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Parkinson’s disease (PD) is a prevalent motor disease caused by the accumulation of mutated α-synuclein (α-Syn), however, its early stages are also characterized by non-motor symptoms, such as olfactory loss, cognitive decline, depression, and anxiety. The therapeutic effects of environmental enrichment (EE) on motor recovery have been reported, but its effects on non-motor symptoms remain unclear. Herein, we reveal the beneficial effects of EE on PD-related non-motor symptoms and changes in synaptic plasticity in the nucleus accumbens. To investigate its therapeutic effects in the early phase of PD, we randomly assigned eight-month-old mice overexpressing human A53T (hA53T) α-Syn to either the EE or standard condition groups for two months. Next, we performed behavioral tests and biochemical and histological analyses at 10 months of age. EE significantly alleviated locomotor hyperactivity and anxiety during the early stages of PD. It normalized the levels of tyrosine hydroxylase, phosphorylated and oligomeric α-Syn, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex-forming proteins, including synaptosomal-associated protein, 25 kDa, syntaxin1, and vesicle-associated membrane protein 2 (VAMP2). Moreover, the interactions between VAMP2 and pSer129 α-Syn were markedly reduced following EE. The restoration of synaptic vesicle transportation status may underlie the neuroprotective effects of EE in hA53T α-Syn mice.

Epichlorohydrin (ECH) is an antifertility agent that acts both as an epididymal toxicant and an agent capable of directly affecting sperm motility. This study identified the time course of apoptotic cell death in rat epididymides after ECH treatment. Rats were administrated with a single oral dose of ECH (50 mg/kg). ECH-induced apoptotic changes were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and its related mechanism was confirmed by Western blot analysis and colorimetric assay. The TUNEL assay showed that the number of apoptotic cells increased at 8 h, reached a maximum level at 12 h, and then decreased progressively. The Western blot analysis demonstrated no significant changes in proapoptotic Bcl-2-associated X (Bax) and anti-apoptotic Bcl-2 expression during the time course of the study. However, phospho-p38 mitogen-activated protein kinase (p-p38 MAPK) and phospho-c-Jun amino-terminal kinase (p-JNK) expression increased at 8–24 h. Caspase-3 and caspase-8 activities also increased at 8–48 h and 12–48 h, respectively, in the same manner as p-p38 MAPK and p-JNK expression. These results indicate that ECH induced apoptotic changes in rat epididymides and that the apoptotic cell death may be related more to the MAPK pathway than to the mitochondrial pathway.

Background A high prevalence of cholestatic disease, including gallbladder mucocele (GBM), has been reported in dogs with naturally occurring pituitary-dependent hyperadrenocorticism (PDH). Hypothesis/Objectives Differences exist in the clinical features of dogs with PDH and concurrent cholestatic disease, and also is the management of these dogs with trilostane. Animals Sixty-five client-owned dogs with naturally occurring PDH. Methods This was a retrospective, observational case series. Each dog was treated with trilostane for at least 3 months before the study, and had a good clinical response, as determined by owners. Statistical comparisons of clinical signs, results of routine blood tests, basal and post-ACTH cortisol concentration, and optimal trilostane dosage were made after dogs were separated into the following 3 groups by ultrasonographic imaging: normal on ultrasound (NOU) group, cholestasis group, and GBM group. Results The GBM group had more severe clinical signs and significantly different total serum cholesterol concentration and post-ACTH stimulation cortisol concentration at the time of diagnosis. Dogs that weighed

To develop a method that can maintain egg freshness during practical storage conditions, eggs were coated with chitosan and stored with or without dry ice. The physicochemical and microbiological qualities of eggs were evaluated during 14 d of storage at 4 and 23°C without dry ice and at 23°C with dry ice. The combination of chitosan coating and dry ice significantly inhibited a Haugh unit decrease during storage at 23°C. No difference in functional properties, such as foaming ability, foam stability, and viscosity, among treatments was observed, but chitosan coating and storage with dry ice decreased the rate of pH increase and moisture loss in albumen at d 7 and 14. The eggs treated with chitosan coating and storage with dry ice had a significantly lower number of Salmonella Typhimurium inoculated on the egg surface than did control eggs during storage at 23°C. Results revealed that the combination of chitosan coating and storage with dry ice limited the moisture loss, CO2 emission, and pH increase, which helped maintaining the freshness of eggs. Microbial growth was also inhibited during storage at 23°C.

Determinants of antagonism of NMDA and calcium permeable AMPA receptor channels by organic cations were studied using several homologous series of mono- and dicationic derivatives of adamantane, phenylcyclohexyl, triphenylmethane, diphenylmethane. Antagonism by these drugs was studied on native receptors of isolated rat brain neurons and on recombinant GluR1 receptors expressed by Xenopus oocytes. The major action of these compounds was on the open channel, although minor competitive or closed channel antagonism cannot be ruled out. Analysis of structure-activity relationships suggests that all organic monocations are selective antagonists of NMDA receptors. Compounds exhibiting trapping block are more potent than those exhibiting weakly-trapping block. AMPA and NMDA receptor channels are blocked by dicationic organic compounds, the former requiring a certain distance between the hydrophobic moiety and the terminal charged group. Variations of their terminal ammonium group demonstrated that trimethylammonium derivatives are the most potent antagonists of AMPA receptors, whereas the terminal amino group is optimal for block of NMDA receptors. Based on the action of 38 compounds, topographical models of the binding sites of these compounds on NMDA and AMPA receptor channels are presented. These models will help to design channel-blocking drugs with defined potency and selectivity of action.

Udgivelsesdato: Aug Dlk1 (Pref-1) is a transmembrane and secreted protein, which is a member of the epidermal growth factor-like family, homologous to Notch/Delta/Serrate. We have found by real-time RT-PCR that Dlk1 mRNA levels were high in CD34(+) cells in 10 of 12 MDS samples compared with CD34(+) cells from 11 normals. Also, Dlk1 mRNA was elevated in mononuclear, low density bone marrow cells from 11/38 MDS patients, 5/11 AML M6 and 2/4 AML M7 samples. Furthermore, 5/6 erythroleukemia and 2/2 megakaryocytic leukemia cell lines highly expressed Dlk1 mRNA. Levels of Dlk1 mRNA markedly increased during megakaryocytic differentiation of both CMK megakaryoblasts as well as normal CD34(+) hematopoietic stem cells. High serum levels of Dlk1 occurred in RA (4/10) and essential thrombocythemia (2/10) patients. Functional studies showed that forced expression of Dlk1 enhanced proliferation of K562 cells growing in 1% fetal bovine serum. Analysis of hematopoiesis of Dlk1 knockout mice suggested that Dlk1 contributed to granulocyte, megakaryocyte and B-cell clonogenic growth and was needed for generation of splenic B-cells. In summary, Dlk1 is overexpressed in selected samples of MDS (especially RA and RAEB) and AML (particularly M6, M7), and it appears to be associated with normal development of megakaryocytes and B cells.

