Effects of a combination of fibrolytic and amylolytic enzymes on ruminal enzyme activities, bacterial diversity, blood profile and milk production in dairy cows.

We hypothesised that adding a combination of fibrolytic and amylolytic enzymes to the diet of early-lactation dairy cows would improve rumen enzyme activity and bacterial diversity, promote energy metabolism, and benefit milk production in cows. Twenty multiparous early-lactation (90 ±â€¯5 d) Holstein cows with similar body conditions were randomly allocated to control (CON, n = 10) and experimental (EXP, n = 10) groups in a completely randomised single-factor design. The CON was fed only a basal total mixed ration diet, and the diet of the EXP was supplemented with a combination of fibrolytic and amylolytic enzymes at 70 g/cow/d (cellulase 3 500 CU/g, xylanase 2 000 XU/g, ß-glucanase 17 500 GU/g, and amylase 37 000 AU/g). The experiment lasted 28 days, with 21 days for adaptation and 7 days for sampling. Enzyme addition increased the activity levels of α-amylase and xylanase, and the ammonia-N concentration (P < 0.05) tended to increase the activity of ß-glucanase (P = 0.08) in rumen fluid. However, there was no significant difference in the rumen bacterial richness and diversity, phylum (richness > 0.1%) or genus (richness > 1%) composition between the CON and EXP groups (P > 0.05). A tendency of difference was found between CON and EXP (R = 0.22, P = 0.098) in principal component analysis. Ten genera showed different abundances across the CON and EXP groups (linear discriminant analysis effect size, linear discriminant analysis > 2). EXP increased the ratio of albumin to globulin and the concentrations of total cholesterol and low-density lipoprotein cholesterol (P < 0.05) and tended to increase triglycerides (P = 0.09) in blood. Milk yield, 3.5% fat-corrected milk yield and energy-corrected milk yield increased with enzyme supplementation (P < 0.05). The production levels of milk fat and lactose increased, but the percentage of solids, not fat and protein, decreased in EXP (P < 0.05). Although the DM intake was not affected, the feed efficiency tended to increase (P = 0.07) in EXP. In conclusion, dietary supplementation with a mixture of fibrolytic and amylolytic enzymes on multiparous early-lactation dairy cows increased α-amylase and xylanase activity levels in rumen fluid, enhanced milk performance and tended to improve the feed efficiency in cows.

Leucine regulates α-amylase and trypsin synthesis in dairy calf pancreatic tissue in vitro via the mammalian target of rapamycin signalling pathway.

Starch digestion in the small intestines of the dairy cow is low, to a large extent, due to a shortage of syntheses of α-amylase. One strategy to improve the situation is to enhance the synthesis of α-amylase. The mammalian target of rapamycin (mTOR) signalling pathway, which acts as a central regulator of protein synthesis, can be activated by leucine. Our objectives were to investigate the effects of leucine on the mTOR signalling pathway and to define the associations between these signalling activities and the synthesis of pancreatic enzymes using an in vitro model of cultured Holstein dairy calf pancreatic tissue. The pancreatic tissue was incubated in culture medium containing l-leucine for 3 h, and samples were collected hourly, with the control being included but not containing l-leucine. The leucine supplementation increased α-amylase and trypsin activities and the messenger RNA expression of their coding genes (P <0.05), and it enhanced the mTOR synthesis and the phosphorylation of mTOR, ribosomal protein S6 kinase 1 and eukaryotic initiation factor 4E-binding protein 1 (P <0.05). In addition, rapamycin inhibited the mTOR signal pathway factors during leucine treatment. In sum, the leucine regulates α-amylase and trypsin synthesis in dairy calves through the regulation of the mTOR signal pathways.

Evaluation of magnetic resonance imaging in staging of rectal cancer and its relationship with P16 expression.

