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Summary: Medulloblastoma (MB) is a malignant pediatric brain tumor arising in the cerebellum. Although abnormal GABAergic receptor activation has been described in MB, studies have not yet elucidated the contribution of receptor-independent GABA metabolism to MB pathogenesis. We find primary MB tumors globally display decreased expression of GABA transaminase (ABAT), the protein responsible for GABA metabolism, compared with normal cerebellum. However, less aggressive WNT and SHH subtypes express higher ABAT levels compared with metastatic G3 and G4 tumors. We show that elevated ABAT expression results in increased GABA catabolism, decreased tumor cell proliferation, and induction of metabolic and histone characteristics mirroring GABAergic neurons. Our studies suggest ABAT expression fluctuates depending on metabolite changes in the tumor microenvironment, with nutrient-poor conditions upregulating ABAT expression. We find metastatic MB cells require ABAT to maintain viability in the metabolite-scarce cerebrospinal fluid by using GABA as an energy source substitute, thereby facilitating leptomeningeal metastasis formation.

An outbreak of enterohaemorrhagic Escherichia coli O157 occurred in multiple prefectures of Japan in November 2009. We conducted two case–control studies with trace-back and trace-forward investigations to determine the source. The case definition was met by 21 individuals; 14 (66.7%) were hospitalised, but no haemolytic uraemic syndrome, acute encephalopathy or deaths occurred. Median age was 23 (range 12–48) years and 14 cases were male (66.7%). No significant associations with food were found in a case–control study by local public health centres, but our matched case–control study using Internet surveys found that beef hanging tender (or hanger steak), derived from the diaphragm of the cattle, was significantly associated with illness (odds ratio = 15.77; 95% confidence interval, 2.00–124.11). Pulsed-field gel electrophoresis analysis of isolates from patients and the suspected food showed five different patterns: two in faecal and food samples, and another three in patient faecal samples only, although there were epidemiological links to the meat consumed at the restaurants. Trace-back investigation implicated a common food processing company from outside Japan. Examination of the logistics of the meat processing company suggested that contamination did not occur in Japan. We concluded that the source of the outbreak was imported hanging tender. This investigation revealed that Internet surveys could be useful for outbreak investigations.

Polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene (BaP) are ubiquitous environmental pollutants found in tobacco smoke, air pollution, and grilled foods. Prenatal exposure to BaP causes premature reproductive senescence in mice, and other PAHs are transplacental ovarian carcinogens. Glutathione (GSH) is critical for detoxification of the reactive metabolites of PAHs. Therefore, we hypothesized that mice that are genetically deficient in GSH synthesis, due to deletion of the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have increased destruction of oogonia, premature ovarian failure, and ovarian tumorigenesis after transplacental BaP exposure compared with Gclm+/+ females. Gclm+/− female and male mice were mated, and dams were treated with 0, 2, or 10 mg/kg/d BaP in sesame oil by gavage from gestational days 7 to 16. Compared with oil-treated F1 females of the same genotype, Gclm−/− prenatally BaP-treated females had significantly greater decrements in offspring production than Gclm+/+ BaP-treated females. Similarly, we observed significant BaP dose × Gclm genotype interactions on ovarian follicle counts and ovarian tumor multiplicity at 7.5 months of age, with Gclm−/− females having greater decrements in follicle numbers and more ovarian tumors in response to prenatal BaP exposure than Gclm+/+ females. The ovarian tumors were positive for the epithelial marker cytokeratin. Our results show that prenatal exposure of females to BaP causes premature ovarian failure and ovarian tumorigenesis and that embryonic GSH deficiency due to deletion of Gclm increases sensitivity to these transplacental ovarian effects of BaP. Cancer Res; 73(2); 908–17. ©2012 AACR.

The present study described the neuro-anatomy of a larval coral reef fish Amphiprion ocellaris and hypothesized that morphological changes during the transition from the oceanic environment to a reef environment (i.e. recruitment) have the potential to be driven by changes to environmental conditions and associated changes to cognitive requirements. Quantitative comparisons were made of the relative development of three specific brain areas (telencephalon, mesencephalon and cerebellum) between 6 days post-hatch (dph) larvae (oceanic phase) and 11 dph (at reef recruitment). The results showed that 6 dph larvae had at least two larger structures (telencephalon and mesencephalon) than 11 dph larvae, while the size of cerebellum remained identical. These results suggest that the structure and organization of the brain may reflect the cognitive demands at every stage of development. This study initiates analysis of the relationship between behavioural ecology and neuroscience in coral reef fishes.

Polycyclic aromatic hydrocarbons (PAHs), like benzo[a]pyrene (BaP), are ubiquitous environmental pollutants formed by the incomplete combustion of organic materials. The tripeptide glutathione (GSH) is a major antioxidant and is important in detoxification of PAH metabolites. Mice null for the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH concentrations. We investigated the effects of Gclm deletion alone on male fertility and spermatogenesis and its effect on the sensitivity of male embryos to the transplacental testicular toxicity of BaP. Gclm-/- males had dramatically decreased testicular and epididymal GCL enzymatic activity and total GSH concentrations compared with Gclm+/+ littermates. Ratios of reduced to oxidized GSH were significantly increased in Gclm-/- testes. GSH reductase enzymatic activity was increased in Gclm-/- epididymides. We observed no changes in fertility, testicular weights, testicular sperm head counts, or testicular histology and subtle changes in cauda epididymal sperm counts, motility, and morphology in Gclm-/- compared with Gclm+/+ males. Prenatal exposure to BaP from gestational day 7 to 16 was dose dependently associated with significantly decreased testicular and epididymal weights, testicular and epididymal sperm counts, and with vacuolated seminiferous tubules at 10 weeks of age. Gclm-/- males exposed prenatally to BaP had greater decreases in testicular weights, testicular sperm head counts, epididymal sperm counts, and epididymal sperm motility than Gclm+/+ littermates. These results show no effects of Gclm deletion alone on male fertility and testicular spermatogenesis and subtle epididymal effects but support increased sensitivity of Gclm-/- males to the transplacental testicular toxicity of BaP. © The Author 2012. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.

The relationship between shear-force value and collagen architecture of connective tissue of the longissimus thoracis (LT) muscle of Japanese Black (n = 10) and Brown (Kumamoto) (n = 5) steers (body weight: 688.4 +/- 8.6 kg as average and standard error) was investigated. There were negative correlations between the shear-force value and lipid content (n = 15, R(2)= 0.3709, P < 0.01) and protein content and lipid content (n = 15, R(2)= 0.6748, P < 0.01). Shear-force value and collagen content (n = 15, R(2)= 0.4344, P < 0.01) were positively correlated. In scanning electron microscopic photographs of the macerated preparation, the perimysium of the high-lipid LT muscle was broken down compared with the low-lipid LT muscle. The endomysium in all LT muscle fibers showed similar architecture. The fine surface cover of reticular collagen fibers around an adipocyte was observed in the high-lipid LT muscle perimysium. These results suggested that the shear-force value of the LT muscle was related to change in collagen architecture and of the perimysium in particular.