Adipose tissue is the source of a wide array of factors of great biological significance that are involved in many aspects of organism physiology, including appetite control and peripheral metabolism. Here, we describe two secreted factors from adipose tissue that inhibit adipogenesis. Pref-1 is a preadipocyte secreted factor synthesized as a transmembrane protein that undergoes proteolitic cleavage to generate two distinct soluble forms. In vitro assays have demonstrated that only the large soluble form of Pref-1 is biologically active and inhibits adipocyte differentiation. In vivo, mice lacking Pref-1 expression show accelerated fat deposition, perinatal mortality and growth retardation as well as distinct skeletal malformations, highlighting the importance of Pref-1 during mouse development in addition to its role in adipose tissue development. ADSF/resistin is secreted by adipocytes and inhibits adipose cells differentiation in vitro. Its function is still unclear, but its expression and high circulating levels have been associated with an impairment of insulin action. The findings show that Pref-1 and possibly ADSF/resistin secretion control fat cell differentiation and adipose tissue development.

Pseudotyped retroviral vectors combine the advantages of broad host range, high expression, stable chromosomal integration, and ease of preparation. These vectors greatly facilitate delivery into mammalian cells of sequences encoding individual peptide inhibitors—including those with therapeutic utility—and inhibitor libraries. However, retroviral vectors vary in behavior, particularly with respect to expression levels in different cell lines. Expression level is especially important in transdominant experiments because the concentration of an inhibitor (for example, an expressed peptide) is one of the key determinants in the degree of complex formation between the inhibitor and its target. Thus, inhibitor concentration should have an impact on the expressivity and/or penetrance of an induced phenotype. Here, we compare several retroviral vectors and human cell lines for relative expression levels using a green fluorescent protein reporter. We show for a subset of these lines that cellular protein concentrations produced by single-copy vectors range up to about 2 μM. We also examine other variables that contribute to expression level, such as the nature of the expressed protein's carboxy terminus. Finally, we test the effect of increased concentration on phenotype with a nine-amino-acid peptide derived from the human papilloma virus protein E7 which overcomes E7-mediated cell growth.

The response to standard chloroquine treatment was evaluated, by microscopical examination of blood-smears, among 81 soldiers diagnosed with Plasmodium vivax malaria in South Korea in 1996. The smears were prepared pre-treatment and 3, 14 and 28 days after starting chemotherapy. Parasitaemias were determined after staining the smears with Giemsa's stain. Blood samples from the patients who were not smear-negative by day 3 were carefully checked for parasites, by staining smears with Acridine Orange and by a PCR-based assay. Only two of the patients appeared to be parasitaemic on day 14 and were therefore considered treatment failures. Although both were apparently cured after additional therapy with the same regimen, one had a recurrence 8 months later. Most cases of recent, resurgent malaria in South Korea therefore appear to sensitive to chloroquine.

We hypothesized that supplementing finishing diets with palm oil would promote adipocyte differentiation in subcutaneous adipose tissue of feedlot steers, and that soybean oil supplementation would depress adipocyte differentiation. Twenty-eight Angus steers were assigned randomly to 3 groups of 9 or 10 steers and fed a basal diet without additional fat (control), with 3% palm oil (rich in palmitic acid), or with 3% soybean oil (rich in polyunsaturated fatty acids), for 10 wk, top-dressed daily. Palm oil had no effect (P0.05) on ADG, food intake, or G:F, whereas soybean oil depressed ADG (P = 0.02), food intake (P = 0.04), and G:F (P = 0.05). Marbling scores tended (P = 0.09) to be greater in palm oil-fed steers (Modest(09)) than in soybean oil-fed steers (Small(55)). Subcutaneous adipocyte mean volume was greater in palm oil-fed steers (515.9 pL) than in soybean-supplemented cattle (395.6 pL; P = 0.01). Similarly, glucose and acetate incorporation into total lipids in vitro was greater in subcutaneous adipose tissue of palm oil-fed steers (119.9 and 242.8 nmol·3h(-1)·10(5) cells, respectively) than adipose tissue of soybean oil-fed steers in (48.9 and 95.8 nmol·3h(-1)·10(5) cells, respectively). Glucose-6-phosphate dehydrogenase and NADP-malate dehydrogenase activities were greater (P ≤ 0.05) in subcutaneous adipose tissue of palm oil-fed steers than in adipose tissue of control steers. Palm oil did not increase palmitic acid or decrease oleic acid in subcutaneous adipose tissue or LM, but decreased (P ≤ 0.05) myristoleic, palmitoleic, and cis-vaccenic acid in adipose tissue, indicating a depression in stearoyl-coenzyme A desaturase activity. Soybean oil increased the proportion of α-linolenic acid in adipose tissue and muscle and increased linoleic acid and 18:1trans-10 in muscle. We conclude that palm oil supplementation promoted lipid synthesis in adipose tissue without depressing feed efficiency or increasing the palmitic acid content of beef.

Malonyl-CoA, which is the unique product of acetyl-CoA carboxylase (ACC), may serve as a metabolic coupler in glucose-stimulated insulin secretion by pancreatic β-cells. Therefore we examined if and how ACC is affected by glucose in association with insulin secretion. Glucose induces a rapid increase in ACC activity which is closely related to insulin secretion in a dose- and time-dependent manner. The acute effect of glucose in increasing ACC activity is caused by dephosphorylation of existing ACC molecules, without the production of new enzyme. Inhibition of ACC dephosphorylation and activation by the use of okadaic acid led to diminished glucose-mediated insulin secretion. Likewise, when ACC phosphorylation and inactivation were induced by the use of 5-amino 4-imidazole-carboxamide ribotide, an AMP analog and activator of 5′-AMP protein kinase, the glucose-induced insulin secretion was virtually nil. In the long term, glucose induced ACC and increased insulin secretion. In β-cells, ACC gene expression is controlled by promoter II and glucose activated promoter II expression. ACC promoter I is not expressed in β-cells. Maximum activation of ACC and insulin secretion by glucose in the short term occurred at 5 mm glucose. On the other hand, activation of the expression of ACC promoter II occurred when the cells were exposed to high glucose concentrations for a long period of time. Thus, we have shown that ACC, the only enzyme that produces malonyl-CoA, is activated by glucose; activation of ACC is accomplished by dephosphorylation in the short term and by induction of ACC by gene activation in the long term. Journal of Endocrinology (1995) 147, 33–41

1. Two experiments were conducted to evaluate a multi-microbe probiotic formulation processed at low (LT) or high (HT) drying temperature. 2. In both the experiments, 640 d-old Ross male chicks were randomly allotted to 4 treatments on the basis of initial BW for 35 d experiments. 3. In experiment one, dietary treatments were a negative control (NC; basal diet without any antimicrobial); positive control (PC; basal diet +10 mg/kg avilamycin); basal diet with 0·3% probiotic LT; and basal diet with 0·3% probiotic HT. 4. Improved overall weight gain, FCR and retention of CP were observed in birds fed the PC and probiotic diets when compared with birds fed the NC diet. At d 21, birds fed the probiotic and NC diets had more caecal Bifidobacterium and total anaerobes than birds fed the PC diet; while birds fed the PC and probiotic diets had fewer caecal Clostridium than birds fed the NC diet at d 35. 5. In experiment two, a 2 × 2 factorial arrangement of treatments was employed to evaluate the effects of two concentrations of probiotic HT (0·30 or 0·60%) and avilamycin (0 or 10 mg/kg). 6. Birds fed the 0·60% probiotic HT diet showed improved overall weight gain and CP retention, higher Lactobacillus and Bifidobacterium in the caecum, and reduced Clostridium and coliforms in the caecum. Inclusion of avilamycin improved the overall weight gain and feed intake, and reduced the caecal Clostridium and Bifidobacterium population. 7. In conclusion, high drying temperature had no effect on the efficacy of the multi-microbe probiotic formulation; while the probiotic HT formulation was more effective at the 0·60% level. Moreover, inclusion of avilamycin improved performance of birds but did not have any interaction with probiotics.

