Hepatitis B Surface Antigen S Gene is an Effective Carrier Molecule for Developing GnRH DNA Immunocastration Vaccine in Mice.

Relatively molecular mass of GnRH antigens is small and hence needs to couple to a large carrier molecule to enhance its immunogenicity. This study investigated whether hepatitis B surface antigen S (HBsAg-S) gene can be used as an effective carrier molecule for developing GnRH DNA immunocastration vaccine. Two copies of human GnRH gene were fused with HBsAg-S gene for constructing a recombinant plasmid pVAX-HBsAg-S-2GnRH that coded for 27 kDa target fusion protein. Ten male mice were divided into two equal groups, treatment and control. The vaccine (50 µg/mice) prepared in saline solution was injected into male mice at weeks 0, 1, 2, 4 and 7 of the experiment. Vaccine's efficacy was evaluated in terms of GnRH-specific IgG antibody response, plasma testosterone levels, testicular weight and extent of the testicular tissue damage. The specific anti-GnRH antibody titre in vaccinated animals was significantly higher than in controls in only 4th week of immunization (p < 0.05). In addition, vaccinated animals showed lower testicular weight than those of the controls (p < 0.05). Spermatogenesis in seminiferous tubules in vaccinated animals was suppressed. In conclusion, in this study, the engineered plasmid to be used as a GnRH DNA vaccine induced antibody response and suppressed spermatogenesis in mice. This suggests that HBsAg-S gene can be an effective carrier molecule for developing GnRH DNA immunocastration vaccine when relatively molecular mass of the aimed antigens is small.

Differential expression of Toll-like receptors in goat dominant and nondominant follicles.

The mechanism of dominant follicle selection is unclear because of its physiological complexity. However, some studies have reported that the immune system plays an important role in reproductive physiology. The objective of the current study was to investigate the differential expression of Toll-like receptors (TLRs) in the dominant (DFs) and nondominant follicles (NFs), and to determine the correlation between the expression of TLRs and the related genes, such as WNT4 and FOXL2. In this comparative study, the expression levels of TLRs, WNT4, and FOXL2 genes of DFs and NFs were obtained from three Dazu black goats were estimated using the real-time PCR. Our results showed no significant difference in the expression of seven TLRs (excluding TLR2, TLR5, and TLR8), WNT4, and FOXL2 between the DFs and NFs. In addition, the mRNA expression levels of WNT4 significantly correlated with the relative expression of TLR6 (r = 0.949739, P < 0.01); however, no significant expression of the TLR genes was found to be associated with FOXL2 mRNA expression. Our results support the fact that TLRs are not involved in the process of dominant follicle selection; however, TLR6 might play a role in the development of follicles by interacting with WNT4.

Efficacy and safety of an oral somatostatin DNA vaccine without antibiotic resistance gene in promoting growth of piglets.

This study aimed to evaluate the efficacy and safety of an oral DNA vaccine against somatostatin (SS) (pGS/2SS-asd, encoding two copies of somatostatin genes) mediated by attenuated Salmonella choleraesuis C500 without antibiotic resistance gene on piglets growth. A total of 50 piglets were uniformly divided into five groups. The animals in the first three groups were orally given vaccine in dose of either 5 9 1010, 5 9 109 or 5 9 108 colony-forming units (CFU).The remaining two groups were orally administered with either bacteria C500(containing pVAX-asd plasmid without somatostatin gene) or phosphate buffered saline (PBS) as controls. The results indicated that the vaccine induced SS-specific antibodies in a dose-dependent pattern. Compared with the PBS control, animals in the high-dose group showed lower SS levels and higher growth hormone (GH) levels in sera. Average daily gain of animals in the high dose group was increased by 32.88% and 26.46% during 4 and 8 weeks,respectively. Anti-SS antibodies were positively correlated with either GH levels or average daily gain at week 8 after primary immunization (P < 0.05). Faecal,soil and water samples originating from immunized piglets and surrounding environment were collected. The target gene (the fusion gene GS/2SS) of C500(pGS/2SS-asd) was not detected by PCR amplification in these samples,indicating that the surrounding environment was not contaminated by residual recombinant bacteria. In conclusion, the vaccine without antibiotic resistance gene is attributable to improve growth performance of piglets through an influence on GH secretion. Moreover, the immunization did not contaminate the surrounding environment of animals.

Effects of atorvastatin in combination with ezetimibe on carotid atherosclerosis in elderly patients with hypercholesterolemia.