Changes in ileal bacterial populations of Salmonella-infected turkeys fed different diets were analyzed by using 16S-V3 PCR denaturing gradient gel electrophoresis. Turkeys raised on litter flooring were fed wheat- and corn-based diets with and without enzyme preparations (XY1 and XY2, respectively) from 0 to 126 d. Preparation XY1 contained exclusively endoxylanase, whereas preparation XY2 contained endoxylanase, protease, and alpha-amylase (Danisco, , Wiltshire, UK). The dietary activity levels of XY1 and XY2 were 2,500 and 650 endo-1,4-beta-xylanase units/kg of feed, respectively. Microbial DNA was extracted from the ileal content of 16-wk-old turkeys, and the 16S rDNA gene was amplified by PCR and analyzed by denaturing gradient gel electrophoresis. Diversity indexes, including richness (number of species, S), evenness (relative distribution of species, EH), diversity (using Shannon's index, H'), and Sorenson's pairwise similarities coefficient (measures the species in common between different habitats, Cs) were calculated. Additionally, diversity indexes were associated with Salmonella prevalence determined from fresh fecal droppings collected from each pen. On the basis of contrast analysis, the wheat-based diets resulted in higher microbial diversity indexes than the corn-based diets (S = 10 vs. 12; EH = 0.9 vs. 0.8; H' = 2.2 vs. 1.9, P < 0.05). Likewise, enzyme supplementation stimulated growth of the microbiota and increased the diversity indexes in comparison with unsupplemented treatments (S = 13 vs. 10; EH = 0.9 vs. 0.8; H' = 2.2 vs. 1.9, P < 0.05). Salmonella prevalence was higher (P < 0.05) at 15 wk in turkeys fed the corn-based diet (Salmonella prevalence = 50%) than in turkeys fed the corn-enzyme (Salmonella prevalence = 13%) and wheat-based (Salmonella prevalence = 0%) dietary treatments. Therefore, contrast analysis showed that birds fed the corn control diet had lower microbiota diversity but higher Salmonella prevalence than birds fed the enzyme-supplemented and wheat-based diets. In contrast, birds fed the wheat-based diets had higher diversity but lower Salmonella prevalence than birds fed the corn-based diets. High dietary nonstarch polysaccharides from wheat and dietary exogenous enzyme supplementation promoted microbial community diversity and apparently discouraged Salmonella colonization through competitive exclusion. Nonstarch polysaccharides and dietary exogenous enzyme supplementation may be practical tools to control enteric pathogens and benefit the intestinal health and food safety of the birds.

We investigated changes in connective tissue components of masseter (MA) muscle in Japanese black heifers (n= 6) in concentrate- and roughage-fed groups (groups C and R, respectively). Body weight, at slaughter, of experimental heifers in group C (272.3 +/- 22.3 kg) was higher (P < 0.05) than that of group R (213.8 +/- 27.5 kg). However, muscle weight and myofiber diameter (superficial and deep layers) of MA muscle did not differ between groups C and R. In contrast, total mastication duration of group R was longer (P < 0.05) than that of group C. MA muscle of groups C and R was composed only of type I myofiber. Using immunohistochemical/confocal laser-scanning microscopy, type I collagen was observed mainly in perimysium, and type V and VI collagen were observed in perimysium and endomysium of both groups. Type IV collagen and laminin were observed only in the endomysium in both groups. However, type III collagen and fibronectin were strongly apparent in the perimysium and endomysium in group R. Connective tissue components in the perimysium of groups C and R were observed to form plate-shaped layers. On the other hand, honeycomb-shaped connective tissue components were seen in the endomysium-surrounded muscle fibers. In particular, fibronectin was strongly observed in the perimysium and endomysium in group R. These results indicate that there are different developmental changes among connective tissue components in MA muscle in response to mastication. The immunohistochemical/confocal laser-scanning microscopic method is useful to investigate the structural relationship among connective tissue components in skeletal muscle.

© 2015 Elsevier Inc.. Polycyclic aromatic hydrocarbons, like benzo[. a]pyrene (BaP), are ubiquitous environmental pollutants and potent ovarian toxicants. The transcription factor NRF2 is an important regulator of the cellular response to electrophilic toxicants like BaP and to oxidative stress. NRF2 regulates transcription of genes involved in the detoxification of reactive metabolites of BaP and reactive oxygen species. We therefore hypothesized that Nrf2-/- mice have accelerated ovarian aging and increased sensitivity to the ovarian toxicity of BaP. A single injection of BaP dose-dependently depleted ovarian follicles in Nrf2+/+ and Nrf2-/- mice, but the effects of BaP were not enhanced in the absence of Nrf2. Similarly, Nrf2-/- mice did not have increased ovarian BaP DNA adduct formation compared to Nrf2+/+ mice. Ovarian follicle numbers did not differ between peripubertal Nrf2-/- and Nrf2+/+ mice, but by middle age, Nrf2-/- mice had significantly fewer primordial follicles than Nrf2+/+ mice, consistent with accelerated ovarian aging.

Copyright © 2015 by the Endocrine Society. Glutathione (GSH) is the one of the most abundant intracellular antioxidants. Mice lacking the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH. Our prior work showed that GSH plays antiapoptotic roles in ovarian follicles. We hypothesized that Gclm-/-mice have accelerated ovarian aging due to ovarian oxidative stress. We found significantly decreased ovarian GSH concentrations and oxidized GSH/oxidized glutathione redox potential in Gclm-/-vs Gclm+/+ovaries. Prepubertal Gclm-/-and Gclm+/+mice had similar numbers of ovarian follicles, and as expected, the total number of ovarian follicles declined with age in both genotypes. However, the rate of decline in follicles was significantly more rapid in Gclm-/-mice, and this was driven by accelerated declines in primordial follicles, which constitute the ovarian reserve. We found significantly increased 4-hydroxynonenal immunostaining (oxidative lipid damage marker) and significantly increased nitrotyrosine immunostaining (oxidative protein damage marker) in prepubertal and adult Gclm-/-ovaries compared with controls. The percentage of small ovarian follicles with increased granulosa cell proliferation was significantly higher in prepubertal and 2-month-old Gclm-/-vs Gclm+/+ovaries, indicating accelerated recruitment of primordial follicles into the growing pool. The percentages of growing follicles with apoptotic granulosa cells were increased in young adult ovaries. Our results demonstrate increased ovarian oxidative stress and oxidative damage in young Gclm-/-mice, associated with an accelerated decline in ovarian follicles that appears to be mediated by increased recruitment of follicles into the growing pool, followed by apoptosis at later stages of follicular development.

Kit and its ligand stem cell factor (SCF) play a fundamental role in hematopoiesis, melanogenesis and gametogenesis. Homozygous W(v) mutant mice with a mutation in kit show abnormalities in these cell lineages. Fas is a member of the death receptor family inducing apoptosis. In this study, we generated double-mutant mice (W(v)/W(v):Fas(-/-)) and analyzed histologically their reproductive organs. In testes and ovaries of the double-mutant mice, testicular germ cells and oocytes were detected, respectively, whereas the same-aged W(v)/W(v) mice contained neither cells. In addition, inhibition of Kit signals by administration of anti-Kit mAb, which induces degeneration of testicular germ cells in vivo in wild-type mice, did not cause degeneration in Fas-deficient mice. In testicular germ cells of W(v)/W(v) mutant mice, an increase of Fas expression was observed in spermatogonia. Further, in vitro treatment with SCF was shown to downregulate Fas on fibroblasts expressing exogenous Kit through activation of PI3-kinase/Akt. All the results clearly indicate that Fas-mediated apoptosis is involved in germ cell degeneration accompanied by defects in Kit-mediated signals, and Kit signaling negatively regulates Fas-mediated apoptosis in vivo.