Two experiments were conducted to determine the effects of dietary supplementation of exogenous enzymes on growth performance, apparent total tract digestibility (ATTD) of energy and nutrients, blood metabolites, fecal VFA, and fecal ammonia-N in growing pigs (Sus scrofa) fed a corn (Zea mays L.)- and soybean [Glycine max (L.) Merr.] meal (SBM)-based diet. In Exp. 1, 240 growing barrows (initial BW: 55.6 ± 0.9 kg) were randomly allotted to 5 treatments on the basis of BW. There were 4 replicates in each treatment with 12 pigs per replicate. The 5 treatments consisted of a corn-SBM-based control diet and 4 additional diets were similar to the control diet, with the exception that 0.05% β-mannanase (M), α-amylase + β-mannanase (AM), β-mannanase + protease (MPr), or α-amylase + β-mannanase + protease (AMP) was added to the diets, which were fed for 28 d. Pigs fed the AM, MPr, or AMP diet had greater (P0.05) ADG than pigs fed the control diet. Pigs fed the AMP diet also had greater (P0.05) ADG than pigs fed the M, AM, or MPr diet. Pigs fed the AMP diet had greater (P0.05) G:F than pigs fed the control diet. The G:F of the pigs fed the M, AM, or MPr diet were not different (P0.05) from the G:F in pigs fed the AMP or control diet. The ADFI, ATTD of nutrients, blood metabolites, and fecal VFA and ammonia-N concentrations were not different among treatments. In Exp. 2, 192 growing barrows (initial BW: 56.9 ± 1.0 kg) were allotted to 4 treatments. There were 4 replicates in each treatment with 12 pigs per replicate. Pigs were fed a corn-SBM-based diet (CSD) or a complex diet (CD) that contained corn, SBM, 3% rapeseed (Brassica napus L.) meal, 3% copra (Cocos nucifera L.) meal, and 3% palm (Elaeis guineensis Jacq.) kernel meal. Each diet was prepared without exogenous enzymes or with 0.05% AMP and all diets were fed for 28 d. The ADG and G:F of pigs fed the CSD were greater (P0.05) than pigs fed the CD. However, the type of diet had no effect on the ATTD of nutrients, blood metabolites, or fecal VFA and ammonia-N, and there was no diet × enzyme interaction for any of the measured variables. Supplementation of diets with exogenous enzymes resulted in greater (P0.05) ADG, G:F, ATTD of DM, GE, and CP, and blood urea nitrogen (BUN) concentration. These results indicate that supplementation of 0.05% of AMP enzymes to a corn-SBM diet or a complex diet may improve the performance of growing pigs.

The aim of this study was to generate temperature-sensitive Edwardsiella tarda mutant and to evaluate potential of the mutant as a vaccine in olive flounder (Paralichthys olivaceus). A temperature-sensitive E. tarda mutant was generated by replacement of alr gene promoter with cI857-λP(R) promoter system plus another CI857 expression cassette that was driven by a constitutive promoter of E. tarda (EtPR C28-1). Growth of the mutant strain was not different to that of wild-type E. tarda under conditions of culture at 39°C. However, growth of the mutant strain was retarded by culturing at 25 or 20°C without d-alanine. To further inhibit leakage of λP(R) promoter, the mutant strain was transformed with a vector harbouring an EtPR C28-1-driven cI857 cassette (pEtPR-cI857), which resulted in more limited growth compared to the mutant without the plasmids. The level of alr gene transcription in the mutant E. tarda and the mutant harbouring pEtPR-cI857 was well coincided with the result of bacterial growth. Olive flounder fingerlings immunized with the E. tarda mutant showed decreased mortality, and a boost immunization induced 100% protection against E. tarda infection. The protection rate of fish was proportional to the serum agglutination titre against E. tarda. An attenuated E. tarda mutant induced by shifting down temperature below 30°C was firstly generated. Immunization of fish with the mutant led to protection against virulent E. tarda challenge. The results suggest that the present temperature-sensitive E. tarda mutant can be a good candidate for effective vaccines for prophylaxis of edwardsiellosis. Moreover, as the present E. tarda mutant can be cultured without supplementation of the specific nutrient and can be attenuated just by decreasing temperatures below 30°C, vaccines based on the present E. tarda mutant would be advantageous in an economical aspect.

We have demonstrated that among carcass adipose tissue depots, brisket subcutaneous adipose tissue contains the greatest concentration of MUFA and lowest concentration of SFA. Therefore, we hypothesized that brisket subcutaneous adipose tissue depots would exhibit greater adipogenic gene expression over time than other major subcutaneous adipose tissue depots. Four Angus steers, each at 9, 12, 14, and 16 mo of age, were harvested and fresh subcutaneous adipose tissue samples were collected from over the brisket, chuck, rib, loin, sirloin, round, flank, and plate. Relative gene expression for C/EBPβ, PPARγ, carnitine palmitoyltransferase-1 beta (CPT-1β), stearoyl-coenzyme A desaturase (SCD), AMP-activated protein kinase alpha (AMPKα), and G-coupled protein receptor 43 (GPR43) was analyzed by quantitative real-time PCR. Expression of C/EBPβ, PPARγ, and CPT-1β was greatest at 12 to 14 mo of age (all P0.0001) and declined to very low abundance by 16 mo of age in all depots. Expression of PPARγ and CPT-1β was greater (P0.03) in flank, rib, and sirloin subcutaneous adipose tissues than in brisket and round adipose tissues. The expression of the SCD gene did not differ among the 4 age groups (P = 0.95). The palmitoleic:stearic acid ratio (an estimate of SCD activity) was greater (P0.001) in the subcutaneous adipose tissues from brisket, plate, and round than in the loin, rib, and sirloin. Conversely, subcutaneous adipose tissue from the loin, rib, and sirloin had greater (P0.001) SCD gene expression than the brisket, plate, and round. In general, subcutaneous adipose tissues with the highest concentration of MUFA and least SFA consistently exhibited the least SCD gene expression and adipogenic gene expression. We conclude that MUFA in the brisket and other depots with large SCD indices were deposited before 9 mo of age, during a time when the subcutaneous adipocytes were highly differentiated.

Nonhuman primates are often used as a model for studying vaccines for humans. However, it is not always clear how closely the antibody responses in these species mimic human responses. Recent studies have characterized the human antibody response to Haemophilus influenzae type b (Hib) in great detail. In this study, we have compared the antibody response to Hib of humans with those of other primates. Studies of isoelectric points and V kappa subgroup usage show that, like humans, nonhuman primates produce oligoclonal antibodies. Also, monkey antibodies to the Hib polysaccharide are as protective as human antibodies in an in vivo model of Hib infection. Thus, we conclude that nonhuman primates produce antibodies to Hib polysaccharide that are structurally and functionally similar to human antibodies and are a good model for testing human vaccines.