OBJECTIVE: To explore the use of magnetic resonance imaging (MRI) in the staging of rectal cancer and its relationship with p16 expression. PATIENTS AND METHODS: A total of 75 patients with rectal cancer treated in Oncology Department of our hospital from March 2013 to March 2017 were randomly included in this study. The entire pelvis was scanned by MRI, and clinicopathological staging was diagnosed. Subsequently, all patients underwent total mesorectal excision (TME). Histopathological gold standard [hematoxylin-eosin (HE) staining] was used to determine the stage. Immunohistochemistry (IHC) was adopted to detect the expression of p16 in cancer tissues and cancer-adjacent tissues. Compared with the results of the pathological examination, the accuracy of MRI diagnosis was analyzed. The relationship between p16 expression and MRI diagnostic materials was analyzed. RESULTS: Compared with the results of the pathological examination, the total accuracy of MRI in the evaluation of T staging was 76.0% (57/75), and the excessive staging rate and insufficient staging rate were 8.0% (6/75) and 16.0% (12/75), respectively in the assessment of tumor T staging. IHC indicated that the positive expression rate of p16 in the tumor tissues was significantly lower than that in the tumor-adjacent tissues [34.67% (26/75) vs. 85.33% (64/75), p<0.05]. The chi-square test showed that the expression of p16 in the tumors was notably correlated with T staging, N staging, and myometrial invasion diagnosed with MRI. CONCLUSIONS: P16 is significantly deficient in the rectal cancer tissues. MRI examination and identification are helpful for clinical diagnosis of rectal cancer staging. The combination of the two items may be helpful for the diagnosis of clinical rectal cancer staging and the establishment of reasonable treatment regimens.

Effects of dietary leucine and phenylalanine on pancreas development, enzyme activity, and relative gene expression in milk-fed Holstein dairy calves.

This study aimed to investigate the effect of dietary supplementation with leucine and phenylalanine on pancreas development, enzyme activity, and related gene expression in male Holstein calves. Twenty male Holstein calves [1 d of age, 38 ± 3 kg of body weight (BW)] were randomly assigned to 1 of the following 4 treatment groups with 5 calves in each group: control, leucine supplementation (1.435 g/L of milk), phenylalanine supplementation (0.725 g/L of milk), and leucine and phenylalanine (1.435 + 0.725 g/L of milk). The diets were made isonitrogenous with the inclusion of alanine in each respective treatment. The feeding trial lasted for 8 wk, including 1 wk for adaption and 7 wk for the feeding experiment. Leucine tended to increase the concentration of total pancreatic protein (mg/kg of BW). Phenylalanine increased the concentrations of plasma insulin, cholecystokinin, and pancreatic DNA (mg/g) and the expression of trypsin gene but decreased the pancreatic protein:DNA ratio and tended to decrease the pancreas weight (g/kg of BW). No differences were observed in total pancreatic DNA (mg/pancreas and mg/kg of BW), pancreatic protein (mg/pancreas), or activities of α-amylase, trypsin, and lipase. The relative expression levels of the genes encoding α-amylase and lipase did not differ among the 4 groups. The supplementation of both leucine and phenylalanine showed an interaction on the pancreas weight (g and g/kg of BW) and a tendency of an interaction on the pancreatic protein concentration (mg/g of pancreas and mg/kg of BW) and the plasma glucose concentration. Leucine tended to increase the size of the pancreatic cells, whereas phenylalanine tended to increase the number of pancreatic cells. However, neither AA affected the activities of the pancreatic enzymes of the calves. These results indicate that leucine and phenylalanine supplementation in milk-fed Holstein calves differentially affect pancreatic growth and development.

Optimization of self-catalyzed InAs Nanowires on flexible graphite for photovoltaic infrared photodetectors.