This paper describes a flexible polyimide microelectrode ribbon with a bending structure for epidural recording. Bending the ribbon establishes good electro-mechanical contact of the electrodes to the cortex. Impedance spectroscopy and noise measurements in vitro were used to characterize the electrodes. Our in vivo experiments then successfully achieved synchronized neuronal activity and evoked potential patterns useful for understanding brain function and for finding cortical function.

In hereditary myopathy of the diaphragmatic muscles in Holstein-Friesian cattle, the largest number of acidophilic intracytoplasmic inclusions was found in the myocardium. These inclusions, which were oval and measured 12-15 microm in the transverse sections, were characterized by a dense, amorphous zone, and a relatively hyalinized sarcous substance in paraffin-embedded hematoxylin and eosin (H & E)-stained sections. Histochemically, each inclusion was stained intense red and dark green with H & E and Gomori's trichrome, respectively. NADH-TR activity was absent. The region surrounding the inclusions was less acidophilic with H & E, and showed an increased activity with NADH-TR. The inclusions showed no immunoreactivity for desmin, vimentin, actin or alpha-actinin, while strong desmin immunoreactivity was observed in the region surrounding the inclusion. Some inclusions showed strong immunoreactivity for ubiquitin, but others reacted only faintly. Ultrastructurally, the inclusion had a dense core composed of myofibrillar aggregations. The periphery of this dense core was surrounded by thin or intermediate-sized filaments, which corresponded to the desmin-positive area. This alteration was sometimes found to be continuous with the Z disk, which showed streaming or disintegration or with the desmosome of the intercalated disk. We discuss here the similarity between this specific inclusion and the other alternative organelles that have been reported previously in cardiomyopathy or in cardiac lesions associated with various myopathies.

We investigated effects of lipo-pro-prostaglandin E1 (lipo-[11alpha, 13E, 15S]-11,15-dihydroxy-9-[1-oxobutoxy]-prosta-8, 13-dien-1-oic acid butyl ester), AS-013 in two models of hearing disturbance and equilibrium dysfunction induced by rat inner ear microcirculatory thrombosis. Inner ear microcirculatory thrombosis was induced by photochemical reaction between systemic injection of Rose Bengal and irradiation of green light to the cochlea and vestibule. Photochemical reaction causes endothelial injury followed by platelet adhesion, aggregation and formation of a platelet- and fibrin-rich thrombus. In the hearing disturbance model, a compound cochlear nerve action potential was recorded by electrocochleography every minute. Photochemical reaction induced inner ear microcirculatory thrombosis, followed by disappearance of the action potential. AS-013 significantly (P

Appropriate structural modification of the difluoromethylene ketone derivatives at both P3 and P' positions led us to the discovery of peptidyl human heart chymase inhibitor 12h which shows potent activity with Ki = 6 nM and high selectivity against closely related serine protease bovine alpha-chymotrypsin (chymotrypsin Ki = > 100 microM). Using the compound 12b, a docking study with human heart chymase was carried out to presume probable interactions.

SUMMARY 1. AE0047 is a new dihydropyridine calcium antagonist with protective effects against cerebral ischaemia and the occurrence of stroke in several animal models. 2. In the present study we investigated whether AE0047 would improve the reduced cerebral blood flow (CBF) and oedema formation in cats subjected to middle cerebral artery (MCA) occlusion and compared it for efficacy with other dihydropyridine calcium antagonists with different moieties, such as nilvadipine an. nicardipine. 3. Middle cerebral artery occlusion reduced local CBF (1CBF), as measured by the hydrogen clearance method, while dry weight measurement showed that water content in the cortical tissues surrounding each 1CBF measurement electrode had increased after 4 h ischaemia. 4. Both AE0047 (10 μg/kg) and nilvadipine (30 μg/kg), given intravenously 20min after MCA occlusion, produced an approximate 10. hypotensive response and significantly increased 1CBF in severely and moderately ischaemic regions, grouped according to the initial reduced flow values. However, nicardipine (5 μg/kg bolus followed by infusion of 3 μg/kg per min for 60 min) failed to mitigate the reduction in 1CBF despite an increase in the ICBF of the contralateral cortex. In addition, AE0047 tended to prevent an increase in cortical water content in severely ischaemic regions, whereas water content in both nilvadipine- and nicardipine-treated groups tended to increase. 5. These results suggest that dihydropyridine calcium antagonists act differently on cerebral ischaemia and oedema formation in a manner dependent on their side-chain structures and that AE0047 effectively attenuates ischaemic brain damage without aggravatin. oedema.

Polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene (BaP), are ubiquitous environmental pollutants found in tobacco smoke, air pollution, and grilled foods. Reactive metabolites and reactive oxygen species generated during PAH metabolism are detoxified by reactions involving glutathione (GSH). Early life exposures to tobacco smoke and air pollution have been linked to increased risk of obesity and metabolic syndrome. We investigated the independent and interactive effects of prenatal exposure to BaP and GSH deficiency due to deletion of the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, on adiposity and hepatic steatosis in adult female F1 offspring. We mated Gclm+/-dams with Gclm+/-males and treated the pregnant dams with 0, 2, or 10mg/kg/day BaP in sesame oil by oral gavage daily from gestational day 7 through 16. We analyzed metabolic endpoints in female Gclm-/-and Gclm+/+littermate F1 offspring. Prenatal BaP exposure significantly increased visceral adipose tissue weight, weight gain between 3 weeks and 7.5 months of age, hepatic lipid content measured by oil red O staining, and hepatic fatty acid beta-oxidation gene expression in Gclm+/+, but not in Gclm-/-, female offspring. Hepatic expression of lipid biosynthesis and antioxidant genes were decreased and increased, respectively, in Gclm-/-mice. Our results suggest that reported effects of pre- and peri-natal air pollution and tobacco smoke exposure on obesity may be mediated in part by PAHs. GSH deficiency is protective against the metabolic effects of prenatal BaP exposure. © 2013 Elsevier Ireland Ltd.

Many eukaryotic cell surface proteins are bound to the membrane via the glycosylphosphatidylinositol (GPI) anchor that is covalently linked to their carboxy-terminus. The GPI anchor precursor is synthesized in the endoplasmic reticulum (ER) and post-translationally linked to protein. We cloned a human gene termed PIG-B (phosphatidylinositol glycan of complementation class B) that is involved in transferring the third mannose. PIG-B encodes a 554 amino acid, ER transmembrane protein with an amino-terminal portion of approximately 60 amino acids on the cytoplasmic side and a large carboxy-terminal portion of 470 amino acids within the ER lumen. A mutant PIG-B lacking the cytoplasmic portion remains active, indicating that the functional site of PIG-B resides on the lumenal side of the ER membrane. The PIG-B gene was localized to chromosome 15 at q21-q22. This autosomal location would explain why PIG-B is not involved in the defective GPI anchor synthesis in paroxysmal nocturnal hemoglobinuria, which is always caused by a somatic mutation of the X-linked PIG-A gene.

