Relationship between body condition score loss and mRNA of genes related to fatty acid metabolism and the endocannabinoid system in adipose tissue of periparturient cows

The endocannabinoid system (ECS) controls feed intake and energy balance in nonruminants. Recent studies suggested that dietary management alters the expression of members of the ECS in the liver and endometrium of dairy cows. The aim of this study was to determine the relationship between body condition score (BCS) loss and the mRNA abundance of genes related to fatty acid metabolism and the ECS in the subcutaneous adipose tissue (AT) of dairy cows. The BCS was determined in multiparous (3.2 ± 0.5 lactations) Holstein cows at −21 and 42 days relative to calving (designated as d = 0). Cows were grouped into three categories according to BCS loss between both assessments as follows: (1) lost ≤0.25 unit (n = 8, low BCS loss (LBL)), (2) lost between 0.5 and 0.75 units (n = 8, moderate BCS loss (MBL)) and (3) lost ≥1 unit (n = 8, high BCS loss (HBL)). Concentrations of haptoglobin and non-esterified fatty acids (NEFAs) were determined in plasma. Real-time PCR was used to determine mRNA abundance of key genes related to fatty acid metabolism, inflammation and ECS in AT. Milk yield (kg/day) between week 2 and 6 post-calving was greater in the LBL group (49.4 ± 0.75) compared to MBL (47.9 ± 0.56) and HBL (47.4 ± 0.62) groups (P < 0.05). The overall mean plasma haptoglobin and NEFA concentrations were greater in MBL and HBL groups compared with the LBL group (P < 0.05). The mRNA abundance of TNF-α, Interleukin-6 (IL-6) and IL-1β was greatest at 21 and 42 days post-calving in HBL, intermediate in MBL and lowest in LBL groups, respectively. Cows in the HBL group had the greatest AT gene expression for carnitine palmitoyltransferase 1A, hormone sensitive lipase and adipose triglyceride lipase at 21 and 42 days post-calving (P < 0.05). Overall, mRNA abundance for very long chain acyl-CoA dehydrogenase and peroxisome proliferator-activated receptor gamma, which are related to NEFA oxidation, were greater in MBL and HBL groups compared to the LBL group at 42 days post-calving. However, mRNA abundance of fatty acid amide hydrolase was lower at 21 and 42 days post-calving in HBL cows than in LBL cows (P < 0.05). In summary, results showed a positive association between increased degree of BCS loss, inflammation and activation of the ECS network in AT of dairy cows. Findings suggest that the ECS might play an important role in fatty acid metabolism, development of inflammation and cow’s adaptation to onset of lactation.     

Effects of pH amendment on metal working fluid wastewater biological treatment using a defined bacterial consortium

The aim of this study was to determine whether pH amendment of a highly alkaline metal working fluid (MWF) wastewater would improve biological treatment in a bioreactor system following introduction of a bacterial inoculum (comprised of the following strains: Agrobacterium radiobacter, Comamonas testosteroni, Methylobacterium mesophilicum, Microbacterium esteraromaticum, and Microbacterium saperdae). The pH values tested were 6, 7, 8, and 9. Three replicate batch mode bioreactors inoculated with the bacterial inoculum (plus an abiotic control bioreactor) were operated for each of the four pH conditions. After 14 days, the final mean chemical oxygen demand (COD) reduction at pH 9 was 50 +/- 1.4%; at pH 8, 58 +/- 1.4%; pH 7, 65 +/- 1.0%; and pH 6, 75 +/- 2.7% of the initial COD (approximately 10,000 mg L(-1)), respectively. Interestingly, within 5 days, the pH in all inoculated bioreactors progressed toward pH 8. However, all abiotic control bioreactors remained at the pH at which they were amended. The fate of the inoculum, determined by denaturing gradient gel electrophoresis (DGGE) and by cluster analysis of the resulting DGGE profiles, revealed that the inocula survived throughout operation of all pH-amended bioreactors. Length-heterogeneity polymerase chain reaction (PCR) was used to track the population dynamics of individual strains. After 7 days of operation, M. esteraromaticum was the most abundant population in all bioreactors, regardless of pH. From our findings, it appears necessary to adjust the MWF wastewater from pH 9 to between 6 and 7, to achieve optimal biological treatment rates.     