The recent discovery of flexible graphene monolayers has triggered extensive research interest for the development of III-V/graphene functional hybrid heterostructures. In order to fully exploit their enormous potential in device applications, it is essential to optimize epitaxial growth for the precise control of nanowire geometry and density. Herein, we present a comprehensive growth study of InAs nanowires on graphitic substrates by molecular beam epitaxy. Vertically well-aligned and thin InAs nanowires with high yield were obtained in a narrow growth temperature window of 420-450 °C within a restricted domain of growth rate and V/III flux ratio. The graphitic substrates enable high nanowire growth rates, which is favourable for cost-effective device fabrication. A relatively low density of defects was observed. We have also demonstrated InAs-NWs/graphite heterojunction devices exhibiting rectifying behaviour. Room temperature photovoltaic response with a cut-off wavelength of 3.4 µm was demonstrated. This elucidates a promising route towards the monolithic integration of InAs nanowires with graphite for flexible and functional hybrid devices.

Optically efficient InAsSb nanowires for silicon-based mid-wavelength infrared optoelectronics.

InAsSb nanowires (NWs) with a high Sb content have potential in the fabrication of advanced silicon-based optoelectronics such as infrared photondetectors/emitters and highly sensitive phototransistors, as well as in the generation of renewable electricity. However, producing optically efficient InAsSb NWs with a high Sb content remains a challenge, and optical emission is limited to 4.0 µm due to the quality of the nanowires. Here, we report, for the first time, the success of high-quality and optically efficient InAsSb NWs enabling silicon-based optoelectronics operating in entirely mid-wavelength infrared. Pure zinc-blende InAsSb NWs were realized with efficient photoluminescence emission. We obtained room-temperature photoluminescence emission in InAs NWs and successfully extended the emission wavelength in InAsSb NWs to 5.1 µm. The realization of this optically efficient InAsSb NW material paves the way to realizing next-generation devices, combining advances in III-V semiconductors and silicon.

MicroRNA analysis in model species based on evolutionary rates.

MicroRNAs (miRNAs) are major post-transcriptional regulators of gene expression. In an attempt to gain insights into miRNAs at the macroevolutionary level, we performed a systematic analysis of miRNAs in six model organisms based on their evolutionary rates. First, we calculated their miRNA evolutionary rates, and found that they did not correlate with the complexity of the organisms. A correlation between evolutionary rates and single nucleotide polymorphisms (SNPs) in the miRNA sequence suggested that slow-evolving miRNAs in humans tolerate more SNPs than miRNAs with similar evolutionary rates in other species. However, fast-evolving miRNAs had lower SNP densities in humans than in the fruit fly. We also found that evolutionary rates were correlated with the proportion of parasite or clustered miRNAs. This correlation exhibited a different pattern in zebrafish, which may be related to significant genome duplication in the early vertebrates. The minimized free energy of the miRNA stem-loop structure was not correlated with the evolutionary rates of any species in our analysis. After evaluating relative miRNA expression levels, we observed that newly emerged miRNAs in complex species would integrate into the gene network at a faster pace and be functionally important; therefore, miRNAs may have accelerated human evolution.

[Comparison of curative effect and prognosis analysis of patients with spinal metastases treated by percutaneous vertebroplasty combined with postoperative radiotherapy and radiotherapy alone].