We describe a family line with an autosomal recessive disease of muscular dystrophy of the diaphragmatic muscles in Holstein-Friesian cattle. Histopathological examination in the present cases revealed various degenerative changes in the diaphragmatic and other thoracic muscles as follows: variation in muscle fiber diameter, fiber splitting, sarcoplasmic masses, ring fiber, vacuolar and hyalinized degeneration of muscle fibers. In addition, central core-like structures were the prominent features in the diaphragmatic muscles, occupying the center of the fiber or scattered within the fiber. These pathological alterations are consistent with the diaphragmatic myopathy previously reported in Meuse-Rhine-Yssel cattle in the Netherlands. The fibers containing core-like structures consisted of three distinct zones which could be well distinguished by NADH-tetrazolium reductase activity. This activity was absent in the innermost zone, decreased in the intermediate zone, and normal or increased in the periphery. Electron microscopically, this structure appeared to be composed of focal myofibrillar degeneration beginning with streaming or disintegration of the Z disk. We discuss here the similarity between this core-like structure and the other alternative organelles that have been reported previously, and a possible defect or storage in the cytoskeleton from the findings of the Z disk abnormalities.

Glutathione (GSH) is the most abundant intracellular thiol and an important regulator of cellular redox status. Mice that lack the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH synthesis. Nicotinamide nucleotide transhydrogenase, an inner mitochondrial membrane protein, catalyzes the interconversion of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate; reduced nicotinamide adenine dinucleotide phosphate is required for reduction of GSH disulfide. Previous work supports roles for GSH in preimplantation development. We hypothesized that Gclm-/- mice have increased preimplantation embryonic mortality and that this effect is enhanced by absence of a functioning Nnt gene. Gclm-/- females produced significantly fewer pups per litter than Gclm-/- littermates. Numbers of oocytes ovulated in a natural estrous cycle or upon superovulation did not differ by genotype. Fewer uterine implantation sites were observed in the Gclm-/- females. Prepubertal Gclm-/- and Gclm+/+ females were superovulated, then mated overnight with a Gclm+/+ male. At 0.5 d postcoitum, Gclm-/- females had significantly lower percentages of zygotes with two pronuclei and higher percentages of zygotes with one pronucleus than Gclm+/+ or Gclm+/- females. At 3.5 d postcoitum, a significantly lower percentage of blastocyst stage embryos was recovered from uteri of Gclm-/-females than Gclm+/+ females. Embryonic development to the blastocyst stage, but not the two-cell stage, was significantly decreased after in vitro fertilization of oocytes from Gclm-/- females compared with Gclm+/+ females. The Nnt mutation did not enhance the effects of Gclm genotype on female fertility. These results demonstrate critical roles for maternal GSH in supporting normal preimplantation development. Copyright © 2011 by The Endocrine Society.

During myocardial infarction leukotriene B4 (LTB4) is probably a major determining factor of tissue damage because it can amplify the inflammatory reaction by recruiting leukocytes and degranulating them. Oral administration of eicosapentaenoic acid (EPA) is known to reduce LTB4 production by polymorphonuclear leukocytes (PMNL). However, it takes several weeks for EPA to take effect. In this study, we formulated a trieicosapentaenoyl-glycerol emulsion and infused it into rabbits (0.8 g EPA/kg). In the ex vivo study, the inhibition of LTB4 production by PMNL from EPA-infused rabbits was maximal (32-60% of preinfusion values, P less than 0.01) 6 h after the infusion. There was also a tendency toward reduced LTB4 production 1, 24, and 168 h after the infusion. A lower dose (0.2 g EPA/kg) also reduced LTB4 production (45% of preinfusion values, P less than 0.02) 6 h after the infusion. There was no significant change in LTB4 production in control groups in which soybean oil emulsion was infused instead of EPA. EPA infusion might be useful for reduction of tissue damage in the acute phase of LTB4-related diseases such as acute myocardial infarction.

Certain mouse strains (e.g., DBA/2) contain two renin genes (termed Ren-1 and Ren-2) and express higher renin levels in nonkidney tissues than strains with a single renin gene. The 5'-flanking regions of the Ren-1 and Ren-2 genes contain several TATA boxes preceding putative transcriptional start sites. These initiators are termed P1a, P1, P2 (from 5' to 3'), and their function (with the exception of P2) is largely unknown. In this study, we mapped the renin transcriptional start sites in renal and extrarenal tissues [adrenal, brain, testis, heart, and submandibular gland (SMG)] and examined the effect of adenosine 3',5'-cyclic monophosphate (cAMP) on tissue specific promoter usage. Our results showed that, in the unstimulated state, P2 (the predicted initiator) is active in all DBA/2 mouse tissues. Additional transcriptional start sites were detected in the adrenal and testis (originated by P1a and P2) and the SMG (originated by P1a, P1, and P2). The administration of 8-bromoadenosine 3',5'-cyclic monophosphate led to selective stimulation of P1a in the adrenal but did not affect the selective usage of initiation sites in other organs. A locus-specific ddNTP primer extension assay was used to verify which renin gene is induced by cAMP. Results indicated that both Ren-1 and Ren-2 responded to cAMP treatment in identical fashion. Taken together, these data indicate that more than one form of renin transcript is present in several mouse tissues. There is tissue specificity in promoter usage in the unstimulated state and in response to cAMP.

The 5' flanking region of the mouse renin genes (Ren-1d and Ren-2d) contains two motifs that are homologous to known negative regulatory elements (NREs). Ren-2d has a 150-base-pair (bp) insertion 5' to the upstream putative NRE (NRE-1), which is lacking in Ren-1d. We tested the functionality of these sequences by using site-directed mutagenesis to delete individually each putative NRE from Ren-1d and to delete the 150-bp insertion from Ren-2d. We examined the effect of these mutations on the expression of the reporter gene chloramphenicol acetyltransferase, which was expressed from a truncated thymidine kinase promoter fused to the renin regulatory region. This plasmid was transfected into human choriocarcinoma JEG-3 cells. Only the upstream NRE (positions -619 to -597) was found to be functional in Ren-1d. The deletion of a 150-bp insertion from Ren-2d resulted in the suppression of chloramphenicol acetyltransferase activity to the level of Ren-1d expression. These data suggest that the upstream NRE that is functional in Ren-1d, but not in Ren-2d, may be partly responsible for differential expression of the renin genes in various tissues. The molecular mechanism of the NRE was examined by studying its interaction with nuclear proteins in submandibular gland and JEG-3 cells by gel-mobility-shift assays. Specific nuclear protein binding was observed only to the upstream NRE and the molecular mass of this protein was approximately 72 kDa as determined by Southwestern blot analysis. Thus our results suggest that both Ren-1d and Ren-2d conserve a cis-acting NRE in the 5' flanking region. In Ren-1d, this NRE could bind a specific nuclear protein resulting in the inhibition of Ren-1d expression in these tissues. On the other hand, the NRE in Ren-2d is nonfunctional due to interference by an adjacent 150-bp insertion.