Crossbred barrows were treated daily with porcine somatotropin (pST; 4 mg/d) from 79 to 127 kg BW to determine whether pST regulates the activity and gene expression of adipose tissue acetyl-CoA carboxylase (ACC), the rate limiting enzyme in de novo fatty acid synthesis. Administration of pST reduced ACC enzyme activity, protein content, and mRNA abundance in adipose tissue by 40 to 50%. When comparisons were made among all pigs, ACC enzyme activity and mRNA abundance were closely associated (r2 = .94). In summary, our results indicate that pST decreases ACC enzyme activity and that this is associated with a significant reduction in ACC mRNA abundance. We speculate that decreased ACC enzyme activity results from a reduction in ACC protein and that this occurs because pST reduces the abundance of mRNA available for translation.

To determine the temporal effect of ractopamine (Rac), a phenethanolamine, on adipose lipogenic enzyme activity and gene expression, 20 crossbred barrows were fed Rac (20 mg/kg of diet) for 0, 1, 8, or 24 d before slaughter (105 +/- 1 kg). Ractopamine had no effect (P > .05) on the activity of acetyl-coenzyme A carboxylase or malic enzyme in either the middle or outer layers of subcutaneous adipose tissue. Similarly, mRNA abundance for acetyl-coenzyme A carboxylase and the glucose transport proteins Glut 1 and Glut 4 were not affected by Rac in either adipose depot. Despite the inability of Rac to affect adipose tissue metabolism, Rac increased nitrogen retention, longissimus muscle area, and alpha-actin gene expression in skeletal muscle. Results indicate that Rac was not a functional beta-adrenergic agonist toward adipose tissue in this study. We suggest that the response to Rac in adipose tissue is masked by a combination of factors including tissue insensitivity, Rac-dose limitation, inherent partial agonism of Rac, and beta-adrenoceptor down-regulation.

Vardenafil hydrochloride (HCl) is a potent and selective phosphodiesterase type-5 (PDE-5) inhibitor that enhances nitric oxide (NO)-mediated relaxation of human corpus cavernosum and NO-induced rabbit penile erection, and enhances erectile function in patients. In the present study, the effect of vardenafil on nitric oxide synthase (NOS) and neuronal NOS expressions in the paraventricular nucleus (PVN) of rats without sexual stimulation was investigated using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and neuronal NOS (nNOS) immunohistochemistry and western blot analysis. The present results showed that NOS and nNOS expression in the PVN was increased by vardenafil treatment as the dose- and duration-dependently without sexual stimulation. The phosphodiesterase type-5 inhibitor, vardenafil, augmented NOS expression in the brain without sexual stimulation. The present study suggests that sexual behaviour can be directly modulated by neurotransmitters such as nitric oxide.

The present study investigated the effect of inclusion of multi-microbe probiotic product on growth performance, apparent digestibility of nutrients, cecal microbiota and small intestinal morphology in broilers. Four hundred days-old Ross chicks were randomly allotted to five treatments on the basis of body weight (BW). Each treatment had four replicates of 20 chicks in each. Experimental diets were fed in two phases, starter (day 0-21) and finisher (day 22-35). Dietary treatments were; basal diet without any antimicrobial (NC), basal diet added with 20 mg Avilamycin/kg of diet (PC), 10(7) cfu multi-microbe probiotic/kg of diet (P1), 10(8) cfu multi-microbe probiotic/kg of diet (P2), and 10(9) cfu multi-microbe probiotic/kg of diet (P3). Overall BW gain and feed conversion ratio were better (p0.05) for treatments PC, P2 and P3 compared with NC and P1, with P1 being better (p0.05) than NC. Overall feed intake in treatments PC, P1, P2 and P3 were greater (p0.05) than NC. Apparent digestibility of dry matter and crude protein were greater (p0.05) in treatments PC, P2 and P3 compared with NC, with P1 being intermediate and not different form NC, PC, P2 and P3. At d 21 and 35, treatments PC, P1, P2 and P3 showed lower (p0.05) cecal Clostridium and Coliforms count in relation to NC. Moreover, cecal Clostridium (d 21) and Coliforms (d 21 and 35) count were lower (p0.05) in treatment PC in relation to P1; with P2 and P3 being intermediate and not different from PC. However, there was no effect of dietary treatments on cecal total anaerobic bacteria and Bifidobacterium spp. count. The villus height of duodenum in treatment PC was greater (p0.05) than NC, with P1, P2 and P3 being intermediate. Villus height of ileum in treatment PC was greater (p0.05) than in treatments P1 and NC, whereas it remained comparable among treatments PC, P2 and P3. Villus height to crypt depth ratio of ileum was greater (p0.05) for treatment PC, P2 and P3 compared with that in P1 and NC. It is concluded that multi-microbe probiotic inclusion at 10(8) and 10(9) cfu/kg diet had beneficial effects on broilers growth performance, apparent digestibility of nutrients and intestinal morphology and can be used as replacement to antibiotics growth promoter in broiler nutrition.

In this study, the effect of a potential multimicrobe probiotic subjected to high-temperature drying was investigated. Potential multimicrobe probiotics produced by solid substrate fermentation were dried at low (LT, 40°C for 72 h) or high (HT, 70°C for 36 h) temperature. In Exp. 1, 288 weaned pigs (BW, 6.43 ± 0.68 kg) were allotted to 4 treatments on the basis of BW (4 pens per treatment with 18 pigs in each pen). Dietary treatments were negative control (NC; basal diet without any antimicrobial), positive control (PC; basal diet + 0.1% chlortetracycline), basal diet with 0.3% probiotic LT, and basal diet with 0.3% probiotic HT. Diets were fed in 2 phases, phase I (d 0 to 14) and phase II (d 15 to 28); and growth performance, apparent total tract digestibility (ATTD, d 28), and fecal microflora (d 14 and 28) were evaluated. Over the 28-d trial, pigs fed PC and probiotic diets had greater ADG (P0.001), ADFI (P0.05), and G:F (P0.01) than pigs fed NC diet. The ATTD of DM and GE was greater (P0.05) in pigs fed probiotic diets when compared with pigs fed the NC diet. At d 28, fewer Clostridia (P0.01) were identified in the feces of pigs fed PC and probiotic diets than pigs fed the NC diet. However, the performance, ATTD of DM and GE, and fecal Clostridia population were similar among pigs fed probiotic LT and HT diets. In Exp. 2, 288 weaned pigs (initial BW, 5.84 ± 0.18 kg) were allotted to 4 treatments in a 2 × 2 factorial arrangement on the basis of BW. The effects of 2 levels of probiotic HT (0.30 or 0.60%), each with or without antibiotic (chlortetracycline, 0 or 0.1%), on performance, ATTD, intestinal morphology, and fecal and intestinal microflora were investigated. Feeding of 0.60% probiotic HT diet improved (P0.05) overall ADG, ATTD of DM and GE, and Lactobacillus population in the feces and intestine, and reduced the population of Clostridium and coliforms in feces (d 14) and ileum. Inclusion of antibiotic improved (P0.05) the overall ADG, ADFI, and ATTD of DM at d 14 and reduced fecal Clostridium population at d 28. Increased (P0.05) villus height at jejunum and ileum, and villus height:crypt depth at the ileum was noticed in pigs fed 0.60% probiotic HT and antibiotic diets. In conclusion, high drying temperature had no effect on the efficacy of potential multimicrobe probiotic product. However, the probiotic product dried at high temperature was more effective at 0.60% inclusion, whereas inclusion of an antibiotic improved pig performance but did not show any interaction with probiotics.

