An alternate high yielding purification method for Clitoria ternatea lectin

In our previous publication we had reported the purification and characterization of Clitoria ternatea agglutinin from its seeds on fetuin CL agarose affinity column, designated CTA [A. Naeem, S. Haque, R.H. Khan. Protein J., 2007]. Since CTA binds beta-d-galactosides, this lectin can be used as valuable tool for glycobiology studies in biomedical and cancer research. So an attempt was made for a high yielding alternative purification method employing the use of asialofetuin CL agarose column for the above-mentioned lectin, designated CTL. The fetuin affinity purified agglutinin was found similar to asialofetuin affinity purified lectin in SDS pattern, HPLC and N-terminal sequence. The content of lectin was found to be 30mg/30g dry weight of pulse. The yield was 2.8% as compared to 0.3% obtained on fetuin column. The number of tryptophan and tyrosine estimated was four and six per subunit.     

Role of Organic Amendments to Mitigate Cd Toxicity and Its Assimilation in <i>Triticum aestivum</i> L.

In soil biota, higher and enduring concentration of heavy metals like cadmium (Cd) is hazardous and associated with great loss in growth, yield, and quality parameters of most of the crop plants. Recently, in-situ applications of eco-friendly stabilizing agents in the form of organic modifications have been utilized to mitigate the adverse effects of Cd-toxicity. This controlled experiment was laid down to appraise the imprints of various applied organic amendments namely poultry manure (PM), farmyard manure (FYM), and sugarcane press mud (PS) to immobilize Cd in polluted soil. Moreover, phytoavailability of Cd in wheat was also accessed under an alkaline environment. Results revealed that the addition of FYM (5–10 ton ha^-1 ) in Cd-contaminated soil significantly increased germination rate, leaf chlorophyll content, plant height, spike length, biological and grain yield amongst all applied organic amendments. Moreover, the addition of FYM (5–10 ton ha^-1 ) also reduced the phytoavailability of Cd by 73–85% in the roots, 57–83% in the shoots, and 81–90% in grains of wheat crop. Thus, it is affirmed that incorporation of FYM (5–10 ton ha^-1 ) performed better to enhance wheat growth and yield by remediating Cd. Thus, the application of FYM (5–10 ton ha^-1 ) reduced the toxicity induced by Cd to plants by declining its uptake and translocation as compared to all other applied organic amendments to immobilize Cd under sandy alkaline polluted soil.     

Ethnic disparity in 21-hydroxylase gene mutations identified in Pakistani congenital adrenal hyperplasia patients

Background

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders caused by defects in the steroid 21 hydroxylase gene (CYP21A2). We studied the spectrum of mutations in CYP21A2 gene in a multi-ethnic population in Pakistan to explore the genetics of CAH.

Methods

A cross sectional study was conducted for the identification of mutations CYP21A2 and their phenotypic associations in CAH using ARMS-PCR assay.

Results

Overall, 29 patients were analyzed for nine different mutations. The group consisted of two major forms of CAH including 17 salt wasters and 12 simple virilizers. There were 14 phenotypic males and 15 females representing all the major ethnic groups of Pakistan. Parental consanguinity was reported in 65% cases and was equally distributed in the major ethnic groups. Among 58 chromosomes analyzed, mutations were identified in 45 (78.6%) chromosomes. The most frequent mutation was I2 splice (27%) followed by Ile173Asn (26%), Arg 357 Trp (19%), Gln319stop, 16% and Leu308InsT (12%), whereas Val282Leu was not observed in this study. Homozygosity was seen in 44% and heterozygosity in 34% cases. I2 splice mutation was found to be associated with SW in the homozygous. The Ile173Asn mutation was identified in both SW and SV forms. Moreover, Arg357Trp manifested SW in compound heterozygous state.

Conclusion

Our study showed that CAH exists in our population with ethnic difference in the prevalence of mutations examined.     

Virulence of an emerging pathogenic lineage of Vibrio nigripulchritudo is dependent on two plasmids

Vibrioses are the predominant bacterial infections in marine shrimp farms. Vibrio nigripulchritudo is an emerging pathogen of the cultured shrimp Litopenaeus stylirostris in New Caledonia and other regions in the Indo‐Pacific. The molecular determinants of V. nigripulchritudo pathogenicity are unknown; however, molecular epidemiological studies have revealed that recent pathogenic V. nigripulchritudo isolates from New Caledonia all cluster into a monophyletic clade and contain a small plasmid, pB1067. Here, we report that a large plasmid, pA1066 (247 kb), can also serve as a marker for virulent V. nigripulchritudo, and that an ancestral version of this plasmid was likely acquired prior to other virulence‐linked markers. Additionally, we demonstrate that pA1066 is critical for the full virulence of V. nigripulchritudo in several newly developed experimental models of infection. Plasmid pB1067 also contributes to virulence; only strains containing both plasmids induced the highest level of shrimp mortality. Thus, it appears that these plasmids, which are absent from non‐pathogenic isolates, may be driving forces, as well as markers, for the emergence of a pathogenic lineage of V. nigripulchritudo.     

High-throughput feature counting and measurement of roots

SUMMARY: The original RootTrace tool has proved successful in measuring primary root lengths across time series image data. Biologists have shown interest in using the tool to address further problems, namely counting lateral roots to use as parameters in screening studies, and measuring highly curved roots. To address this, the software has been extended to count emerged lateral roots, and the tracking model extended so that strongly curved and agravitropic roots can be now be recovered. Here, we describe the novel image analysis algorithms and user interface implemented within the RootTrace framework to handle such situations and evaluate the results. AVAILABILITY: The software is open source and available from http://sourceforge.net/projects/roottrace.     

Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion

Fibroblasts often constitute the majority of the stromal cells within a breast carcinoma, yet the functional contributions of these cells to tumorigenesis are poorly understood. Using a coimplantation tumor xenograft model, we demonstrate that carcinoma-associated fibroblasts (CAFs) extracted from human breast carcinomas promote the growth of admixed breast carcinoma cells significantly more than do normal mammary fibroblasts derived from the same patients. The CAFs, which exhibit the traits of myofibroblasts, play a central role in promoting the growth of tumor cells through their ability to secrete stromal cell-derived factor 1 (SDF-1); CAFs promote angiogenesis by recruiting endothelial progenitor cells (EPCs) into carcinomas, an effect mediated in part by SDF-1. CAF-secreted SDF-1 also stimulates tumor growth directly, acting through the cognate receptor, CXCR4, which is expressed by carcinoma cells. Our findings indicate that fibroblasts within invasive breast carcinomas contribute to tumor promotion in large part through the secretion of SDF-1.     

Spectroscopic studies on the protective effect of a specific sugar on concanavalin A at acidic, neutral and alkaline pH

A Systematic investigation of the effect of pH on concanavalin A in the presence of specific and non-specific sugars is made using CD (circular dichroism) and fluorescence. The specific and non-specific sugars for concanavalin A were methyl alpha-D-glucopyranoside and methyl alpha-D-galactopyranoside respectively. Far-UV CD showed changes in the MRE value at 217 nm in the presence of the above-mentioned sugars. At pH 7, the CD and fluorescence spectra obtained in the presence of methyl alpha-D-glucopyranoside were slightly different from those for the native state and a significant difference was obtained in the presence of methyl alpha-D-galactopyranoside. Near-UV CD spectra showed the retention of a native-like tertiary structure in the presence of the specific sugar upon pH denaturation. Tryptophan fluorescence studies indicated a change in the tryptophan enviornment. The results obtained from our CD data are consistent with those obtained from fluorescence studies. Upon pH exposure of concanavalin A in the presence of methyl alpha-D-glucopyranoside and methyl alpha-D-galactopyranoside, the former acted as a protector preventing conformational alteration at different pH while the presence of latter induced a different stable conformational state and this state persists over the pH range from 2 to 10.     

Presence of a novel phosphopentomutase and a 2-deoxyribose 5-phosphate aldolase reveals a metabolic link between pentoses and central carbon metabolism in the hyperthermophilic archaeon Thermococcus kodakaraensis

Numerous bacteria and mammalian cells harbor two enzymes, phosphopentomutase (PPM) and 2-deoxyribose 5-phosphate aldolase (DERA), involved in the interconversion between nucleosides and central carbon metabolism. In this study, we have examined the presence of this metabolic link in the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. A search of the genome sequence of this strain revealed the presence of a closely related orthologue (TK2104) of bacterial DERA genes while no orthologue related to previously characterized PPM genes could be detected. Expression, purification, and characterization of the TK2104 protein product revealed that this gene actually encoded a DERA, catalyzing the reaction through a class I aldolase mechanism. As PPM activity was detected in T. kodakaraensis cells, we partially purified the protein to examine its N-terminal amino acid sequence. The sequence corresponded to a gene (TK1777) similar to phosphomannomutases within COG1109 but not COG1015, which includes all previously identified PPMs. Heterologous gene expression of TK1777 and characterization of the purified recombinant protein clearly revealed that the gene indeed encoded a PPM. Both enzyme activities could be observed in T. kodakaraensis cells under glycolytic and gluconeogenic growth conditions, whereas the addition of ribose, 2-deoxyribose, and 2'-deoxynucleosides in the medium did not lead to a significant induction of these activities. Our results clearly indicate the presence of a metabolic link between pentoses and central carbon metabolism in T. kodakaraensis, providing an alternative route for pentose biosynthesis through the functions of DERA and a structurally novel PPM.     

Herpesvirus-specific CD8 T cell immunity in old age: cytomegalovirus impairs the response to a coresident EBV infection

Aging in humans is associated with increased infections and the reduced proliferative capacity of T cells, part of the more global phenomenon termed immune senescence. The etiology of immune senescence is unknown but the accumulation of virus-specific memory T cells may be a contributory factor. We have examined CD8 T cell responses to two persistent herpesvirus infections, CMV and EBV, and to a recurrent virus infection, influenza, in different age cohorts of healthy donors using HLA-peptide tetramers and intracellular cytokine detection. Of these, CMV appears to be the most immunogenic, with the CD8 T cell response representing over 10% of the CD8 pool in many elderly donors. Interestingly, the effect of age upon EBV-specific responses depends upon donor CMV sero-status. In CMV seropositive donors, the magnitude of the EBV-specific immune response is stable with age, but in CMV seronegative donors, the response to EBV increases significantly with age. By contrast, the influenza-specific CD8 T cell immune response decreases with age, independent of CMV status. The functional activity of the herpesvirus-specific immune response decreases in elderly donors, although the characteristic phenotypes of CMV- and EBV-specific memory populations are retained. This demonstrates that aging is associated with a marked accumulation of CMV-specific CD8 T cells together with a decrease in immediate effector function. Moreover, infection with CMV can reduce prevailing levels of immunity to EBV, another persistent virus. These results suggest that carriage of CMV may be detrimental to the immunocompetent host by suppressing heterologous virus-specific immunity during aging.