Oxidative stress occurs when generation of reactive oxygen species (ROS) overwhelms antioxidant defenses. Oxidative stress has been associated with male infertility. The transcription factor nuclear factor-erythroid 2-related factor 2 (NRF2) regulates basal and inducible transcription of genes encoding enzymes important for protection against ROS. We hypothesized that deletion of the Nrf2 gene causes testicular and epididymal oxidative stress, which disrupts spermatogenesis. Our results show that male Nrf2-/-mice have decreased fertility compared to wild-type and heterozygous littermates, due to accumulating seminiferous tubule damage with increasing age. Testicular sperm head counts, epididymal sperm counts, and epididymal sperm motility in 2-month-old Nrf2-/-males did not differ from those of wild-type littermates; however, by age 6months, Nrf2-/-males had 44% lower testicular sperm head counts, 65% lower epididymal sperm counts, and 66% lower epididymal sperm motility than wild-type males. Two- to 4-month-old Nrf2-/-males had elevated levels of testicular and epididymal lipid peroxidation and testicular germ cell apoptosis, and decreased levels of antioxidants, compared to wild-type males. These results provide evidence that oxidative stress has deleterious effects on the testis and epididymis and demonstrate a critical role for the transcription factor NRF2 in preventing oxidative disruption of spermatogenesis. © 2010 Elsevier Inc.

Glutathione (GSH), the most abundant intracellular nonprotein thiol, is critical for many cellular functions. The rate-limiting step in GSH synthesis is catalyzed by glutamate cysteine ligase (GCL), a heterodimer composed of a catalytic (GCLC) and a modifier (GCLM) subunit. The tissue-specific regulation of GSH synthesis is poorly understood. We showed previously that gonadotropin hormones regulate ovarian GSH synthesis. In the present study, we sought to clarify the ovarian cell type-specific effects of follicle-stimulating hormone (FSH) and estradiol on GSH synthesis. Immature female rats were treated with estradiol to stimulate development of small antral follicles. Granulosa cells (GCs) from these follicles or whole follicles were cultured in serum-free media, with or without FSH and 17beta-estradiol. The GSH and GCLC protein and mRNA levels increased in GCs treated with FSH alone. The effects of FSH on GCLC and GCLM protein and mRNA levels, GCL enzymatic activity, and GSH concentrations in GCs were significantly enhanced by the addition of estradiol. Estradiol alone had no effects on GSH. Dibromo-cAMP mimicked and protein kinase A (PKA) inhibitors prevented FSH stimulation of GCL subunit protein levels. In cultured small antral follicles, FSH stimulated estradiol synthesis and robustly increased GCL subunit mRNA and protein levels and GSH concentrations. The GCL subunit mRNA expression increased in both the granulosa cells and theca cells of follicles with FSH stimulation. These data demonstrate that maximal stimulation of GSH synthesis by FSH in granulosa cells and follicles requires estradiol. Without estradiol, FSH causes lesser increases in GCL subunit expression via a PKA-dependent pathway.

The transport mechanism of soluble molecules throughout the interstitial matrix is closely associated with human tumor behavior in vivo. However, the examination of soluble components in histological architectures has been hampered by artifacts caused during conventional tissue preparation. In this study, the immunodistribution of intrinsic and extrinsic serum components in tumor tissues was examined in xenografted human tumor cells using 'in vivo cryotechnique' (IVCT) and cryobiopsy, where target tissues are directly cryofixed in vivo. Human lung cancer cells were subcutaneously injected into the dorsal flank of nude mice, and paraffin sections and cryosections of produced tumors were prepared with different methods. Immunolocalization of serum proteins, including albumin, immunoglobulin G (IgG) and IgM, as well as intravenously injected bovine serum albumin (BSA) was examined. Their immunodistribution was more clearly observed in the interstitium by both IVCT and cryobiopsy than conventional methods. IgM was immunolocalized within blood vessels, whereas albumin and IgG were observed in the tumor interstitium. Moreover, intravenously injected bovine serum albumin exhibited leakage from the blood capillaries into surrounding connective tissues in 24 h, but it gradually diffused to the interstitium of the tumor masses during 3 days. These results suggest that molecular leakage from blood capillaries varies significantly in different areas of developing tumors, and that small serum proteins, but not large ones, were abundantly immunolocalized in the tumor interstitium. Both IVCT and cryobiopsy were found to be useful for immunohistochemical studies of soluble molecules in tumors with blood circulation, and may therefore be helpful for further histopathological analyses.

The human genome carries multiple copies of sequences related to endogenous retroviral genomes that include long terminal repeat (LTR) sequences. We used the LTR of one such viral genome, called HERV-A, as a probe in Southern analysis to examine the distribution profiles of the hybridizing DNA in the genomes of twelve human × rodent hybrid cell lines carrying one or a few human chromosomes, and in the DNA samples prepared from six sorted, individual chromosomes. The HERV-A sequence was found to be widely distributed among different chromosomes and the Southern patterns for chromosomes 5, the X, and the Y, each obtained in duplicate from independently prepared cell lines or sorted chromosomes, were matched. Chromosome-specific Southern profiles can be used to monitor chromosomes in hybrid cells or to characterize chromosome aberrations, such as deletions.

An expression vector containing three copies of the AP-1 binding element (TRE) upsteam of a thymidine kinase promotor which controlled the expression of the chloramphenicol acetyl transferase (CAT) gene was transiently transfected into vascular smooth muscle (VSM) cells and a human hepatocarcinoma cell line, Hep G2. Twelve hours of angiotensin (Ang) II exposure stimulated significantly CAT expression by 3.4 fold and 2.7 fold in Hep G2 and VSM cells, respectively. AngII had no effect on CAT expression of a control vector. This AngII-induced stimulation was attenuated significantly by an AngII receptor antagonist, Sar 1 Ile 8 AngII, and abolished completely by a PKC inhibitor, staurosporine. Our data suggest that the TRE plays a crucial role in AngII-induced gene expression that is mediated by PKC. We concluded that TRE is one of the AngII-responsive elements.

The polycyclic aromatic hydrocarbon (PAH) 9,10-dimethyl-1,2-benzanthracene (DMBA) destroys primordial, primary, and secondary ovarian follicles in rodents, but its effects on antral follicles have received limited attention. PAHs are metabolized to reactive species, some of which can undergo redox cycling to generate reactive oxygen species (ROS). We previously showed that ROS initiate apoptosis in preovulatory follicles cultured without gonadotropin support and that glutathione (GSH) depletion induces apoptosis in the presence of gonadotropins. In the present study, we tested the hypothesis that DMBA induces apoptosis in preovulatory follicles, which is mediated by ROS and prevented by GSH. Preovulatory follicles were isolated from ovaries of 25-day-old rats 48 h after the injection of 10 IU of eCG and were cultured with DMBA in the presence of FSH for 2 to 48 h. DMBA induced granulosa cell (GC) and theca cell (TC) apoptosis at 48 h, as judged by TUNEL and activated caspase-3 immunostaining. DMBA treatment also increased the numbers of GCs and TCs that immunostained for the proapoptotic protein BAX. Follicular ROS levels were significantly increased in DMBA-treated follicles at 12, 24, and 48 h. GSH supplementation protected against and GSH depletion enhanced the induction of apoptosis in GCs and TCs by DMBA. These findings suggest that GSH is a critical protective mechanism against DMBA-induced apoptosis in antral follicles and that ROS generation may mediate DMBA-induced GC apoptosis.