Quantitative structure-activity relationships of 57 substituted aminotetralin analogues, an overview of their syntheses, and their pharmacological activity are described in this study. Lipophilic substituents at R3 as well as the overall lipophilicity of the molecule contribute toward increasing the inhibitory potency. An ethyl group is preferred, and a group larger than a propyl is not desirable as a nitrogen substituent. Among the ring substituents examined, an hydroxy group at the R6 position and either an unsubstituted R9 position or a methoxy substituent at the R9 position increase the inhibitory potency, whereas a methoxy group at the R7 position decreases inhibitory potency.

In addition to nuclear-initiated (genomic) responses, estrogen receptors (ERs) have the ability to facilitate rapid, membrane-initiated, estrogen-triggered signaling cascades via a plasma membrane-associated form of the receptor. These rapid responses are dependent on assembly of membrane ER-centered multimolecular complexes, which can transduce ligand-activated signals to affect a variety of enzymatic pathways, often occurring in a cell-type-specific fashion with tissue-specific physiological outcomes. In some instances, cross-talk occurs between these membrane-initiated and nuclear responses, ultimately regulating transcriptional activation. The role of splice variants in membrane-initiated estrogen responses has been described, notably those within the vascular endothelium. In this review, we describe the evidence for membrane ERs, the molecular components of the aforementioned signaling complexes and pathways, the relevance of ER splice variants, and ER-mediated responses in specific tissues. Our growing understanding of ER-mediated actions at a molecular level will provide insight into the controversies surrounding hormone replacement therapy in postmenopausal women.

Anisakis simplex third stage larvae (L3) are parasites that frequently give rise to allergic responses. The larvae molt into fourth stage larvae (L4), and at each stage they produce L3-excretory-secretory products (L3-ESP) and L4-ESP, respectively, which are different in their main protein constituents. Although the allergenicity of L4-ESP has been investigated by several research groups, research on the allergenicity of L3-ESP has not been carried out by any researcher. In this investigation, the allergenicity and antigenicity of L3-ESP were investigated in comparison with L4-ESP, using rat sera.Rat sera were produced by L3 oral infection two times with a 9-week interval. Larvae ESP prepared by culture were concentrated and fractioned using lyophilizer and a centrifugal filter device, respectively. Immunochemical analysis was performed using both indirect ELISA and immunoblot. Biological allergenicity was analyzed by RBL-2H3 exocytosis.With the indirect ELISA, the optical density (OD) value of the nonfractioned (NF)-L3ESP was only one third of that of the NF-L4ESP in both specific IgM and IgG. On measuring specific IgE, the OD of NF-L3ESP was less than one tenth of that of NF-L4ESP. In addition, neither antigen nor allergen was shown in NF-L3ESP, but it was shown in NF-L4ESP with immunoblot. However, the biological allergenicity of NF-L3ESP was comparable to that of NF-L4ESP. To demonstrate the presence of any allergen, L3-ESP was fractioned and found to carry twelve visualized allergen bands from 10 to 186 kDa by immunoblot.These results indicate that L3-ESP may include the important allergens necessary to induce the allergy by L3 oral infection, as compared to L4-ESP.

Increasing evidence suggests that protein-protein interaction is essential in many biological processes including epithelial transport. In this report, we discuss the significance of protein interactions to HCO(3)(-) secretion in pancreatic duct cells. In pancreatic ducts HCO(3)(-) secretion is mediated by cystic fibrosis transmembrane conductance regulator (CFTR) activated luminal Cl(-)/HCO(3)(-) exchange activity and HCO(3)(-) absorption is achieved by Na(+)-dependent mechanisms including Na(+)/H(+) exchanger 3 (NHE3). We found biochemical and functional association between CFTR and NHE3. In addition, protein binding through PDZ modules is needed for this regulatory interaction. CFTR affected NHE3 activities in two ways. Acutely, CFTR augmented the cAMP-dependent inhibition of NHE3. In a chronic mechanism, CFTR increases the luminal expression of Na(+)/H(+) exchange in pancreatic duct cells. These findings reveal that protein complexes in the plasma membrane of pancreatic duct cells are highly organized for efficient HCO(3)(-) secretion.

The spermatozoa of the slender catfish Silurus microdorsalis examined using transmission and scanning electron microscopy are characterized by a tubular structure and the absence of axonemal fins. Their characteristics can be considered to represent a valid symplesiomorphy uniting the Siluridae and taxon-specific for this family. The tubules of Silurus spermatozoon are not observed in the other siluroids. Unusual tubules occur in Citharinus spermatozoa of Characiformes. In addition, there is a considerable similarity in the absence of axonemal fins between Siluridae and Characiformes. Thus the two shared characteristics provide strong evidence that there exists a phylogenetic link between Characiformes and Siluriformes. Silurus appears to be plesiomorphic in having lost the axonemal fins while retaining the tubules in midpiece. The spermatozoa of S. microdorsalis are similar to those of other siluroids having a spherical head with a deep nuclear fossa, a short midpiece and an elongated tail. However, there are some differences between Silurus and other siluroids in the orientation of the centrioles, the number of the mitochondria and the axonemal fins, and minor differences between S. microdorsalis and S. asotus, in particular the orientation of the centriolar complex, and the arrangement of the tubules and the mitochondria in the midpiece. The deep nuclear fossa represents a synapomorphy uniting the Siluriformes, while the shallow nuclear fossa and the absence of axonemal fins are shared by two orders, Characiformes and Cypriniformes.

Many recombinant proteins are produced as a fusion protein tagging hexahistidine, which has been used for studying their biological function both in vitro and in vivo. Unexpectedly, we observed activation of BALB/c mouse splenocytes when treated with hexahistidine-tagged recombinant proteins. This activation was hexahistidine-specific since the anti-pentahistidine antibody completely neutralized the effect, and the bovine serum albumin conjugated with the hexahistidine peptides also showed a similar activation effect. The B cells seemed to be the activated splenocytes, since the cell population stained with the anti-immunoglobulin antibody and anti-CD80 antibody was increased after the treatment. However, the activation signal by hexahistidine was insufficient to fully differentiate the B lymphocytes. This result suggests that caution must be taken in the use of hexahistidine-tagged recombinant proteins, due to their nonspecific activation of B lymphocyte.

Ceramide is a bioactive sphingolipid that mediates a variety of cell functions. However, the effects of ceramide on cell growth and the melanogenesis of melanocytes are not known. In the present study, we investigated the actions of cell-permeable ceramide and its possible role in the signaling pathway of a spontaneously immortalized mouse melanocyte cell line, Mel-Ab. Our results show that C2-ceramide inhibits DNA synthesis in Mel-Ab cells and G361 human melanoma cells in a dose-dependent manner. Cell cycle analysis confirmed the inhibition of DNA synthesis by a reduction in the S phase. To investigate the ceramide signaling pathway, we studied whether C2-ceramide is able to influence extracellular signal-regulated kinase (ERK) and/or Akt/protein kinase B (PKB) activation. We demonstrated that phosphorylated Akt/PKB is decreased by C2-ceramide, whereas phosphorylated ERK was only slightly affected. Therefore, the C2-ceramide-induced inactivation of Akt/PKB may be closely related to the reduced cell proliferation of Mel-Ab cells. Furthermore, we assessed the effects of C2-ceramide on the pigmentation of Mel-Ab cells. The results obtained showed that the melanin content of cells was significantly reduced by C2-ceramide at concentrations in the range of 1-10 microM, and that the pigmentation-inhibiting effect of C2-ceramide is much greater than that of kojic acid at 1-100 microM. In addition, we found that the activity of tyrosinase is reduced by C2-ceramide treatment. Our results demonstrate that C2-ceramide reduces the pigmentation of Mel-Ab cells by inhibiting tyrosinase activity.