Objective: To evaluate the efficacy of percutaneous vertebroplasty(PVP) combined with postoperative radiotherapy and radiotherapy alone in the treatment of spinal metastatic tumors and to evaluate the prognostic factors for survival. Methods: From December 2011 to December 2015, according to the choice of treatment, patients in group A(60 cases) were treated with PVP combined with postoperative radiotherapy and those in group B(50 cases) underwent radiotherapy alone, age, sex, primary tumor type , and other basic characteristics were analyzed in both groups in department of orthopedics and radiotherapy department, 307 Hospital of the People's Liberation Army. The pain visual analogue scale(visual analogue scale, VAS), tumors of the spine instability score(the spinal instability neoplastic score and sins), physical status score(Karnofsky performance score and KPS) were used to evaluate pain, spinal stability improvement and physical condition. Kaplan-Meier was used to analyze the survival rates of two groups of patients and the influence of primary tumor types on the survival of patients; Cox proportional hazard model was used to calculate the correlations between survival and visceral metastases, system medical treatment, vertebral number before treatment and physical condition. Results: There was no significant difference in baseline data between the two groups(P>0.05). The VAS in the group A was significantly lower than the scores in the group B at 1 month, 3 months, 6 months, and 12 months after surgery. The SINS score dropped from(7.8±1.2) to(6.3±0.9)(1 month), (6.1±0.8)(3 months) in patients with PVP combined with postoperative radiotherapy(P<0.05), the SINS score of radiotherapy patients simply dropped from(7.6±0.9) to(7.4±0.7)(1 month), (7.3±0.6)(3 months), and there was no statistically significant difference(P=0.12). The survival rates of 6 months, 1 years, and 3 years were similar between two groups(P>0.05). The influence of different types of primary tumors on the survival time of the patients was statistically significant(P<0.05). Multiple analysis showed that the internal organs metastasis, systemic medical treatment, the number of vertebral bodies and the physical condition were the important prognostic factors of the survival in patients with spinal metastases. Conclusion: PVP combined with postoperative radiotherapy for spinal metastases is better than radiotherapy alone in the treatment of relieving pain, maintaining the stability of vertebral body and improving the quality of life of patients. Survival prognosis was similar in two groups. The types of primary tumors, visceral metastasis, systemic medical treatment, the number of vertebral bodies and the physical condition are important prognostic factors in the survival of patients with spinal metastases.

Relationships between leucine and the pancreatic exocrine function for improving starch digestibility in ruminants.

Four Holstein heifers (215 ± 7 kg; means ± SD), fitted with one pancreatic pouch, duodenal re-entrant cannulas, and duodenal infusion catheters, were used in this experiment. In phase 1, the 24-h profile of pancreatic fluid was determined. Pancreatic fluid flow peaked 1h after feeding, but peaks of similar magnitude also occurred before the morning feed, necessitating 24-h collection of pancreatic fluid to estimate daily excretion. In phase 2, the effects of duodenal infusions of 0, 10, 20, or 30 g of leucine on pancreatic fluid flow were determined in a 4 × 4 Latin square design. The leucine was infused for 12h in 2,500 mL of the infusate, and samples of pancreatic fluid and jugular blood were collected in 1-h intervals from the beginning of the infusion for 36 h. The results showed that the secretion rate of pancreatic fluid (mL/h) was significantly higher in 10-g leucine group than the other groups (mL/h). Protein concentration (mg/mL) in pancreatic fluid was elevated proportional to the amount of leucine infused. Leucine infusions increased both the concentration (U/mL) and secretion rate (U/h) of α-amylase. Infusion of 10 g of leucine also increased the secretion rates (U/h) of trypsin, chymotrypsin, and lipase, but did not change their concentrations. No significant effects of leucine infusions on plasma glucose and insulin concentrations were found. The results indicate that leucine could act as a nutrient signal to stimulate α-amylase production and pancreatic exocrine function in dairy heifers.

Enzymatic characteristics of crude feruloyl and acetyl esterases of rumen fungus Neocallimastix sp. YAK11 isolated from yak (Bos grunniens).