Tissue in body must quickly recognize injury to response to the rapid pace of epidermal growth. In skin, the epidermal cells must also react to danger signals from the surrounding extracellular lipid of the stratum corneum spaces and immediately participate by initiating the wound repair process. The topical administration of the lyotropic liquid crystal nanocube to stratum corneum rapidly broke down the lipid lamella structure which would be recognized as a wound without organ-change. This can activate a variety of biological processes. This study set out to determine whether the phase transition of the lipid to a neighbouring different physicochemical structure can stimulate keratinocyte cells and what mechanism is responsible for this response. Using small angle x-ray scattering (SAXS) analysis, a response to the transient structural change of lipid was detected which might result from the diffusion of oil and/or water from nanocube liquid crystal towards the lipid lamella phase. Simultaneously, a significant increase in growth factors and inflammatory cytokines was detected after administration of nanocube. Not only the excess expression of cytokines but also the extent of TEWL as a barrier marker of skin increased. These observations suggest that a structural change in lipid can stimulate and trigger recognition of a slight injury in the wound defence and a repair response as homeostasis. This method actually succeeded in improving photo-induced hyperpigmentation on a human face.

All-trans retinoic acid (atRA) which could smooth wrinkles and produce less pigmented skin after a few months of treatment has been studied in research into topical treatments for a potent inhibitor of new melanin production. However, the clinical responses of commercial atRA cream predominantly comprise severe inflammation. We report a novel nanotechnology "nanoegg" system giving improved effects of atRA self-assembly which were coated by CaCO3. Dorsal areas of hairless mouse and porcine skin were employed for administration of nanoegg ointment and commercial products. The mRNA for heparin-binding epidermal growth factor-like growth factor (HB-EGF) from tissues was measured by a real-time PCR method. All tissues were stained for detection of hyaluronate and the thickness of the epidermis. A clinical trial in humans was carried out at St. Marianna University in Japan. As a result, the irritation and inflammation associated with atRA molecules were substantially reduced. The physicochemical instability of atRA was also dramatically improved. Furthermore, nanoegg enhanced marked expression of mRNA for HB-EGF from keratinocytes, which is known as one of the markers of keratinocyte turnover. Also, production of hyaluronate was surprisingly in the intercellular spaces of the basal and spinous cell layers 2 days after treatment. Even at the low concentration of atRA in the nanoegg system, the proliferation and differentiation of keratinocyte was somewhat enhanced. A nanoegg may thus not only prevent adverse effects, but also markedly enhance the main effect.

Introduction The protective role of nitric oxide (NO) against hepatic ischemia-reperfusion (I/R) injury remains controversial. In this study we investigated the effect of tetrahydrobiopterin (BH4) on the survival of rats exposed to an hepatic I/R injury. Methods The rats were subjected to 100 minutes of 70% hepatic ischemia 30 minutes after administration of BH4 or saline. A specific inducible NO synthase (iNOS) blocker, 1400W, was used to evaluate endogenous iNOS. NOS protein measured the histological appearance of the liver by Western blotting, and survival was evaluated after reperfusion. Results The 1-week survival rate was 60% among the BH4 group and 10% for the saline group. The serum ALT and bilirubin values in the BH4 group were significantly lower than the saline group. Histological examination of the liver revealed only a small necrotic area in the BH4 group as opposed to massive necrosis and cell infiltration in the saline group. Injection of 1400W significantly decreased the prolongation of survival produced by BH4. Conclusions BH4 significantly improved the survival rate, the histological findings, and the liver function, thereby reducing liver failure. Western blotting showed a higher level of iNOS protein in the BH4 group than the saline group, 1400W suppressed this effect of BH4. Taken together, these observations suggest that NO derived from reactions driven by BH4-induced iNOS exerts a protective effect against reperfusion injury.

Summary Among meat inspectors in Hokkaido, Japan, the term “bovine parasitic hepatitis” (BPH) has long been used to refer to a hepatic disorder characterized by multiple small yellowish lesions. However, the aetiology is unknown. By means of detailed histopathological examination, fragments of parasitic worms resembling Capillaria were detected in nine (2.25%) of 400 livers showing BPH lesions. Histologically, the degenerative lesions showed eosinophilic papillary endothelial hyperplasia of the interlobular veins and eosinophilic membranous structures in the eosinophilic granulomatous areas of inflammation. These characteristic findings differed from those of hepatic capillariasis of other animal species. BPH was found in 5–20% of milk cows examined throughout Hokkaido, and was also detected in cattle from another prefecture and from Australia. Possibly the same parasitic disorder, albeit undiagnosed, is more widespread.

We evaluated and characterized the mouse scratching behavior using a new apparatus, MicroAct. Scratching behavior was evoked in ICR and BALB/c mice by compound 48/80, passive cutaneous anaphylaxis or repeated hapten application. Under the present experimental condition, MicroAct detected consecutive scratching behavior (events) consisting of 3 or more beats. Although the detecting standard of MicroAct was not identical to that of an observer, the number of events detected by MicroAct and by an observer were almost comparable with each other. Frequency of events, total scratching time and total number of beats detected by MicroAct increased depending on the intensity of the causing stimuli for scratching. In contrast, the duration of each event and the number of beats in each event increased only slightly, but the scratching speed was almost constant. The present results demonstrate that MicroAct is a useful tool for evaluating mouse scratching behavior. Mouse scratching behavior seems to have a relatively fixed pattern and the causing stimulus increases mainly in the frequency of event without affecting the scratching speed.

The nucleotide sequences of the coding region of the nucleocapsid (N) gene of 12 mouse hepatitis virus (MHV) strains recently found in animal facilities in Japan were analyzed. Nucleotide sequencing was performed directly on polymerase chain reaction (PCR) products amplified by reverse transcription (RT) and polymerase chain reaction (RT-PCR) analysis from fecal samples or isolated viruses. Phylogenetic analysis of these MHV strains along with those reported previously indicated that sequence analysis of the N gene was a useful tool for differentiation of MHV strains,although most MHV strains in Japanese facilities were phylogenetically close. Results suggested that interchange of mice infected with MHV among facilities provided opportunities of introduction of MHV into otherwise MHV-free facilities and that the source of MHV infection could be traced by use of nucleotide analysis of the N gene.

We investigated the effects of Orengedoku-to (Huanglian-Jie-Du-Tang in Chinese) and Senkanmeimoku-to (Xygan-Ming-Mu-Tang in Chinese), traditional herbal medicines, on experimantal elevation of aqueous flare in pigmented rabbits. To produce the elevation of aqueous flare in rabbits, prostaglandin E2 (PGE2) was applied to the cornea with use of a glass cylinder, or lipopolysaccharides (LPS) were injected into the ear vein. Animals were pretreated by the oral administration of 150 g/day of food containing 0.7%, 0.2% or 0.07% (w/w) Orengedoku-to, or 2%, 0.6% or 0.2% (w/w) Senkanmeimoku-to for 5 days. Aqueous flare was measured with a laser flare-cell meter. Pretreatment with 0.7% or 0.2% Orengedoku-to and 2% Senkanmeimoku-to did suppress significantly (P0.05) elevation of aqueous flare induced by PGE2. Pretreatment with 0.7% or 0.2% Orengedoku-to and 2% or 0.6% Senkanmeimoku-to significantly suppressed (P0.001) elevation of aqueous flare induced by LPS. It is possible that Orengedoku-to and Senkanmeimoku-to may migrate some forms of uveitis.