Sequence analysis of sub-genotype D hepatitis B surface antigens isolated from Jeddah,Saudi Arabia

Little is known about the prevalence of HBV genotypes/sub-genotypes in Jeddah province, although the hepatitis B virus (HBV) was identified as the most predominant type of hepatitis in Saudi Arabia. To characterize HBV genotypes/sub-genotypes, serum samples from 15 patients with chronic HBV were collected and subjected to HBsAg gene amplification and sequence analysis. Phylogenetic analysis of the HBsAg gene sequences revealed that 11 (48%) isolates belonged to HBV/D while 4 (18%) were associated with HBV/C. Notably, a HBV/D sub-genotype phylogenetic tree identified that eight current isolates (72%) belonged to HBV/D1, whereas three isolates (28%) appeared to be more closely related to HBV/D5, although they formed a novel cluster supported by a branch with 99% bootstrap value. Isolates belonging to D1 were grouped in one branch and seemed to be more closely related to various strains isolated from different countries. For further determination of whether the three current isolates belonged to HBV/D5 or represented a novel sub-genotype, HBV/DA, whole HBV genome sequences would be required. In the present study, we verified that HBV/D1 is the most prevalent HBV sub-genotype in Jeddah, and identified novel variant mutations suggesting that an additional sub-genotype designated HBV/DA should be proposed. Overall, the results of the present HBsAg sequence analyses provide us with insights regarding the nucleotide differences between the present HBsAg/D isolates identified in the populace of Jeddah, Saudi Arabia and those previously isolated worldwide. Additional studies with large numbers of subjects in other areas might lead to the discovery of the specific HBV strain genotypes or even additional new sub-genotypes that are circulating in Saudi Arabia.     

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness¹. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)^2,3 and a C-terminal 10xHis tag⁴, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression⁵. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme⁶. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl₂ and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.     

利用单基因组扩增法对马传染性贫血病毒疫苗株异质性的分析

前期研究发现,马传染性贫血病毒（Equine infectious anemia virus EIAV）中国弱毒疫苗株并非单一病毒,而是由多种准种（quasispecies）组成的种群。阐明该疫苗株的具体构成,对于确定优势疫苗株和分析其在体内的进化具有重要意义。本研究比较了传统RNA病毒测序法（即bulk PCR）和单基因组扩增法（Single-genome Amplification, SGA）在扩增EIAV疫苗株囊膜表面蛋白gp90基因 V3~V5区序列上的差异。结果发现,利用SGA法和bulk PCR法获得的序列在组内差异率分别为1.84%和1.88%。进一步序列比较发现,SGA法扩增的序列中除了含有与bulk PCR法中同源性较高的序列外,还存在bulk PCR法未检出的含强毒株LN40特异性位点,以及单个氨基酸缺失的序列。上述序列的存在为该疫苗株“多克隆构成”假说提供了佐证。此外,在对抽样偏差分析中发现,由于疫苗株中各种病毒准种在量上的差异,使得传统bulk PCR法不能有效的扩增组成比例较低的病毒准种,而导致测得的序列组成不能完全代表实际情况。SGA法通过对单基因组分的扩增和测序,可避免bulk PCR法的以上缺陷,在分析以准种形式存在的RNA病毒序列方面具有独特的优势。     

Functional expression of voltage-gated calcium channels in human melanoma

The expression of voltage-gated calcium channels (VGCCs) has not been reported previously in melanoma cells in spite of increasing evidence of a role of VGCCs in tumorigenesis and tumour progression. To address this issue we have performed an extensive RT-PCR analysis of VGCC expression in human melanocytes and a range of melanoma cell lines and biopsies. In addition, we have tested the functional expression of these channels using Ca(2+) imaging techniques and examined their relevance for the viability and proliferation of the melanoma cells. Our results show that control melanocytes and melanoma cells express channel isoforms belonging to the Ca(v) 1 and Ca(v) 2 gene families. Importantly, the expression of low voltage-activated Ca(v) 3 (T-type) channels is restricted to melanoma. We have confirmed the function of T-type channels as mediators of constitutive Ca(2+) influx in melanoma cells. Finally, pharmacological and gene silencing approaches demonstrate a role for T-type channels in melanoma viability and proliferation. These results encourage the analysis of T-type VGCCs as targets for therapeutic intervention in melanoma tumorigenesis and/or tumour progression.     