Rumen fungus Neocallimastix sp. YAK11 was isolated from yak (Bos grunniens), and three consecutive 10-day pure cultures were anaerobically performed at 39 °C in 20-ml Hungate's tubes to explore ferulic acid esterase (FAE) and acetyl esterase (AE) activity profiles of the fungus grown on whole hay fraction of Chinese wildrye grass (Leymus chinensis) (WHOcw , n = 4) and its neutral detergent fibre fraction (NDFcw , n = 4), respectively. An aliquot of 0.7-ml culture was sampled daily using a sterile syringe, and 0.7-ml fresh medium was immediately added to the tubes to compensate for the withdrawn samples. Peak esterase activity occurred for FAE on day 5 (p < 0.001) and for AE on day 6 (p < 0.001). The mean activities of FAE and AE in WHOcw were 2.07 and 1.29 times of those in NDFcw (p < 0.001). Both FAE and AE activities were positively correlated with xylanase (r > 0.65, p < 0.001) and carboxymethyl cellulase (r > 0.57, p < 0.001) activities. Total volatile fatty acid concentration was positively correlated with enzyme activities of AE (r > 0.87, p < 0.001), FAE (r > 0.82, p < 0.001) and xylanase (r > 0.56, p < 0.001). Crude enzyme solution was harvested for the fungus grown on WHOcw , and the pH optimum of FAE activity was 8.0 while the optimum for AE was 9.0. Both FAE and AE had a broad pH stability range. The optimal temperatures for FAE and AE activity were 40 and 50 °C. The Michaelis constant (Km ) and maximum velocity (Vmax ) for FAE against methyl ferulate at pH 6.0 and 39 °C were 0.078 mm and 2.93 mU, respectively. The Km and Vmax for AE against p-nitrophenyl acetate at pH 7.0 and 39 °C were 2.73 mm and 666.67 mU, respectively. Both FAE and AE may have prospective advantages for the enzymatic degradation of roughages in ruminant animals.

Immunopathological effects of ochratoxin A and T-2 toxin combination on broilers.

The purpose of this study was to investigate the immunopathological effects of combinations of ochratoxin A (OTA) and T-2 toxin on broilers. Four hundred eighty 1-d-old broilers were randomly assigned to 4 groups, each group consisting of 4 duplicates each with 30 broilers. The 4 groups were fed the following diets for 4 wk: group 1=basal diet (control, mycotoxin-free); group 2=basal diet+2,000 mg/kg of Mycofix Plus; group 3=basal diet+0.25 mg/kg of OTA and 0.5 mg/kg of T-2; and group 4=basal diet+0.25 mg/kg of OTA and 0.5 mg/kg of T-2+2,000 mg/kg of Mycofix Plus. The feeding of OTA-T-2 toxin diets reduced (P<0.05) the level of anti-Newcastle disease virus antibody titers by 10.4%. When broilers were administered lipopolysaccharide, the results of real-time PCR showed that broilers fed OTA-T-2 toxin reduced the cytokine mRNA expression levels of interleukin-2 and interferon-gamma to some extent but not significantly (P>0.05). The concentrations of interleukin-2 and interferon-gamma in serum were significantly decreased (P<0.05) by OTA-T-2 toxin combination. Histopathological studies demonstrated that OTA-T-2 toxin combination caused abnormalities in the thymus, bursa of Fabricius, spleen, and liver. Ochratoxin A-T-2 toxicity could be counteracted by Mycofix Plus partially but not significantly (P>0.05). The concentrations of OTA and T-2 toxin used in this study are under the maximum tolerated levels recommended by Canadian Food Inspection Agency. Our study clearly put the standard and detoxification method for these toxins into question. We suggest that it may be time to reduce the maximum allowable limits of OTA and T-2 mycotoxins in feeds to improve animal health and the safety of the food chain.

Molecular detection of Muscovy duck parvovirus by loop-mediated isothermal amplification assay.

Muscovy duck parvovirus (MDPV) usually causes high morbidity and mortality in 1- to 3-wk-old Muscovy ducklings due to serious infections, which is an imminent threat to the commercial duck industry in China. The objectives of this study were to develop and evaluate a simple, rapid, and inexpensive loop-mediated isothermal amplification (LAMP) method for specific detection of MDPV and to compare it with the PCR method in rapidity, sensitivity, and accuracy. The novel LAMP assay used a set of 4 specific primers to recognize 6 distinct genomic sequences of capsid protein (VP3) from MDPV, which could be completed within 50 min at 63 degrees C in a simple water bath. The diagnostic results demonstrated that the LAMP assay detected all 7 preserved MDPV isolates, had no cross-reactivity with other duck pathogens (i.e., goose parvovirus, duck plague virus, H9N2 avian influenza virus, duck hepatitis type virus I, and Muscovy duck reovirus). The LAMP assay was at least 10-fold more sensitive than the routine PCR assay and obtained more sensitivity in 61 clinical samples. Therefore, the newly developed LAMP assay provides a specific and sensitive means for detecting MDPV and can be simply applied both in field conditions and in laboratory operations in a cost-effective manner with primary care facilities.

