The N-terminal SH2 domains of Syk and ZAP-70 mediate phosphotyrosine-independent binding to integrin beta cytoplasmic domains

Syk and ZAP-70 form a subfamily of nonreceptor tyrosine kinases that contain tandem SH2 domains at their N termini. Engagement of these SH2 domains by tyrosine-phosphorylated immunoreceptor tyrosine-based activation motifs leads to kinase activation and downstream signaling. These kinases are also regulated by beta3 integrin-dependent cell adhesion via a phosphorylation-independent interaction with the beta3 integrin cytoplasmic domain. Here, we report that the interaction of integrins with Syk and ZAP-70 depends on the N-terminal SH2 domain and the interdomain A region of the kinase. The N-terminal SH2 domain alone is sufficient for weak binding, and this interaction is independent of tyrosine phosphorylation of the integrin tail. Indeed, phosphorylation of tyrosines within the two conserved NXXY motifs in the integrin beta3 cytoplasmic domain blocks Syk binding. The tandem SH2 domains of these kinases bind to multiple integrin beta cytoplasmic domains with varying affinities (beta3 (Kd = 24 nm) > beta2 (Kd = 38 nm) > beta1 (Kd = 71 nm)) as judged by both affinity chromatography and surface plasmon resonance. Thus, the binding of Syk and ZAP-70 to integrin beta cytoplasmic domains represents a novel phosphotyrosine-independent interaction mediated by their N-terminal SH2 domains.     

Development of hammerhead ribozymes to modulate endogenous gene expression for functional studies

Hammerhead ribozymes are catalytic RNAs that are being used to inhibit endogenous gene expression to study key components of basic biochemical pathways such as angiogenesis. In addition, these ribozymes have the potential to be used as components of gene therapy protocols for the treatment of disease states. We detail here a set of protocols for the design and testing of hammerhead ribozymes that will efficiently inhibit gene expression both in cell culture and in vivo.     

Structure and function of Plasmodium falciparum malate dehydrogenase: Role of critical amino acids in co-substrate binding pocket

The malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our laboratory have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal glycine motif, which forms a characteristic Rossman dinucleotide-binding fold in the co-substrate binding pocket, differentiates PfMDH (GlyXGlyXXGly) from other eukaryotic and prokaryotic malate dehydrogenases (GlyXXGlyXXGly). The amino acids lining the co-substrate binding pocket are completely conserved in MDHs from different species of human, primate and rodent malaria parasites. Based on this knowledge and conserved domains among prokaryotic and eukaryotic MDH, the role of critical amino acids lining the co-substrate binding pocket was analyzed in catalytic functions of PfMDH using site-directed mutagenesis. Insertion of Ala at the 9th or 10th position, which converts the N-terminal GlyXGlyXXGly motif (characteristic of malarial MDH and LDH) to GlyXXGlyXXGly (as in bacterial and eukaryotic MDH), uncoupled regulation of the enzyme through substrate inhibition. The dinucleotide fold GlyXGlyXXGly motif seems not to be responsible for the distinct affinity of PfMDH to 3-acetylpyridine-adenine dinucleotide (APAD, a synthetic analog of NAD), since Ala9 and Ala10 insertion mutants still utilized APADH. The Gln11Met mutation, which converts the signature glycine motif in PfMDH to that of PfLDH, did not change the enzyme function. However, the Gln11Gly mutant showed approximately a 5-fold increase in catalytic activity, and higher susceptibility to inhibition with gossypol. Asn119 and His174 participate in binding of both co-substrate and substrate. The Asn119Gly mutant exhibited approximately a 3-fold decrease in catalytic efficiency, while mutation of His174 to Asn or Ala resulted in an inactive enzyme. These studies provide critical insights into the co-substrate binding pocket of PfMDH, which may be important in design of selective PfMDH/PfLDH inhibitors as potential antimalarials.     

Non-linear regression models for time to flowering in wild chickpea combine genetic and climatic factors

Background

Accurate prediction of crop flowering time is required for reaching maximal farm efficiency. Several models developed to accomplish this goal are based on deep knowledge of plant phenology, requiring large investment for every individual crop or new variety. Mathematical modeling can be used to make better use of more shallow data and to extract information from it with higher efficiency. Cultivars of chickpea, Cicer arietanum, are currently being improved by introgressing wild C. reticulatum biodiversity with very different flowering time requirements. More understanding is required for how flowering time will depend on environmental conditions in these cultivars developed by introgression of wild alleles.

Results

We built a novel model for flowering time of wild chickpeas collected at 21 different sites in Turkey and grown in 4 distinct environmental conditions over several different years and seasons. We propose a general approach, in which the analytic forms of dependence of flowering time on climatic parameters, their regression coefficients, and a set of predictors are inferred automatically by stochastic minimization of the deviation of the model output from data. By using a combination of Grammatical Evolution and Differential Evolution Entirely Parallel method, we have identified a model that reflects the influence of effects of day length, temperature, humidity and precipitation and has a coefficient of determination of R²=0.97.