The aim of this study was to observe the effects of atorvastatin combined with ezetimibe on carotid atherosclerosis in elderly patients with hypercholesterolemia. A total of 84 elderly hypercholesterolemic patients complicated with carotid atherosclerosis were divided into control group (atorvastatin alone) and combined group (atorvastatin combined with ezetimibe) and treated for 12 months. Carotid atherosclerosis-related indicators including blood lipid and high-sensitivity C-reactive protein (hsCRP) were determined before and after treatment. The levels of carotid intima-media thickness (CIMT), serum low density lipoprotein cholesterol (LDL-C) and hsCRP were markedly decreased (P < 0.05) after treatment in the two groups, while the reduction of the levels of CIMT, serum LDL-C and hsCRP was more significant in the combined group (P < 0.01). After treatment, the levels of CIMT, serum LDL-C and hsCRP were distinctly different between combined and control group (P < 0.05). The combination of atorvastatin with ezetimibe could further decrease LDL-C and hsCRP levels and have certain effects on the progression of carotid atherosclerosis in elderly patients with hypercholesterolemia.

Polarization-dependent refractometer for discrimination of temperature and ambient refractive index.

Transmission characteristics of polarization-dependent refractometer based on a surface long-period grating (SLPG) inscribed in a D-shaped photonic crystal fiber (PCF) are investigated. The birefringence of SLPG produces the separation of transmission spectra for TE and TM polarization modes. We also measure the sensitivities of PCF-based SLPG to temperature and external refractive index change depending on the input polarization states. The SLPG-based sensor exhibits different temperature and ambient index sensitivities corresponding to TE and TM polarization modes. Therefore, the SLPG inscribed in D-shaped PCFs can effectively discriminate temperature and ambient index sensitivities.

Primary cilia are required for cerebellar development and Shh-dependent expansion of progenitor pool.

Cerebellar granule cell precursors (GCPs), which give rise to the most abundant neuronal type in the mammalian brain, arise from a restricted pool of primary progenitors in the rhombic lip (RL). Sonic hedgehog (Shh) secreted by developing Purkinje cells is essential for the expansion of GCPs and for cerebellar morphogenesis. Recent studies have shown that the primary cilium concentrates components of Shh signaling and that this structure is required for Shh signaling. GCPs have a primary cilium on their surface [Del Cerro, M.P., Snider, R.S. (1972). Studies on the developing cerebellum. II. The ultrastructure of the external granular layer. J Comp Neurol 144, 131-64.]. Here, we show that 1) this cilium can be conditionally ablated by crossing Kif3a(fl/-) mice with hGFAP-Cre mice, 2) removal of Kif3a from GCPs disrupts cerebellar development, and 3) these defects are due to a drastic reduction in Shh-dependent expansion of GCPs. A similar phenotype is observed when Smoothened (Smo), an essential transducer of Shh signaling, is removed from the same population of GCPs. Interestingly, Kif3a-Smo double conditional mutants show that Kif3a is epistatic to Smo. This work shows that Kif3a is essential for Shh-dependent expansion of cerebellar progenitors. Dysfunctional cilia are associated with diverse human disorders including Bardet-Biedl and Joubert syndromes. Cerebellar abnormalities observed in these patients could be explained by defects in Shh-induced GCP expansion.

In vitro study on high rotation deep removal of ceramic prostheses in dental surgery.

In vitro study on high rotation (up to over 300,000 rpm) deep removal (up to 150 microm) of ceramic prostheses, made of a machinable ceramic, Vita Mark II, was performed in dental surgery using a high-speed dental handpiece. Dental clinical removal relevance, including tangential and normal grinding forces, specific grinding energy, and surface roughness, was investigated to establish the relationships among the surgery parameters, chip geometry, and fracture mechanism. The results show that both the tangential and normal grinding forces increased with increases in both depth of cut and maximum undeformed chip thickness, but decreased with an increase in grinding speed. Specific grinding energy decreased with increase in the depth of cut and the maximum undeformed chip thickness, but increased with an increase in grinding speed. Surface roughness and morphology appeared to be independent of the increases in depth of cut, grinding speed, and maximum chip thickness. The limitation for deep removal using the dental handpiece was found that the operation at the depth of cut of 150 microm or beyond resulted in a huge normal force exertion of 3 N with a great variation. The microfracture, the lateral fracture, and the ductile microcutting were found to occur simultaneously in dental surgery to remove the ceramic prostheses.

Effect of Dietary sugar beet pulp supplementation on growth performance, nutrient digestibility, fecal Microflora, blood profiles and Diarrhea incidence in weaning pigs.