The pancreatic duct expresses cystic fibrosis transmembrane conductance regulator (CFTR) and HCO3- secretory and salvage mechanisms in the luminal membrane. Although CFTR plays a prominent role in HCO3- secretion, the role of CFTR in HCO3- salvage is not known. In the present work, we used molecular, biochemical, and functional approaches to study the regulatory interaction between CFTR and the HCO3- salvage mechanism Na+/H+ exchanger isoform 3 (NHE3) in heterologous expression systems and in the native pancreatic duct. We found that CFTR regulates NHE3 activity by both acute and chronic mechanisms. In the pancreatic duct, CFTR increases expression of NHE3 in the luminal membrane. Thus, luminal expression of NHE3 was reduced by 53% in ducts of homozygote DeltaF508 mice. Accordingly, luminal Na+-dependent and HOE694- sensitive recovery from an acid load was reduced by 60% in ducts of DeltaF508 mice. CFTR and NHE3 were co-immunoprecipitated from PS120 cells expressing both proteins and the pancreatic duct of wild type mice but not from PS120 cells lacking CFTR or the pancreas of DeltaF508 mice. The interaction between CFTR and NHE3 required the COOH-terminal PDZ binding motif of CFTR, and mutant CFTR proteins lacking the C terminus were not co-immunoprecipitated with NHE3. Furthermore, when expressed in PS120 cells, wild type CFTR, but not CFTR mutants lacking the C-terminal PDZ binding motif, augmented cAMP-dependent inhibition of NHE3 activity by 31%. These findings reveal that CFTR controls overall HCO3- homeostasis by regulating both pancreatic ductal HCO3- secretory and salvage mechanisms.

To investigate the effects of basement membrane proteins on the reconstruction of mucosa equivalent, oral mucosa substitute were cultured on (1) type I collagen gels, (2) type IV collagen-coated type I collagen gels, (3) laminin-coated type I collagen gels, and (4) type I collagen gels containing both type IV collagen and laminin. H/E and PAS staining showed that the characteristics of the oral mucosa were preserved under all the experimental conditions. However, the basal keratinocytes appeared cuboidal when the type I collagen gels were coated with type IV collagen plus laminin. The expression of the differentiation markers was similar, but weak staining of filaggrin, K13, and involucrin was observed with the type IV collagen plus laminin coating. Furthermore, electron microscopy revealed that the size of the basal keratinocytes was relatively small and uniform when both type IV collagen and laminin were used. These findings suggested that these two major basement membrane proteins are important in the process of differentiation in mucosal keratinocytes.

We have developed a transgenic female goat harboring goat beta-casein promoter/human granulocyte colony stimulating factor (G-CSF) fusion gene by microinjection into fertilized one-cell goat zygotes. Human G-CSF was produced at levels of up to 50 microg/ml in transgenic goat milk. Its biological activity was equivalent to recombinant human G-CSF expressed from Chinese hamster ovary (CHO) cell when assayed using in vitro HL-60 cell proliferation. Human G-CSF from transgenic goat milk increased the total number of white blood cells in C57BL/6N mice with leucopenia induced by cyclophosphamide (CPA). The secreted human G-CSF was glycosylated although the degree of O-glycosylation was lower compared to CHO cell-derived human G-CSF.

The effect of combining adenoviral vector and cationic liposomes on the efficiency of gene transfer to head and neck tumor cells was investigated. Two human and two murine cell lines were used for the screening of gene transfer efficiency using an adenoviral vector. Cationic liposome-enhanced gene transfer was checked using a murine squamous carcinoma cell line, SCCVII/SF. A considerable difference in the efficiency of gene transduction was observed among the cell lines. The combination of DOSPER and adenoviral vector containing human alkaline phosphatase showed a remarkable enhancing effect in gene transfer in vitro and in vivo, compared to the adenovirus alone or control groups. With an improvement in the efficiency of gene transfer, it may be possible not only to enhance the expression of transduced genes, but also to deliver a smaller amount of virus, as a result, reducing toxicity and the immune response against adenovirus.

We previously reported that the glycoprotein extracted from aloe strongly inhibited the mediator releases caused by the activation of guinea pig lung mast cells. Therefore, this study aimed to purify a single component that has an antiallergic effect from crude aloe extract and then to assess the effects of aloe single component (alprogen) on the mechanism of mediator releases caused by the mast cell activation. We purified aloe extracts by using various columns. We also purified mast cells from guinea pig lung tissues by using enzyme digestion, rough and discontinuous density Percoll gradient. Mast cells were sensitized with IgG(1) (anti-ovalbumin) and challenged with ovalbumin. Histamine was assayed by using a fluorometric analyzer and leukotrienes by radioimmunoassay. [Ca(2+)](i) level was analyzed by using a confocal laser scanning microscope. Protein kinase activity was determined by the protein phosphorylated with [gamma-(32)P]ATP. The phospholipase D activity was assessed by the labeled phosphatidylalcohol. The amount of mass 1,2-diacylglycerol (DAG) was measured by the [(3)H]DAG produced when prelabeled with [(3)H]myristic acid. Phospholipase A(2) activity was determined by measuring the lyso-phosphatidylcholine released from the labeled phospholipids. Alprogen significantly decreased histamine and leukotriene releases and blocked completely Ca(2+) influx during mast cell activation. The protein kinase C and phospholipase D activities were decreased by alprogen in dose-dependent manner. Alprogen inhibited mass DAG formation and the phospholipase A(2) activity during mast cell activation. The data suggest that alprogen purified from aloe inhibits multiple signals as well as blocking Ca(2+) influx caused by mast cells activated with specific antigen-antibody reactions and that then the inhibition of histamine and leukotriene release follows.

Vitamin A is required in the testis for germ cell development. It acts through two families of retinoid receptors, retinoic acid receptors (RAR) and retinoid X receptors (RXR), each with three subtypes alpha, beta, and gamma. These receptors are postulated to dimerize and regulate the transcription of retinoid-responsive genes that are crucial for germ cell development. In this study, we determined the cellular and subcellular localization of six retinoid receptors in the developing rat testis to identify the specific cellular sites and times of receptor expression. Immunohistochemical results revealed the expression of RARalpha, RARbeta, RXRalpha, and RXRgamma proteins in somatic and germ cells throughout postnatal development. In contrast, the expression of RARgamma and RXRbeta did not increase until 30-35 days of age in somatic cells from the testis. Interestingly, RARalpha and RXRalpha had a similar subcellular localization pattern in Sertoli cells throughout postnatal testis development, while RARalpha and RXRgamma were both present in the nucleus of spermatocytes and elongating spermatids. These results suggest that RARalpha may potentially dimerize with RXRalpha in Sertoli cells and with RXRgamma in germ cells. In addition, we demonstrate that the only RAR in the nucleus of early meiotic germ cells is RARalpha.

