N-acetyltransferase polymorphism among northern Sudanese

Interindividual and interethnic differences in allele frequencies of N-acetyltransferase (NAT2) single nucleotide polymorphisms (SNPs) are responsible for phenotypic variability of adverse drug reactions and susceptibility to cancer. We genotyped the seven NAT2 common SNPs in 127 randomly selected unrelated northern Sudanese subjects using allele-specific and RFLP polymerase chain reaction (PCR) based methods. Molecular genotyping was enough to designate alleles for 41 individuals unambiguously, whereas 63 individuals' alleles were inferred from haplotypes previously described. In the remaining 23 individuals, however, the phase of the SNPs could not be decided because of multiple SNP heterozygotes. Using computational methods in the HAP and Phase programs, we confirmed the inferred alleles of the 62 individuals and predicted the remaining 23 ambiguous alleles. Twelve NAT2 alleles were identified. Four alleles coded for rapid acetylators (18%), and eight alleles coded for slow acetylators (82%). Two genotypes coded for rapid acetylation (3.9%), 10 for intermediate acetylation (27.6%), and 13 for slow acetylation (68.5%). The G191A African SNP and the G857A predominantly Asian SNP were each detected at a low frequency of 3.1%. The combination of molecular and computational analysis was useful in resolving ambiguous genotypes of NAT2 in multiple SNP heterozygotes. Among the northern Sudanese the SNPs associated with slow acetylation are more prevalent than in Caucasians and Asians. This and other African studies are suggestive of an African origin for NAT2-associated polymorphism.     

Nitrification Inhibitor and Plant Growth Regulators Improve Wheat Yield and Nitrogen Use Efficiency

Journal of Plant Growth Regulation - The field experiment was conducted to investigate the effects of applying urea with nitrification inhibitor (NI) (Nitrapyrine) alone or in combination with...     

Reproductive biology of the Suez Canal spider crab Schizophrys aspera (H. Milne Edwards, 1834: Crustacea: Brachyura: Majidae)

A reproductive biology study of the spider crab Schizophrys aspera (H. Milne Edwards, 1834) was conducted in the Suez Canal from July 2012 to June 2013. The annual sex ratio (Male:Female) of S. aspera was female biased with values of 1:1.25. Out of the four ovarian development stages of this crab, two stages were observed in the Suez Canal throughout the whole year. The ovigerous crab’s carapace width varied from 28 to 52 mm. This crab species can spawn during most of the year in the canal water, with a peak during late spring and early winter. The fecundity of ovigerous females ranged between 2349 and 13600 eggs with a mean of 5494 ± 1486 eggs. Female crabs that reached sexual maturity exhibited a minimum carapace width varying between 22 and 46 mm, and fifty percentage of all ovigerous females showed a carapace width of 36 mm.     

Conserved cysteine residues in the mammalian lamin A tail are essential for cellular responses to ROS generation

Pre-lamin A and progerin have been implicated in normal aging, and the pathogenesis of age-related degenerative diseases is termed 'laminopathies'. Here, we show that mature lamin A has an essential role in cellular fitness and that oxidative damage to lamin A is involved in cellular senescence. Primary human dermal fibroblasts (HDFs) aged replicatively or by pro-oxidants acquire a range of dysmorphic nuclear shapes. We observed that conserved cysteine residues in the lamin A tail domain become hyperoxidized in senescent fibroblasts, which inhibits the formation of lamin A inter- and intramolecular disulfide bonds. Both in the absence of lamin A and in the presence of a lamin A cysteine-to-alanine mutant, which eliminates these cysteine residues (522, 588, and 591), mild oxidative stress induced nuclear disorganization and led to premature senescence as a result of decreased tolerance to ROS stimulators. Human dermal fibroblasts lacking lamin A or expressing the lamin A cysteine-to-alanine mutant displayed a gene expression profile of ROS-responsive genes characteristic of chronic ROS stimulation. Our findings suggest that the conserved C-terminal cysteine residues are essential for lamin A function and that loss or oxidative damage to these cysteine residues promotes cellular senescence.     