BACKGROUND: In 2006, the European Union (EU) has decided to forbid use of antibiotics as growth promoters. Although many researches had been conducted about fiber source as alternatives of antibiotics, there are still lack of reports in the literature about the optimum level of sugar beet pulp supplementation, affecting growth performance and nutrient digestibility in weaning pigs. Therefore, different level of sugar beet pulp was added to diets to determine the effects of sugar beet pulp supplementation on growth performance, nutrient digestibility, fecal microflora, blood profile and incidence of diarrhea in weaning pigs. METHODS: A total of 200 weaning pigs [(Yorkshire × Landrace) × Duroc], averaging 9.01 ± 1.389 kg of initial body weight were, allotted to 5 treatments in a randomized complete block (RCB) design. Each treatment was composed of 4 replicates with 10 pigs per pen. The treatments were control treatment: Corn-SBM basal diet + ZnO (phase 1: 0.05%; phase 2; 0.03%) and four different levels of sugar beet pulp were supplemented in Corn-SBM basal diet (3, 6, 9 or 12%). Two phase feeding programs (phase 1: 1-2 weeks; phase 2: 3-5 weeks) were used for 5 week of growth trial. RESULTS: In feeding trial, there were no significant differences in growth performance and incidence of diarrhea among treatments. The E.coli counts were not significantly different among dietary treatments but linear response was observed in Lactobacillus counts as sugar beet pulp supplementation increased (P < 0.05). In addition, IGF-1, IgA and IgG were not affected by dietary treatments. However, the BUN concentration was decreased when pigs were fed the treatments of diets with SBP compared to that of control treatment (P < 0.05). In nutrient digestibility, crude fiber and NDF digestibilities were improved as the sugar beet pulp increased (P < 0.05). However, digestibilities of crude ash, crude fat, crude fiber and nitrogen retention were not affected by dietary sugar beet pulp levels. CONCLUSION: This experiment demonstrated that sugar beet pulp can be supplemented in weaning pigs' diet instead of ZnO to prevent postweaning diarrhea without any detrimental effect on growth performance.

Surface integrity and removal mechanism in simulated dental finishing of a feldspathic porcelain.

Dental abrasive finishing of a fine-grained feldspathic porcelain was performed on a computer-assisted apparatus for simulation of a 2-degrees-of-freedom restorative operation with a dental handpiece and a coarse diamond bur of grit size of 106-125 mum. Finishing forces, surface roughness, and morphology were investigated as functions of finishing conditions. The tangential and normal forces were measured using a piezoelectric dynamometer and a data processing system. The results indicated that these forces increased with either the depth of cut or with the feed rate, in the ranges of 0.12-0.31 N and 0.45-1.09 N, respectively. However, an increase in either depth of cut or feed rate affected neither the surface roughness measured using a stylus profilometer nor the morphology observed under a scanning electron microscope. The finished porcelain surfaces were found to consist of the microfracture and chipping areas, ductile removal areas, smeared areas, and debris. Irregular fracture and chipping resulted from the extension of lateral/median cracks; ductile micromachining was attributed to the plastic deformation accompanied by distributed microcracks. It was determined that a combination of the microfracture and ductile micromachining was the primary mechanism for material removal.

A novel protein phosphatase inhibitor, tautomycin. Effect on smooth muscle.

The antibiotic, tautomycin, was found to be a potent inhibitor of protein phosphatases and equally effective for the type-1 and type-2A enzymes. For the catalytic subunits of the type-1 and type-2A phosphatases the IC50 value was 22 to 32 nM. For the phosphatase activity present in chicken gizzard actomyosin the IC50 value was 6 nM. Tautomycin had no effect on myosin light chain kinase activity. Tautomycin induced a Ca(2+)-independent contraction of intact and permeabilized smooth muscle fibers and this was accompanied by an increase in the level of myosin phosphorylation. Thus, tautomycin by virtue of its ability to inhibit phosphatase activity is a valuable addition for studying the role of protein phosphorylation.

Differential regulation of gonadotropin-releasing hormone (GnRH) receptor expression in the posterior mediobasal hypothalamus by steroid hormones: implication of GnRH neuronal activity.