Preparation and immune activity analysis of H5N1 subtype avian influenza virus recombinant protein-based vaccine.

Avian influenza is a severe disease among farmed poultry and free-living birds and a constant threat to the commercial chicken industry around the world. Hemagglutinin (HA) is the major immunogen on the envelope of influenza A virus and is the predominant inducer of neutralizing antibody. To obtain the bioactive antigen proteins in large quantities, a new protein expression vector pBCX was constructed, which is based on the pET32a vector. The HA gene of the H5N1 subtype of avian influenza virus (AIV) was inserted into the pBCX vector and expressed efficiently in Escherichia coli BL21 (DE3). Fused expression of the exogenous gene and msyB produced a 97-kDa msyB-HA fusion protein. Sodium dodecyl sulfate-PAGE combined with scanning analysis demonstrated that the msyB-HA fusion protein accounted for 29.5% of the total bacterial protein, 90.5% being soluble. The msyB-HA fusion protein was purified with nondenaturing 50% Ni-NTA column chromatography, and the result showed that 24 mg of purified msyB-HA fusion protein could be obtained from 1 L of induced expression bacterial culture medium. The comparative results in the present study showed that pBCX was superior to pET32a as a protein expression vector. Western blotting showed the recombinant msyB-HA (rHA) to have better antigenic activity, which may be the result from the better posttranslation protein modification and folding in the pBCX expression system. With the rHA fusion protein as antigen, we successfully prepared and screened specific monoclonal antibodys against the H5N1 subtype AIV, which indicated that the rHA had antigen epitopes and biofunctions. The immune test confirmed that the rHA protein vaccine could also induce high neutralizing antibodies, and the AIV challenge test proved that the rHA protein-based vaccine could prevent the corresponding infection. This study demonstrates that the recombinant HA protein produced by the pBCX expression system could be used as a recombinant protein-based vaccine and has potential for further development for diagnosis.

Effects of combinations of ochratoxin A and T-2 toxin on immune function of yellow-feathered broiler chickens.

The study was to investigate the effects of combinations of ochratoxin A (OTA) and T-2 toxin on immune function of yellow-feathered broiler chickens. Three-hundred sixty 21-d-old broiler chickens were randomly assigned to 3 groups, each group consisting of 4 duplicates each with 30 chickens. The 3 groups were fed the following diets for 3 wk: C, basal diet (control, mycotoxin-free); L, basal diet + 0.25 mg/kg of OTA, 0.5 mg/kg of T-2 toxin; and H, basal diet + 0.5 mg/kg of OTA, 1 mg/kg of T-2 toxin. Body weight and feed consumption of chickens in the H group decreased significantly (P < 0.05) during the study, but their efficiency of feed utilization was not affected. The feeding of OTA-T-2 toxin diets decreased not only the relative weight of spleen, thymus, and bursa of Fabricius, but also serum concentrations of total protein, albumin, and globulin. Meanwhile, the feeding of OTA-T-2 toxin diets elevated the activities of serum gamma-glutamyltransferase, asparate aminotransferase, and alanine aminotransferase. The results of methyl thiazolyl tetrazolium reduction assay indicated that the mitogenic responses of peripheral blood lymphocytes were diminished significantly (P < 0.05 for L group; P < 0.01 for H group). Flow cytometry was employed to determine 3 indexes in peripheral blood lymphocyte of broilers, including CD4(+)/CD3(+), CD8(+)/CD3(+), and CD4(+)/CD8(+). Both toxin treatments significantly decreased (P < 0.01) CD4(+)/CD3(+) and CD4(+)/CD8(+) ratios. In summary, the combination of OTA and T-2 toxin impaired chick immune function even at combined concentrations as low as 0.25 mg/kg of OTA and 0.5 mg/kg of T-2 toxin.