Autophagy-independent function of MAP-LC3 during intracellular propagation of Chlamydia trachomatis

Microtubule-associated protein 1 (MAP1) light chain 3 (LC3) has proven useful as autophagosomal marker in studies on the interaction between pathogens and the host autophagic machinery. However, the function of LC3 is known to extend above and beyond its role in autophagosome formation. We previously reported that intrinsic LC3 is associated with the intracellular Chlamydia trachomatis inclusion in human epithelial cells. Here we show that LC3, most likely the cytoplasmic nonlipidated form, interacts with the C. trachomatis inclusion as a microtubule-associated protein rather than an autophagosome-associated component. In contrast, N-terminally GFP-tagged LC3 exclusively targets autophagosomes rather than chlamydial inclusions. Immunofluorescence analysis revealed an association of LC3 and MAP1 subunits A and B with the inclusion as early as 18 h post infection. Inclusion-bound LC3 was connected with the microtubular network. Depolymerization of the microtubular architecture disrupted the association of LC3/MAP1s with the inclusion. Furthermore, siRNA-mediated silencing of the MAP1 and LC3 proteins revealed their essential function in the intracellular growth of C. trachomatis. Interestingly, defective autophagy remarkably enhanced chlamydial growth, suggesting a suppressive effect of the autophagic machinery on bacterial development. However, depletion of LC3 in autophagy-deficient cells noticeably reduced chlamydial propagation. Thus, our findings demonstrate a new function for LC3, distinct from autophagy, in intracellular bacterial pathogenesis.     

Spatial distribution of predators and prey affect biological control of twospotted spider mites by Phytoseiulus persimilis in greenhouses

We evaluated the effects of predator release pattern and prey distribution on rate of suppression of the twospotted spider mite, Tetranychus urticae Koch (Acari, Tetranychidae) and visual damage to the ornamental plant, Impatiens wallerana Hook.f., in a greenhouse. Sixteen impatiens plants were arranged in a square and infested with the same total number of spider mites distributed either evenly (equal numbers on all plants) or clumped (divided equally among the 4 central plants), simulating a “hot spot.” The predatory mite, Phytoseiulus persimilis Athias-Henriot, was released at a 1:4 predator:prey ratio based on total spider mites in the experimental unit, but the pattern of release was either even or clumped, which simulated broadcast or point-release strategies, respectively. Nine days after predator release, spider mite populations were reduced in all treatments, but only in the clumped pest-clumped predator treatment were spider mites undetectable. Poorest pest suppression occurred in the clumped spider mite-even predator treatment. Eighteen days after predator release, spider mites were eliminated in all treatments, but a reduction in average plant damage occurred only in treatments in which the predator release pattern matched the spider mite distribution (i.e., even-even or clumped-clumped) with the greatest reduction in the even-even treatment. Results suggest that there is an advantage to releasing predators in “hot spots” provided that the recommended predator:prey ratio is maintained within infested patches. If more uniform predator releases are planned, overall predator numbers need to be kept sufficiently high so that the predator:prey ratio of 1:4 shown to prevent damage on impatiens is achieved in higher-density spider mite patches.     

An effective extracellular protein secretion by an ABC transporter system in <Emphasis Type="Italic">Escherichia coli</Emphasis>: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production

Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 μM IPTG, 1.0% (w/v) arabinose and 34.7°C post-induction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage.     

Characterisation and carriage ratio of Clostridium difficile strains isolated from a community-dwelling elderly population in the United Kingdom

Background

Community-associated Clostridium difficile infection (CDI) appears to be an increasing problem. Reported carriage rates by C.difficile are debatable with suggestions that primary asymptomatic carriage is associated with decreased risk of subsequent diarrhoea. However, knowledge of potential reservoirs and intestinal carriage rates in the community, particularly in the elderly, the most susceptible group, is limited. We have determined the presence of C.difficile in the faeces of a healthy elderly cohort living outside of long-term care facilities (LCFs) in the United Kingdom.