The present study is designed to evaluate the relationship between gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) gene expression during the steroid-induced LH surge. One week after ovariectomy (OVX), a capsule containing 17beta-estradiol (E) or vehicle (V) was implanted into OVX rats, and 2 days later a single injection of progesterone (P) or V was administered s.c. at 10:00 h. Poly(A)-rich RNA samples were isolated from the micropunches of the preoptic area (POA) and the posterior mediobasal hypothalamus (pMBH) from both sides of individual brain slices. Using competitive reverse transcription-polymerase chain reaction (RT-PCR) procedures, three parameters (POA GnRH, pMBH GnRHR) and pituitary GnRHR mRNA levels were simultaneously determined in each individual animal. POA GnRH mRNA and pituitary GnRHR mRNA levels were decreased by treatment with E, but increased by a combination of E and P. In contrast, pMBH GnRHR mRNA levels were clearly augmented by treatment with E, and decreased by the combination of E and P. Temporal changes in such parameters were determined in OVX+E+V- and OVX+E+P-treated rats. P augmented POA GnRH mRNA levels at the time of the LH surge (17:00 h) and the increased GnRH mRNA levels were remained until 22:00 h, while E alone failed to alter POA GnRH mRNA levels. In the pMBH micropunch samples, P substantially decreased E-induced increase in GnRHR mRNA levels at 17:00 h and further lowered those until 22:00 h. Antisense oligonucleotides of GnRHR mRNA administered into the lateral ventricle of OVX+E-treated rats blocked the E-induced increase in pMBH GnRHR mRNA levels. The antisense oligonucleotides also prevented the LH surge as well as the increase in pituitary GnRHR mRNA levels in the OVX+E+P-treated group. However, administration of this antisense oligonucleotides failed to alter POA GnRH mRNA levels. In conclusion, the present study demonstrated that there is an inverse relationship between POA GnRH mRNA levels and pMBH GnRHR mRNA levels in response to E and/or P, and that the blockade of the E-induced increase in pMBH GnRHR mRNA levels effectively nullified the P-induced LH surge. These results indicate that pMBH GnRHR gene expression is involved in synchronizing the GnRH neuronal activity, which is crucial for the generation of the LH surge.

Negative regulation of gonadotropin-releasing hormone and gonadotropin-releasing hormone receptor gene expression by a gonadotrophin-releasing hormone agonist in the rat hypothalamus.

There exists evidence for the presence of ultrashort loop feedback circuits of gonadotropin-releasing hormone (GnRH) secretion in the hypothalamus. It is, however, uncertain whether a similar mechanism is involved in the regulation of GnRH gene expression in vivo. Furthermore, little is known about the regulation of GnRH receptor (GnRHR) expression in the brain. In the present study, we examined the regulation of GnRH and its receptor gene expression by GnRH in vivo. A GnRH agonist, [D-Ala6, des-Gly10]GnRH-ethylamide (des-Gly GnRH), was administered by intracerebroventricular (i.c.v.) injection via the lateral ventricle of ovariectomized and estradiol (OVX + E)-treated rats. The amounts of GnRH and GnRHR mRNA were measured in the preoptic area (POA) and posterior mediobasal hypothalamus (pMBH) micropunch samples from individual rat brain slices by respective competitive reverse transcription-polymerase chain reactions. The i.c.v. administration of des-Gly GnRH significantly decreased GnRH and GnRHR mRNA expression in a dose-and time-related manner: des-Gly GnRH (6 ng) suppressed GnRH and GnRHR mRNA expression within 2 h, and the suppression was maintained without significant variation until 8 h after treatment. Treatment with Antide, [N-Ac-D-Nal(2)1, pCl-D-Phe2, D-Pal(3)3, Lys(Nic)5, D-Lys(Nic)6, Lys(iPR)8, D-Ala10]GnRH (10 ng), a potent GnRH antagonist, did not alter GnRH mRNA expression, but prevented des-Gly GnRH-induced suppression of GnRH mRNA expression. Antide alone decreased GnRHR mRNA expression, but failed to alter agonist-induced suppression of GnRHR mRNA expression. These results demonstrate the existence of an ultrashort loop feedback mechanism for GnRH gene expression in the POA, along with homologous down-regulation of GnRHR mRNA expression in the pMBH.

Hepatocyte deformation induced by cyanobacterial toxins reflects inhibition of protein phosphatases.

The cyclic peptide hepatotoxins microcystin-LR, 7-desmethyl-microcystin-RR and nodularin are potent inhibitors of the protein phosphatases type 1 and type 2A. Their potency of inhibition resembles calyculin-A and to a lesser extent okadaic acid. These hepatotoxins increase the overall level of protein phosphorylation in hepatocytes. Evidence is presented to indicate that in hepatocytes the morphological changes and effects on the cytoskeleton are due to phosphatase inhibition. The potency of these compounds in inducing hepatocyte deformation is similar to their potency in inhibiting phosphatase activity. These results suggest that the hepatotoxicity of these peptides is related to inhibition of phosphatases, and further indicate the importance of the protein phosphorylation in maintenance of structural and homeostatic integrity in these cells.