Conclusions

We used our model to test two important hypotheses. We propose that chickpea phenology may be strongly predicted by accession geographic origin, as well as local environmental conditions at the site of growth. Indeed, the site of origin-by-growth environment interaction accounts for about 14.7% of variation in time period from sowing to flowering. Secondly, as the adaptation to specific environments is blueprinted in genomes, the effects of genes on flowering time may be conditioned on environmental factors. Genotype-by-environment interaction accounts for about 17.2% of overall variation in flowering time. We also identified several genomic markers associated with different reactions to climatic factor changes. Our methodology is general and can be further applied to extend existing crop models, especially when phenological information is limited.

     

Alcohol and Tobacco Increases Risk of High Risk HPV Infection in Head and Neck Cancer Patients: Study from North-East Region of India

Background

Human papilloma virus (HPV) associated Head and Neck Cancers (HNCs) have generated significant amount of research interest in recent times. Due to high incidence of HNCs and lack of sufficient data on high-risk HPV (hr-HPV) infection from North -East region of India, this study was conceived to investigate hr-HPV infection, its types and its association with life style habits such as tobacco, alcohol consumption etc.

Methods

A total of one hundred and six primary HNC tumor biopsy specimens were collected. These samples were analyzed for hr-HPV DNA (13 HPV types) using hybrid capture 2 (HC2) assay and genotyping was done by E6 nested multiplex PCR (NMPCR).

Results

The presence of hr-HPV was confirmed in 31.13% (n = 33) and 24.52% (n = 26) of the HNC patients by nested multiplex PCR (NMPCR) and HC2 assay respectively. Among hr-HPV positive cases, out of thirteen hr- HPV types analyzed, only two prevalent genotypes, HPV-16 (81.81%) followed by HPV-18 (18.18%) were found. Significant association was observed between hr-HPV infection with alcohol consumption (p <0.001) and tobacco chewing (p = 0.02) in HNC cases. Compared to HPV-18 infection the HPV-16 was found to be significantly associated with tobacco chewing (p = 0.02) habit.

Conclusions

Our study demonstrated that tobacco chewing and alcohol consumption may act as risk factors for hr-HPV infection in HNCs from the North-East region of India. This was the first study from North-East India which also assessed the clinical applicability of HC2 assay in HNC patient specimens. We suggest that alcohol, tobacco and hr- HPV infection act synergistically or complement each other in the process of HNC development and progression in the present study population.     

Interaction pattern for the complex of B-DNA Fullerene compounds with a set of known replication proteins using docking study

Fullerenes have attracted considerable attention due to their unique chemical structure and potential applications which has opened wide venues for possible human exposure to various fullerene types. Therefore, in depth knowledge of how fullerene may interfere with various cellular processes becomes quite imperative. The present study was designed to investigate how the presence of fullerene affect the binding of DNA with different enzymes involved in replication process. Different fullerenes were first docked with DNA and then binding scores of different enzymes was analyzed with fullerene docked DNA. C30, C40 & C50 once docked with DNA, reduced the binding score of primase, whereas no significant change in the binding score was observed with the helicase, ssb protein, dna pol δ, dna pol ε, ligase, DNA clamp, and topoisomerases. On the contrast, the binding score of RPA14 decreases in fluctuating manner while interacting with increasing molecular weight of fullerene bound single-stranded DNA complex. The study revealed the affect of fullerene family interacting with DNA on the binding pattern of enzymes involved in replication process. Study suggests that the presence of most of fullerenes may not affect the activity of these enzymes necessary for replication process whereas C30, C40 & C50 may disrupt the activity of primase, (strating point for DNA polymerase) its docking score decreases from 13820 to 10702.     

Hybrid histidine kinases in pathogenic fungi

Histidine kinases (HK) sense and transduce via phosphorylation events many intra‐ and extracellular signals in bacteria, archaea, slime moulds and plants. HK are also widespread in the fungal kingdom, but their precise roles in the regulation of physiological processes remain largely obscure. Expanding genomic resources have recently given the opportunity to identify uncharacterised HK family members in yeasts and moulds and now allow proposing a complex classification of Basidiomycota, Ascomycota and lower fungi HK. A growing number of genetic approaches have progressively provided new insight into the role of several groups of HK in prominent fungal pathogens. In particular, a series of studies have revealed that members of group III HK, which occur in the highest number of fungal species and contain a unique N‐terminus region consisting of multiple HAMP domain repeats, regulate morphogenesis and virulence in various human, plant and insect pathogenic fungi. This research field is further supported by recent shape‐function studies providing clear correlation between structural properties and signalling states in group III HK. Since HK are absent in mammals, these represent interesting fungal target for the discovery of new antifungal drugs.     

Dysbiosis of salivary microbiome and cytokines influence oral squamous cell carcinoma through inflammation

Archives of Microbiology - Advanced combinatorial treatments of surgery, chemotherapy, and radiotherapy do not have any effect on the enhancement of a 5-year survival rate of oral squamous cell...