Cloning and expression of a new rat procarboxypeptidase B gene in Escherichia coli and purification of recombination carboxypeptidase B

A new coding sequence of the procarboxypeptidase B gene was obtained from SD rat fresh pancreas by RT-PCR and highly expressed in Escherichia coli in inclusion bodies. The folded procarboxypeptidase B was subjected to trypsin enzymatic cleavage to produce active carboxypeptidase B, subsequently, carboxypeptidase B was effectively purified with anion exchange chromatography DEAE-FF and hydrophobic interaction chromatography Octyl FF, as a result, 40 mg carboxypeptidase B per litre cell culture with specific activity 7.42 u/mg was achieved. Further research showed that the obtained recombinant carboxypeptidase B could substitute carboxypeptidase B isolated from pancreas.     

Expression of baculovirus anti-apoptotic genes p35 and op-iap in cotton (Gossypium hirsutum L.) enhances tolerance to verticillium wilt

Background

Programmed cell death plays an important role in mediating plant adaptive responses to the environment such as the invasion of pathogens. Verticillium wilt, caused by the necrotrophic pathogen Verticillium dahliae, is a serious vascular disease responsible for great economic losses to cotton, but the molecular mechanisms of verticillium disease and effective, safe methods of resistance to verticillium wilt remain unexplored.

Methodology/Principal Findings

In this study, we introduced baculovirus apoptosis inhibitor genes p35 and op-iap into the genome of cotton via Agrobacterium-mediated transformation and analyzed the response of transgenic plants to verticillium wilt. Results showed that p35 and op-iap constructs were stably integrated into the cotton genome, expressed in the transgenic lines, and inherited through the T₃ generation. The transgenic lines had significantly increased tolerance to verticillium wilt throughout the developmental stages. The disease index of T₁–T₃ generation was lower than 19, significantly (P<0.05) better than the negative control line z99668. After treatment with 250 mg/L VD-toxins for 36 hours, DNA from negative control leaves was fragmented, whereas fragmentation in the transgenic leaf DNA did not occur. The percentage of cell death in transgenic lines increased by 7.11% after 60 mg/L VD-toxin treatment, which was less than that of the negative control lines''s 21.27%. This indicates that p35 and op-iap gene expression partially protects cells from VD-toxin induced programmed cell death (PCD).

Conclusion/Significance

Verticillium dahliae can trigger plant cells to die through induction of a PCD mechanism involved in pathogenesis. This paper provides a potential strategy for engineering broad-spectrum necrotrophic disease resistance in plants.     

Major effect of retinal short-chain dehydrogenase reductase (RDHE2) on bovine fat colour

Beef with yellow fat is considered undesirable by consumers in most European and Asian markets. β-Carotene is the major carotenoid deposited in the adipose tissue and milk fat of cattle (Bos taurus), which can result in the yellowness. The effects of retinal short-chain dehydrogenase reductase (RDHE2) and β, β-carotene 9',10-dioxygenase (BCO2) were considered jointly as major candidate genes for causing the yellow fat colour, based on their genomic locations in the fat colour quantitative trait loci (QTL) and their roles in the metabolism of β-carotene. In a secondary pathway, BCO2 cleaves β-carotene into retinoic acid, the most potent form of vitamin A. RDHE2 converts trans-retinol to trans-retinal, a less active form of vitamin A. We evaluated the effects of two amino acid variants of the RDHE2 gene (V6A and V33A) along with a mutation in the BCO2 gene that results in a stop codon (W80X) in seven cattle populations. The RDHE2 V6A genotype affected several fat colour traits but the size of the effect varied in the populations studied. The genotype effect of the RDHE2 V33A variant was observed only in New Zealand samples of unknown breed. In general, the individual effects of RDHE2 V6A and V33A SNPs genotypes were greater in the random New Zealand samples than in samples from pedigreed Jersey-Limousin backcross progeny, accounting for 8-17 % of the variance in one population. Epistasis between the BCO2 W80X and RDHE2 variants was observed, and in some populations this explained more of the variation than the effects of the individual RDHE2 variants.     

Protective Effects of Lactobacillus plantarum CCFM8610 against Chronic Cadmium Toxicity in Mice Indicate Routes of Protection besides Intestinal Sequestration

Our previous study confirmed the ability of Lactobacillus plantarum CCFM8610 to protect against acute cadmium (Cd) toxicity in mice. This study was designed to evaluate the protective effects of CCFM8610 against chronic Cd toxicity in mice and to gain insights into the protection mode of this strain. Experimental mice were divided into two groups and exposed to Cd for 8 weeks via drinking water or intraperitoneal injection. Both groups were further divided into four subgroups, control, Cd only, CCFM8610 only, and Cd plus CCFM8610. Levels of Cd were measured in the feces, liver, and kidneys, and alterations of several biomarkers of Cd toxicity were noted. The results showed that when Cd was introduced orally, cotreatment with Cd and CCFM8610 effectively decreased intestinal Cd absorption, reduced Cd accumulation in tissue, alleviated tissue oxidative stress, reversed hepatic and renal damage, and ameliorated the corresponding histopathological changes. When Cd was introduced intraperitoneally, administration of CCFM8610 did not have an impact on tissue Cd accumulation or reverse the activities of antioxidant enzymes. However, CCFM8610 still offered protection against oxidative stress and reversed the alterations of Cd toxicity biomarkers and tissue histopathology. These results suggest that CCFM8610 is effective against chronic cadmium toxicity in mice. Besides intestinal Cd sequestration, CCFM8610 treatment offers direct protection against Cd-induced oxidative stress. We also provide evidence that the latter is unlikely to be mediated via protection against Cd-induced alteration of antioxidant enzyme activities.