Methods

Faecal samples from 149 community-based healthy elderly volunteers (median age 81 years) were screened for C.difficile using direct (Brazier''s CCEY) and enrichment (Cooked Meat broth) culture methods and a glutamate dehydrogenase (GDH) immunoassay. Isolates were PCR-ribotyped and analysed for toxin production and the presence of toxin genes.

Results

Of 149 faecal samples submitted, six (4%) were found to contain C.difficile. One particular sample was positive by both the GDH immunoassay and direct culture, and concurrently produced two distinct strain types: one toxigenic and the other non-toxigenic. The other five samples were only positive by enrichment culture method. Overall, four C.difficile isolates were non-toxigenic (PCR-ribotypes 009, 026 (n = 2) and 039), while three were toxigenic (PCR-ribotypes 003, 005 and 106). All individuals who had a positive culture were symptom-free and none of them had a history of CDI and/or antibiotics use in the 3 month period preceding recruitment.

Conclusions

To our knowledge, this is the first study of the presence of C.difficile in healthy elderly community-dwelling individuals residing outside of LCFs. The observed carriage rate is lower than that reported for individuals in LCFs and interestingly no individual carried the common epidemic strain PCR-ribotype 027 (NAP1/BI). Further follow-up of asymptomatic carriers in the community, is required to evaluate host susceptibility to CDI and identify dynamic changes in the host and microbial environment that are associated with pathogenicity.     

Development and inter-rater reliability of the Liverpool adverse drug reaction causality assessment tool

Aim

To develop and test a new adverse drug reaction (ADR) causality assessment tool (CAT).

Methods

A comparison between seven assessors of a new CAT, formulated by an expert focus group, compared with the Naranjo CAT in 80 cases from a prospective observational study and 37 published ADR case reports (819 causality assessments in total).

Main Outcome Measures

Utilisation of causality categories, measure of disagreements, inter-rater reliability (IRR).

Results

The Liverpool ADR CAT, using 40 cases from an observational study, showed causality categories of 1 unlikely, 62 possible, 92 probable and 125 definite (1, 62, 92, 125) and ‘moderate’ IRR (kappa 0.48), compared to Naranjo (0, 100, 172, 8) with ‘moderate’ IRR (kappa 0.45). In a further 40 cases, the Liverpool tool (0, 66, 81, 133) showed ‘good’ IRR (kappa 0.6) while Naranjo (1, 90, 185, 4) remained ‘moderate’.

Conclusion

The Liverpool tool assigns the full range of causality categories and shows good IRR. Further assessment by different investigators in different settings is needed to fully assess the utility of this tool.     

Rat Sertoli cells express epithelial but also mesenchymal genes after immortalization with SV40

A new immortal Sertoli cell line from pubertal rat testis was established and characterized. We have generated the clonal line SCIT-C8 expressing established markers for Sertoli cells (SC) like transferrin, clusterin and steel factor/stem cell factor (SCF). Additionally, the immortalized cells express afadin, a protein which is a member of tight and adherens junctions, therefore the cells may be useful for studies of the blood-testis barrier (BTB) in vitro. In contrast to primary SC, the immortalized cells lost expression of androgen receptor and responsiveness to androgens and follicle-stimulating hormone. Surprisingly, we found mRNA expression and protein secretion of the mesenchymal markers, fibronectin and entactin-1, which we also observed for the immortalized SC lines, ASC-17D and 93RS2. In comparison to primary SC, the immortalized cells demonstrated enhanced adhesion in vitro. This correlated with the expression of entactin-1 because adhesion was strongly reduced by antibody perturbation experiments. Additionally, we found the alternatively spliced and primarily muscle cell-specific long variant of TGF-beta2 not only in peritubular cells (PC), but also in the primary and immortalized SC. Furthermore, all immortalized cell lines secreted higher amounts of TGF-beta2 than primary SC. In conclusion, the immortalized SC lines from different developmental stages showed a similar pattern of epithelial and mesenchymal markers.