Since most brain glycoproteins from beta-1,4-galactosyltransferase (beta-1,4-GalT) I knockout mice were galactosylated without apparent reduction the gene expression of novel beta-1,4-GalTs II and V which are involved in N-linked oligosaccharide biosynthesis in addition to beta-1,4-GalT I was studied during mouse brain development. Isolation and characterization of beta-1,4-GalT II and V cDNAs from mouse brains indicates that they are also functioning in the brain. Northern blot analysis revealed that the beta-1,4-GalT I gene is expressed mainly in mid-embryonic stages, while the expression level of beta-1,4-GalT II transcript remains constant and of beta-1,4-GalT V transcript increases during mouse brain development after birth. In situ hybridization revealed that beta-1,4-GalT II and V signals are present in most neural cells, with a marked difference between them in the hippocampus of adult mouse brain tissue. The differential gene expression of beta-1,4-GalTs I, II and V during mouse brain development could affect the differential galactosylation of brain glycoproteins, as revealed by lectin blot analysis.

The brain of a 6-year-old Holstein cow, which showed progressive neurologic symptoms during several months, was examined by histopathologic methods. Many round or oval-shaped cytoplasmic inclusions were observed, mainly in neurons of the temporal lobe and the hippocampus. Those inclusions were faintly eosinophilic with hematoxylin and eosin and positive with Bielschowsky's silver stain. Immunohistochemically, the inclusions were recognized by antiubiquitin and antiphosphorylated tau antibodies. Ultrastructurally, the inclusions were globular and well demarcated from the rest of the cytoplasm, lacked limiting membranes, and were mainly composed of straight fibrils about 15 nm in width. The structure of the inclusions was similar to that of Pick bodies in Pick's disease of humans. The pathogenesis of this bovine condition is not known.

ERM (ezrin, radixin, and moesin) proteins function as membrane-cytoskeletal linkers, and are known to be localized at filopodia and microvilli-like structures. We have shown that Rho-associated kinase (Rho-kinase)/ROKalpha/ROCK II phosphorylates moesin at Thr-558 at the lower stream of Rho, and the phosphorylation is crucial to the formation of microvilli-like structures (Oshiro, N., Fukata, Y.Kaibuchi, K. (1998) Phosphorylation of moesin by Rho-associated kinase (Rho-kinase) plays a crucial role in the formation of microvilli-like structures. J. Biol. Chem. 273, 34663- 34666). However, the role of ERM proteins in the formation of filopodia is less well characterized.Here we examined the phosphorylation state of ERM during filopodia formation induced by Cdc42 using the antibody recognizing ERM proteins phosphorylated at COOH (C)-terminal threonine. When NIH 3T3 cells were transfected with constitutively active Cdc42 (Cdc42V12), filopodia formation was induced and phosphorylation of ERM at C-terminal threonine was observed at the tip of filopodia, while the phosphorylation levels of ERM were lower and phosphorylated ERM was distributed throughout the cytoplasm in the control cells. We also showed that Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) which has been identified as an effector of Cdc42, phosphorylated moesin at C-terminal threonine in a cell-free system. Coexpression of the dominant negative form of MRCK inhibited both the formation of filopodia and accumulation of C-terminal threonine-phosphorylated ERM proteins at filopodia induced by Cdc42V12.The formation of filopodia induced by Cdc42 is accompanied by phosphorylation of ERM proteins, and MRCK is a candidate for the kinase that phosphorylates ERM proteins at filopodia.

We previously identified Rho-associated protein kinase (Rho-kinase) as a specific effector of Rho. In this study, we identified collapsin response mediator protein-2 (CRMP-2), as a novel Rho-kinase substrate in the brain. CRMP-2 is a neuronal protein whose expression is up-regulated during development. Rho-kinase phosphorylated CRMP-2 at Thr-555 in vitro. We produced an antibody that specifically recognizes CRMP-2 phosphorylated at Thr-555. Using this antibody, we found that Rho-kinase phosphorylated CRMP-2 downstream of Rho in COS7 cells. Phosphorylation of CRMP-2 was observed in chick dorsal root ganglion neurons during lysophosphatidic acid (LPA)-induced growth cone collapse, whereas the phosphorylation was not detected during semaphorin-3A-induced growth cone collapse. Both LPA-induced CRMP-2 phosphorylation and LPA-induced growth cone collapse were inhibited by Rho-kinase inhibitor HA1077 or Y-32885. LPA-induced growth cone collapse was also blocked by a dominant negative form of Rho-kinase. On the other hand, semaphorin-3A-induced growth cone collapse was not inhibited by a dominant negative form of Rho-kinase. Furthermore, overexpression of a mutant CRMP-2 in which Thr-555 was replaced by Ala significantly inhibited LPA-induced growth cone collapse. These results demonstrate the existence of Rho-kinase-dependent and -independent pathways for growth cone collapse and suggest that CRMP-2 phosphorylation by Rho-kinase is involved in the former pathway.

Some plasma membrane receptors in yeast are known to be internalized and degraded in lysosomes upon ligand-dependent ubiquitination. However, the role of ubiquitination in endocytosis and lysosomal degradation in higher eukaryotes has been controversial. In order to directly assess this question, we investigated the fate of chimeric molecules in which ubiquitin moiety was fused in-frame to the cytoplasmic region of membrane proteins. The chimeric proteins with the wild-type ubiquitin were endocytosed and delivered to lysosomes efficiently. Mutant ubiquitin with lysine-to-arginine substitution could still mediate endocytosis, suggesting that polyubiquitination is not required for the endocytosis. We next searched for the existence of an endocytosis signal(s) in the ubiquitin moiety, and identified a di-leucine signal, Leu(43)-Ile(44). The Leu(43)-Ile(44) sequence mediated endocytosis and lysosomal sorting in a Leu(43)-dependent manner. These results suggest that the di-leucine signal in ubiquitin can be involved in ubiquitination-mediated endocytosis and lysosomal targeting of membrane proteins.

Hsp70-2 is a unique member of the mouse 70-kDa heat shock protein family that is synthesized during meiosis in spermatogenic cells. Germ cells in male mice homozygous for a targeted mutation in the Hsp70-2 gene (Hsp70-2(-/-)) arrest in development and undergo apoptosis at the end of the pachytene spermatocyte stage of meiotic prophase. However, cells with a putative acrosome were present occasionally in histological sections of the testes of juvenile and adult Hsp70-2(-/-) mice. This study verified that acrosomes were present and investigated the relationship between acrosome formation and the process of meiosis. Histochemistry with the periodic acid-Schiff procedure and immunostaining with monoclonal antibody MN7 verified that acrosomes were present in Hsp70-2(-/-) mice, and electron microscopy showed that some of these cells had condensing nuclei characteristic of step 8-9 spermatids. The frequency of acrosome-containing cells in Hsp70-2(-/-) mice was less than 0.01% of that in wild-type mice. Propidium iodide staining and cytophotometry indicated that the average DNA content of nuclei in MN7-positive cells in Hsp70-2(-/-) mice was usually about twice, or occasionally the same as, that of nuclei in round spermatids of wild-type mice. Meiotic metaphase I and II chromosome spreads were observed in spermatogenic cells from Hsp70-2(-/-) mice but at a much lower frequency than in wild-type mice. These results indicate that not all pachytene spermatocytes in Hsp70-2(-/-) mice arrest in meiosis, but they may divide once or sometimes twice and begin acrosome formation and nuclear condensation. This demonstrates that some aspects of spermatid development can occur without the completion of meiosis in mice, as has been reported recently for Drosophila.