Effects of different sources of nitrogen on performance,relative population of rumen microorganisms,ruminal fermentation and blood parameters in male feedlotting lambs

Slow-release urea (SRU) can substitute dietary protein sources in the diet of feedlotting ruminant species. However, different SRU structures show varying results of productive performance. This study was conducted to investigate the effect of different sources of nitrogen on performance, blood parameter, ruminal fermentation and relative population of rumen microorganisms in male Mehraban lambs. Thirty-five male lambs with an average initial BW of 34.7 ± 1.8 kg were assigned randomly to five treatments. Diets consisted of concentrate mixture and mineral and vitamin supplements plus (1) alfalfa and soybean meal, (2) wheat straw and soybean meal, (3) wheat straw and urea, (4) wheat straw and Optigen® (a commercial SRU supplement) and (5) wheat straw and SRU produced in the laboratory. No statistical difference was observed in animal performance and DM intake among treatments. The mean value of ruminal pH and ammonia was higher (P < 0.05) for the SRU diet compared with WU diet. The difference in pH is likely to be due to the higher ammonia level as VFAs concentrations were unchanged. The level of blood urea nitrogen (BUN) was different among treatments (P = 0.065). The highest concentration of BUN was recorded in Optigen diet (183.1 mg/l), whereas the lowest value was recorded in wheat straw-soybean meal diet (147 mg/l). The amount of albumin and total protein was not affected by the treatments. The relative population of total protozoa, Fibrobacter succinogenes, Ruminococcus flavefaciens and Ruminococcus albus in the SRU treatment was higher (P < 0.01) than that in urea treatment at 3 h post-feeding. During the period of lack of high-quality forage and in order to reduce dietary costs, low-quality forage with urea sources can be used in the diet. Results of microbial populations revealed that SRU can be used as a nitrogen source which can sustainably provide nitrogen for rumen microorganism without negative effects on the performance of feedlotting lambs.     

木耳属真菌rDNA特异性扩增片段的RFLP研究

对木耳属8个种25个菌株的ITS和28SrDNA5’端两个区域分别进行了PCR扩增和限制酶切片段长度多态性（RFLP）研究。ITS－RFMP研究结果表明,HaeⅢ可将黑木耳与其它种区分开,MspⅠ可将盾形木耳、角质木耳、琥珀木耳和黑木耳4个种区分开,而供试的HaeⅢ、TaqⅠ、HinfⅠ和MaPⅠ这四种限制酶均不能将皱木耳、大木耳、网脉木耳及毛木耳4个种区分开,表明它们之间的亲缘关系较近;结果还表明,ITS—rDNA拷贝在毛木耳和琥珀木耳种内是异质性的,而在黑木耳种内是同质性的。285rDNA-RFLP研究结果表明,供试的4种限制酶中,仅MspⅠ可将盾形木耳和角质木耳区分开,而不能将其它种区分开,这显示了28SrDNA序列在木耳属不同种间的保守性。     

Agrobacterium-mediated transformation of African violet

A method for genetic transformation of Saintpaulia ionantha by co-cultivation of in vitro-grown leaves and petioles with Agrobacterium tumefaciens is described. Two bacterial strains, EHA105 and A281 both harbouring the binary plasmid pKIWI105 carrying the genes uidA and nptII, were used in the experiments. Regenerants were not obtained using the disarmed strain EHA105. The oncogenic strain A281 resulted in efficient transient and stable expression of the transferred traits for petiole explants only. After transformation and regeneration, the integration of the transgenes in the plant genome was confirmed by PCR analysis and Southern hybridization.