To investigate the potential application of allergen gene immunization in the modulation of food allergy, C3H/HeSn (C3H) mice received i.m. injections of pAra h2 plasmid DNA encoding one of the major peanut allergens, Ara h2. Three weeks following pDNA immunization, serum Ara h2-specific IgG2a, IgG1, but not IgE, were increased significantly in a dose-dependent manner. IgG1 was 30-fold higher in multiply compared with singly immunized mice. Ara h2 or peanut protein injection of immunized mice induced anaphylactic reactions, which were more severe in multiply immunized mice. Heat-inactivated immune serum induced passive cutaneous anaphylaxis, suggesting that anaphylaxis in C3H mice was mediated by IgG1. IgG1 responses were also induced by intradermal injection of pAra h2, and by i.m. injection of pOMC, the plasmid DNA encoding the major egg allergen protein, ovomucoid. To elucidate whether the pDNA immunization-induced anaphylaxis was a strain-dependent phenomenon, AKR/J and BALB/c mice also received multiple i.m. pAra h2 immunizations. Injection of peanut protein into these strains at weeks 3 or 5 following immunization did not induce reactions. Although IgG2a was increased significantly from week 2 in AKR/J mice and from week 4 in BALB/c mice and remained elevated for at least 6 wk, no IgG1 or IgE was detected. These results indicate that the type of immune responses to pDNA immunization in mice is strain dependent. Consequently, models for studying human allergen gene immunization require careful selection of suitable strains. In addition, this suggests that similar interindividual variation is likely in humans.

A series of 2-amino-9-(1,3-dihydroxy-2-propoxymethyl)-6-fluoropurine mono- and diesters, 6a-h, were synthesized as potential prodrugs of ganciclovir and evaluated for their oral ganciclovir bioavailability in rats. Treatment of 2-amino-6-chloro-9-(1, 3-dihydroxy-2-propoxymethyl)purine (4) with Me3N in DMF/THF (1/4) followed by the reaction of the resulting trimethylammonium chloride salt 5 with KF in DMF gave 2-amino-9-(1, 3-dihydroxy-2-propoxymethyl)-6-fluoropurine (2) in 83% yield. Esterification of 2 with an appropriate acid anhydride (Ac2O, (EtCO)2O, (n-PrCO)2O, or (i-PrCO)2O) (6 equiv for 6a-d or 1 equiv for 6e-h) in DMF in the presence of a catalytic amount of DMAP produced the diesters 6a-d in 92-98% yields and the monoesters 6e-h in 37-44% yields. Of the prodrugs tested in rats, the monoisobutyrate 6h achieved the highest ganciclovir bioavailability (45%) that is 15-fold higher than that from ganciclovir (3%), followed in order by the diisobutyrate 6d (42%), the diacetate 6a (41%), the monobutyrate 6g (41%), the monopropionate 6f (39%), the dipropionate 6b (35%), the dibutyrate 6c (35%), and the monoacetate 6e (29%). The prodrugs 6e-h were found to be quite stable at pH 6.0 (t1/2 =29 days), 7.4 (t1/2 =7 days), and 8.0 (t1/2 =2 days) but had relatively short half-lives at pH 1.2 (t1/2 = 60-83 min).

cDNAs for the Xenopus laevis homologue of the endo/exonuclease FEN-1 (DNase IV) have been cloned using a polymerase chain reaction strategy. Products were obtained from two nonallelic Xenopus genes (xFEN-1a and xFEN-1b) that differ from each other by 4.5% in amino acid sequence. Both are 80% identical to mammalian FEN-1 proteins and 55% identical to the yeast homologues. When expressed in Escherichia coli, the Xenopus enzymes showed flap endonuclease activity, a unique feature of this class of nucleases. In addition, expression from the Xenopus cDNAs complemented the temperature and methyl methanesulfonate sensitivity of a yeast rad27 deletion, which eliminates the endogenous FEN-1 gene product. Antiserum raised against xFEN-1 was used to show that the protein accumulates during the middle and late stages of oogenesis, in parallel with other DNA metabolic activities, and that it is localized to the oocyte nucleus. Flap endonuclease activity was demonstrated in oocyte nuclear extracts, and this was inhibited by the anti-xFEN-1 antiserum. The antiserum did not inhibit the major oocyte 5' --3' exonuclease activity. DNA synthesis in oocyte extracts was blocked by the antiserum, and the nature of this inhibition suggests that xFEN-1 may be part of a large complex of replication factors. Chromatographic evidence was obtained for the existence of a complex that forms during DNA synthesis and includes proliferating cell nuclear antigen in addition to xFEN-1. These observations support a critical role for xFEN-1 in DNA replication, but indicate that another enzyme must be responsible for the exonuclease function required for homologous recombination in Xenopus oocytes.

Studies of defective interfering (DI) RNAs of mouse hepatitis virus (MHV), suggest that a 69 nt-long packaging signal, which is located about 20 kb from the 5'-end of the 31 kb-long MHV genomic RNA, is necessary and sufficient for MHV genomic RNA packaging into MHV particles. We demonstrated that use of a low pH culture medium combined with subsequent ultrafiltration increased MHV infectivity about 60 times over MHV preparations grown in neutral medium. Using this virus concentration procedure, we successfully prepared DI particle-rich MHV preparations. Characterization of virus samples released from the cells infected with DI particle-rich MHV revealed that infectious MHV genomic RNA was not required for packaging of DI RNAs. These data suggested that interaction of the DI packaging signal with an unidentified region(s) of helper virus genomic RNA is unlikely, and therefore unlikely to facilitate the packaging of MHV DI RNA into the MHV virion. Rather, both DI RNA and MHV genomic RNA probably use the packaging signal for RNA packaging.

Four Lepidoptera-specific reference strains of Bacillus thuringiensis, belonging to serovars sumiyoshiensis (H3a:3d), fukuokaensis (H3a:3d:3e), darmstadiensis (H10a:10b) and japonensis (H23), which produce spherical parasporal inclusions, were examined for comparative characterization of delta-endotoxins. SDS-PAGE profiles of the alkali-solubilized parasporal inclusions revealed the presence of single major protein bands of 130 kDa in the four strains. Chymotrypsin and trypsin treatment of the proteins gave profiles different from those of the strains HD-1 (serovar kurstaki, H3a:3b:3c) and T84 A1 (serovar sotto, H4a:4b). Also, minor variations were observed in proteolysis profiles among the four strains. The LC50 values of purified parasporal inclusions for the silkworm (Bombyx mori) larvae were 7.35, 6.45, 3.08 and 2.63 micrograms g-1 diet, respectively, showing that their toxicity levels were 5-15 times lower than that of the strain HD-1 (0.49 microgram g-1 diet). Analysis by immunodiffusion and immunoblotting with polyclonal antisera revealed that parasporal inclusion proteins of the four strains are highly related, whereas they shared few or no common antigens with those of the strains HD-1, T84 A1 and Buibui (serovar japonensis).

The present study aimed to investigate the role of nitric oxide on the oxidative damage in isolated rabbit gastric cells exposed to hypoxia-reoxygenation. Nitric oxide synthesis modulators such as L-arginine and NG-nitro-L-arginine methyl ester, a nitric oxide donor, sodium nitroprusside, and superoxide dismutase were used to treat the cells, and the synthesis and secretion of mucus, lipid peroxide production, and glutathione contents of the cells were determined. As a result, hypoxia-reoxygenation decreased nitric oxide production and the synthesis and secretion of mucus, as well as glutathione contents of gastric cells, but hypoxia-reoxygenation increased lipid peroxide production. Pretreatment with L-arginine, a substrate for nitric oxide synthase, sodium nitroprusside, and superoxide dismutase prevented the increase in lipid peroxide production and the decreases in glutathione contents, as well as the synthesis and secretion of mucus induced by hypoxia-reoxygenation. However, NG-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, had no effect on these alterations. In conclusion, nitric oxide has an antioxidant defensive role on gastric cells by maintaining mucus and glutathione.

