adult alligators, tested in outdoor semi-natural enclosures, removed more cheesecloth-wrapped meat presented under water, and more of both meat and raccoon (Procyon lotor) carcasses placed under perforated baskets on land, than control materials. Juvenile alligators, tested in tanks partially filled with water, exhibited more lateral head movements and mouth-openings to an aqueous beef extract than to water alone. Juvenile alligators, tested in an olfactometer, exhibited more gular pumps to airborne chemicals from beef than to distilled water. These experiments indicate that alligators may use chemical cues to locate food both on land and under water, and that they detect both water- and airborne chemicals from meat.