Water and methanol extracts of 30 traditional medicinal plants, collected in Indonesia, were tested for their anti HSV-1 activity. The extracts of eight plant species showed potent activity on the plaque assay at a concentration of 100 micrograms/mL. The therapeutic efficacy of seven selected plants was demonstrated by using a mouse HSV-1 infection assay, both the methanol extracts of the fruit of Melaleuca leucadendron (Myrtaceae) and the pericarp of Nephelium lappaceum (Sapindaceae) significantly prolonged the development of skin lesions and reduced the mortality.

Manipulation of ligament healing has been a major focus of orthopaedic research. In recent years, gene transfer to healing ligament appears to be a feasible method for manipulating the healing process. In this study, we investigated the feasibility of gene transfer to healing rat patellar ligament by intra-arterial delivery.An attempt was made to transfer a reporter gene (Escherichia coli, beta-galactosidase gene) to healing rat patellar ligament using the haemagglutinating virus of Japan (HVJ) liposome-mediated gene transfer method. Three days after cutting the patellar tendons of 25 14-week-old male Wistar rats, HVJ-liposome complexes containing beta-galactosidase (beta-gal) cDNA were injected into the femoral artery of 15 Wistar rats as the experimental group. HVJ liposomes without DNA were injected into the femoral artery of 10 Wistar rats as the control group. Three rats from the experimental group and two control rats were killed 3, 7, 14, 28 and 56 days after the injection.After X-gal staining, the rate of transfection in the experimental group (mean +/- SEM) was found to be 12.1% +/- 0.590%, 8.7% +/- 0.217%, 10.2% +/- 0.227%, 3.2% +/- 0.247% and 0.7% +/- 0.060% at post-injection days 3, 7, 14, 28 and 56 respectively. In control sections the number of blue-stained cells were very few at any point.We succeeded in introducing a reporter gene into healing rat patellar ligament by infra-arterial delivery of HVJ-liposome complexes. This method appears to have the potential to be applicable for soft-tissue healing studies and also healing studies of other tissues and organs.

Peptidyl difluoromethylene ketone derivatives were designed to take advantage of probable additional interactions with the S′ subsite of human heart chymase. They showed potent inhibitory activities against human heart chymase and were more efficient than bovine chymotrypsin.

1. To investigate the effectiveness of recombinant human serum albumin (rHSA) in the treatment of ascites in liver cirrhosis, we examined its effect on rats with carbon tetrachloride-induced liver cirrhosis. 2. Twenty-five percent rHSA was administered intravenously at a dose of 0.25 to 1.0 g/kg for 2 days to rats with liver cirrhosis accompanied by ascites retention and hypoalbuminemia. 3. rHSA dose dependently decreased abdominal circumference, a clinical index of ascites, with significant difference at a dose of 1.0 g/kg. 4. Although there was no significant difference, rHSA increased blood colloid osmotic pressure (b-COP) and urine volume (UV) in a nearly dose-dependent manner, with significant negative correlation between changes from baseline value in these parameters and in abdominal circumference. 5. These findings suggest that rHSA has abdominal circumference-decreasing action associated with b-COP improvement and UV increase and that it could be effective as a therapeutic drug for ascites in patients with liver cirrhosis accompanied by hypoalbuminemia.

Two H+/peptide cotransporters, PEPT1 and PEPT2, are expressed in the kidney, mediating the renal tubular reabsorption of oligopeptides and beta-lactam antibiotics. We examined the interactions of beta-lactam antibiotics with peptide transporters in rat renal brush-border membranes by evaluating the inhibitory potencies of the antibiotics against glycylsarcosine transport. Western blot analysis revealed that PEPT1 and PEPT2 were expressed in the renal brush-border membranes with the apparent molecular masses of 75 and 105 kDa, respectively. Using renal brush-border membrane vesicles, the uphill transport of glycylsarcosine was observed in the presence of an inward H+ gradient and an inside-negative membrane potential. Two transport systems with high affinity (Km of 50 microM) and low affinity (Km of 1.2 mM) appeared kinetically to mediate the glycylsarcosine uptake. The inhibition constants of the antibiotics for glycylsarcosine transport were more closely correlated with those in stable LLC-PK1 cells transfected with rat PEPT2 rather than PEPT1 cDNA. The beta-lactam antibiotics with an alpha-amino group showed trans-stimulation effects on the glycylsarcosine uptake, suggesting that these antibiotics and glycylsarcosine share a common peptide transporter. However, the antibiotics lacking an alpha-amino group failed to show the trans-stimulation effect. It is concluded that amino-beta-lactam antibiotics at therapeutic concentrations interact predominantly with PEPT2 localized in the brush-border membranes of rat kidney.

Unique type 1 hexokinase (HK1) mRNAs are present in mouse spermatogenic cells (mHk1-s). They encode a spermatogenic cell-specific sequence region (SSR) but not the porin-binding domain (PBD) necessary for HK1 binding to porin on the outer mitochondrial membrane. This study determined the origin of the multiple Hk1-s transcripts in mouse spermatogenic cells and verified that they are translated in mouse spermatogenic cells. It also showed that a single mHk1 gene encodes the mHk1 transcripts of somatic cells and the mHk1-sa and mHk1-sb transcripts of spermatogenic cells, that alternative exons are used during mHk1 gene expression in mouse spermatogenic cells, and that mHK1-S is translated in mouse spermatogenic cells and is localized mainly with the fibrous sheath in the tail region, not with the mitochondria in the midpiece of mouse sperm.

We evaluated the effect of AE0047, a dihydropyridine-type calcium antagonist, on the plasma lipid levels of obese Zucker rats. In rats treated orally with 3 to 10 mg/kg/day AE0047 for 7 days, plasma triglyceride (TG) and TG-rich lipoprotein levels dose-dependently decreased, whereas those of high-density lipoprotein cholesterol increased. Total cholesterol and low-density lipoprotein levels did not change. To elucidate the mechanism by which AE0047 decreases plasma TG levels, we examined the effect of AE0047 on the synthesis and secretion of TG-rich lipoproteins in human intestinal cell line Caco-2, as well as on the association and degradation of very low density lipoprotein (VLDL) in human hepatoblastoma cells HepG2. When Caco-2 cells were grown on a membrane filter and 14C-oleic acid was added to the apical side, 10(-5) and 10(-6) M AE0047 inhibited basolateral secretion of 14C-TG. AE0047 also suppressed the basolateral secretion of apolipoprotein B. In HepG2 cells, AE0047 increased the cellular uptake of 125I-VLDL. These results suggested that AE0047 decreased plasma TG level by the inhibition of intestinal chylomicron secretion and the enhancement of hepatic uptake of VLDL. AE0047 may be beneficial for the treatment of hypertensive patients with hypertriglyceridemia to reduce the risk factors of coronary heart disease.