Orally delivered foot-and-mouth disease virus capsid protomer vaccine displayed on T4 bacteriophage surface: 100% protection from potency challenge in mice.

An orally delivered foot-and-mouth disease (FMD) vaccine has not previously been reported. By using a T4 bacteriophage nanoparticle surface gene-protein display system (T4-S-GPDS), we created a foot-and-mouth disease virus (FMDV) entire capsid protein vaccine candidate. On the T4 phage surface SOC site, a full length FMDV capsid precursor polyprotein (P1, 755 aa) and proteinase 3C (213 aa) derived from an infected pig of serotype O strain GD-10 (1999), were separately displayed on different T4 phage particle surfaces through inserting their coding region DNAs into the T4 phage genome, yielding phage strains T4-P1 and T4-3C. We also constructed a series of FMDV sub-full length capsid structural protein (subunit) containing T4 phage recombinant vaccines. Both sucking and young BALB/c mice were used as two kinds of FMDV vaccine potency evaluation models. Many groups of both model mice were vaccinated orally or by subcutaneous injection with varying FMDV-T4 phage recombinant vaccines, with and without addition of adjuvant, then challenged with a lethal dose of cattle source virulent FMDV. In the case of immunization with a mixture of phage T4-P1 and phage T4-3C particles without any adjuvant added, all mice were 100% protected following either oral or injection immunization, whereas 100% of the control, non-immunized mice and mice immunized with only T4 phage vector Z1/Zh(-) or wild-type T4(+)D phage died; in contrast, with FMDV subunit vaccine, less than 75% protection followed the same potency challenge in both mice model groups. In addition, two pigs immunized with a phage T4-P1 and phage T4-3C mix were protected upon housing together with infected pigs. This study represents a clear example of how FMD and other pathogenic disease vaccines can be prepared by a simple and efficient bacteriophage route.

[Cloning and sequencing of prolactin gene cDNA in three chicken breeds].

The total RNA was extracted from the pituitary of two Chinese native chicken breeds, Yuehuang and Taihe Silkies, and one layer Isa, using RNeasy Kit (QIAGEN, Germany). The total RNA was used to synthesize a specific fragment with RT-PCR, in which the primers were designed based on the sequence of broiler prolactin gene cDNA. The specific fragment was ligated to a linear plasmid pBSSK and cloned with XL1-Blue. The sequencing of prolactin cDNA was carried out with ABI PRISH 377XL DNA Sequencer after cloning. The cDNA sequences and deduced amino acid sequences of prolactin gene of two Chinese native chicken breeds and one layer were compared with that of broiler, dwarf chicken and turkey. There was 93.97%-99.89% cDNA sequence homology among Chinese native, layer, broiler and dwarf chickens, in which there was the highest (99.87%) between those of Taihe and dwarf chickens. There was 98.25%-100% deduced amino acid sequence homology among Chinese native, layer, broiler and dwarf chickens, in which there was the highest (100%) between those of Taihe Silkies and dwarf chickens. It was found that Yuehuang and Taihe Silkies had the same signal peptide cleavage site Leu-Pro-Ile-Cys among amino acids sequence deduced from pre-prolactin cDNA as broiler, dwarf and turkey, while layer Isa had a different cleavage site Pro-Pro-Ile-Cys. Such difference might cause a different translation processing of pre-prolactin, which could make layer Isa non-broody. There were different amino acids in the positions 71, 141, 150 and 175 of deduced prolactin amino acid sequences among Yuehuang, Taihe Silkies, layer, broiler and dwarf chickens. There was a heparin-binding site in positions 175-181 (L-R-R-D-S-H-K) among prolactin amino acid sequences of broiler and Taihe Silkies.

Therapeutic potential of curcumin in human prostate cancer. III. Curcumin inhibits proliferation, induces apoptosis, and inhibits angiogenesis of LNCaP prostate cancer cells in vivo.