The development of pulmonary allergic responses was examined in mice following pulmonary transfer of Ag (conalbumin)-specific Th2 cells. The levels of serum-specific IgE, cellular infiltrates, airway mucus goblet cells, and airway responsiveness were analyzed and compared with those in Ag-sensitized and -challenged mice. Pulmonary transfer of the conalbumin-specific Th2 clone (D10) induced, in an Ag-specific manner, high levels of the Th2 cytokines IL-4 and IL-5 in the bronchoalveolar lavage fluids and mucosal eosinophils, concomitant with an increase in airway responsiveness. The D10 cell-induced responses were seen in the absence of serum specific IgE. In the presence of Ag, the transferred D10 cells not only remained in the lungs, but also increased in number 72 h post-cell transfer. Although significantly higher levels of IL-4 and IL-5 in the bronchoalveolar lavage fluids were found in D10-transferred mice, the levels of pulmonary eosinophilia, mucus goblet cells, and airway responsiveness were significantly lower than those in Ag-sensitized and -challenged mice. These results demonstrate that although Ag-specific activation of Th2 cells at mucosal sites is able to mediate the recruitment of eosinophils and the subsequent induction of airway hyper-responsiveness, the more severe pulmonary allergic responses were observed only in mice sensitized and challenged with Ag.

Germ cell nuclear factor (GCNF/RTR), a novel orphan receptor in the nuclear receptor superfamily of ligand-activated transcription factors, is expressed predominantly in developing germ cells. In several mammalian species two GCNF/RTR mRNAs are present in the testis, with the smaller 2.3-kb transcript generally expressed at higher levels than the larger 7.4- or 8.0-kb transcript. In both the mouse and rat, the 2.3- and 7.4-kb GCNF/RTR transcripts were detected in isolated spermatogenic cells, but not in Sertoli cells. Expression of these transcripts is differentially regulated, with the larger 7.4-kb mRNA appearing earlier during testicular development. The major 2.3-kb transcript is expressed predominantly in round spermatids in the mouse and rat. In situ hybridization studies in the rat demonstrated that GCNF/RTR transcripts reach maximal steady-state levels in round spermatids at stages VII and VIII of the spermatogenic cycle, and then decline abruptly as spermatids begin to elongate. RNase protection assays were used to predict the 3' termination site of the 2.3-kb transcript. An alternative polyadenylation signal (AGUAAA) was identified just upstream of this termination site. These studies suggest that GCNF/RTR may regulate transcription during spermatogenesis, particularly in round spermatids just prior to the initiation of nuclear elongation and condensation.

We compared the dose-response relationship and the neuromuscular blocking effects of mivacurium during infusions of esmolol in 40 anaesthetised rabbits. Train-of-four stimuli were applied every 10 s to the common peroneal nerve and the force of contraction of the tibialis anterior muscle was measured. Plasma cholinesterase activity decreased by 13% after esmolol infusion. The ED95 of mivacurium increased significantly from 29 (4.8) micrograms.kg-1 with placebo to 61 (9.8) micrograms.kg-1 during esmolol 100 micrograms.kg-1.min-1, 49 (8.2) micrograms.kg-1 during esmolol 300 micrograms.kg-1.min-1 and 54 (7.3) micrograms.kg-1 during esmolol 500 micrograms.kg-1.min-1, respectively (p < 0.001). The duration of neuromuscular block with mivacurium 0.16 mg.kg-1 was prolonged by 30% with esmolol due to diminished plasma cholinesterase activity (p < 0.05). Heart rate and mean arterial blood pressure decreased by 15% with esmolol (p < 0.05). The results of this study show that, in rabbits, esmolol decreased plasma cholinesterase activity, antagonised the neuromuscular blocking potency of mivacurium and prolonged its neuromuscular blocking effect.

The present study was performed to evaluate the ability of KR-30026 and KR-30031 to overcome multidrug resistance (MDR) by measuring the cytotoxicity of paclitaxel and the rate of rhodamine accumulation, which were then compared with verapamil. KR-30026 potentiated the paclitaxel-induced cytotoxicity of HCT15 to over 60 fold greater than that of verapamil, and KR-30031 was equipotent with verapamil (EC50: 0.00066, 0.04 and 0.05 nM at 4.0 micrograms/ml, respectively). KR-30026 and KR-30031 were without effect on paclitaxel-induced cytotoxicity to SK-OV-3 cells, as well as on tamoxifen-induced cytotoxicity to HCT15, HCT15/CL02 and SK-OV-3 cells. Maximal rhodamine accumulation by KR-30026, KR-30031 and verapamil were similar in HCT15 cells, while KR-30026 was more potent than verapamil in HCT15/CL02 cells (721 and 440%, respectively). To evaluate the cardiac toxicity of KR-30026 and KR-30031, the changes of tension in isolated rat aorta and left ventricular pressure (LVP) in guinea pig heart were determined; KR-30026 and KR-30031 were 15-40 and 25-70 fold less potent than verapamil, respectively. These results suggest that KR-30026 and KR-30031 are active modulators of MDR with potentially minimal cardiovascular toxicity.

The pharmacokinetic study on cis-(glycolato-O,O') [(4R,5R)-4,5-bis(aminomethyl)-2-isopropyl-1,3-dioxolane]-platinum(II) (SKI 2034R, NSC D642489) was performed in beagle dogs. A single dose of 2.0 mg/kg of SKI 2034R was given by i.v. bolus to three beagle dogs. Plasma samples were analyzed for platinum by flameless atomic absorption spectrophotometry. Plasma concentrations of total and ultrafiltrable platinum for SKI 2034R declined in a biexponential fashion. The mean area under the concentration-time curve (AUC0--infinity) determined for ultrafiltrable platinum derived from SKI 2034R, as an active component, was 2.76 +/- 0.13 micrograms.h/ml (mean +/- S.D.), with an initial half-life of 0.15 +/- 0.09 hour, a terminal half-life of 0.96 +/- 0.12 hour, a total clearance of 12.07 +/- 0.67 ml/min/kg, and a steady-state volume of distribution of 0.82 +/- 0.06 1/kg.

cdc25A and cdc25C, which encode threonine/tyrosine phosphatases involved in cell cycle regulation, were found to be differentially localized in rat testicular germ cells during spermatogenesis. Northern blot analysis identified two cdc25A transcripts. A transcript of 3.8 kilobases (kb) was expressed in the early germ cells including spermatogonia, and a 3.3-kb transcript was highly expressed in spermatocytes and round spermatids. In situ hybridization showed that the levels of mRNA for cdc25A were highest in diplotene spermatocytes and round spermatids but were undetectable in Sertoli cells. Both transcripts for cdc25A increased with retinol treatment in the vitamin A-deficient testis. Immunolocalization showed that cdc25A protein was distributed in the cytoplasm of germ cells, whereas cdc25C was localized to the nucleus of late spermatocytes and round spermatids. Previous studies have identified two cdc25C transcripts of 2.1 kb and 1.9 kb, which were expressed in spermatocytes and round spermatids, respectively. These results suggest that cdc25A may function during mitosis of spermatogonia. Both cdc25A and cdc25C may function in the regulation of the meiotic cell cycle, although in different manners, as indicated by their subcellular localization.