BACKGROUND: Earlier work from our laboratory highlighted the therapeutic potential of curcumin (turmeric), used as a dietary ingredient and as a natural anti-inflammatory agent in India and other Southeast Asian countries. This agent was shown to decrease the proliferative potential and induce the apoptosis potential of both androgen-dependent and androgen-independent prostate cancer cells in vitro, largely by modulating the apoptosis suppressor proteins and by interfering with the growth factor receptor signaling pathways as exemplified by the EGF-receptor. To extend these observations made in vitro and to study the efficacy of this potential anti-cancer agent in vivo, the growth of LNCaP cells as heterotopically implanted tumors in nude mice was followed. METHODS: The androgen-dependent LNCaP prostate cancer cells were grown, mixed with Matrigel and injected subcutaneously into nude mice. Experimental group received a synthetic diet containing 2% curcumin for up to 6 weeks. At the end point, sections taken from the excised tumors were evaluated for pathology, cell proliferation, apoptosis, and vascularity. RESULTS: Curcumin causes a marked decrease in the extent of cell proliferation as measured by the BrdU incorporation assay and a significant increase in the extent of apoptosis as measured by an in situ cell death assay. Moreover, a significant decrease in the microvessel density as measured by the CD31 antigen staining was also seen. CONCLUSIONS: Curcumin could be a potentially therapeutic anti-cancer agent, as it significantly inhibits prostate cancer growth, as exemplified by LNCaP in vivo, and has the potential to prevent the progression of this cancer to its hormone refractory state.

The in vivo and in vitro effects of chicken interferon alpha on infectious bursal disease virus and Newcastle disease virus infection.

The in vitro and in vivo effects of chicken interferon alpha on infectious bursal disease virus (IBDV) infection were investigated in this study. A cDNA of interferon alpha was first cloned from a Chinese strain chicken Shiqi by reverse transcription-polymerase chain reaction. The deduced amino acid sequence has one amino acid substitution with chicken interferon alpha 1 at residue 65 (N to S) and two amino acid substitutions with chicken interferon alpha 2 at residues 50 (N to S) and 58 (P to L), respectively. A prokaryotic expression system was employed to produce a large quantity of recombinant protein. Recombinant interferon was purified in a one-step process, and an optimal refolding process was devised. About 51% recombinant protein from inclusion bodies was refolded, and the final yield of the recombinant interferon reached 24.66 mg/liter culture. The recombinant interferon suppressed IBDV plaque formation in a dose-dependent manner and ameliorated IBDV and Newcastle disease virus infection in both specific-pathogen-free (SPF) and commercial chickens. The antiviral effect of interferon alpha is more significant in commercial chickens than in SPF chickens, and the route of administration affects the efficacy of interferon therapy. This is the first reported study of the effects of interferon alpha on IBDV infection.

[On molecular identification and taxonomic status of Anopheles lesteri and Anopheles anthropophagus in China (Diptera: Culicidae)].

OBJECTIVE: To clarify the taxonomic status of Anopheles lesteri and An. anthropophagus in China. METHODS: Using molecular identification (PCR assay and rDNA-ITS2 sequencing) to examine the field anopheline mosquito specimens from Liaoning and Shandong. According to the ITS2 sequences, molecular phylogenetic tree was made. RESULTS: According to the molecular identification, An. lesteri and An. anthropophagus were distributed both in Liaoning Province and Shandong Province. The length and GC content of rDNA-ITS2 sequence were 451 bp, 46.2% in An. lesteri (n = 6), and 448 bp, 46.0% in An. anthropophagus (n = 10), respectively. The ITS2 sequences from presentation sites were same in An. lesteri, while the intraspecies difference in An. anthropophagus was 0.88%. The specific difference between An. lesteri and An. anthropophagus was 25.7%. By analyzing molecular phylogenetic tree, the relationship between An. lesteri and An. sinensis, An. anthropophagus and An. liangshanensis was found to be closer. CONCLUSION: According to the molecular identification, it was defined that An. lesteri and An. anthropophagus were sympatric independent species in China.