Modeling the structure of the type I peritrophic matrix: characterization of a Mamestra configurata intestinal mucin and a novel peritrophin containing 19 chitin binding domains

Twelve to fourteen integral proteins were found to reside in the Type I peritrophic matrix (PM) of Mamestra configurata (bertha armyworm) larvae. Several methods were employed, including de novo peptide sequencing, the generation of a midgut-specific EST database and immunological screening, which led to the isolation of cDNAs encoding two integral PM proteins. McPM1, the largest PM protein described to date at 202 kDa, was comprised of a concatamer of 19 chitin binding domains (CBD), 12 of which resided within a central repetitive region consisting of six iterations of a two CBD module. The protein was found to reside within the PM primarily as several lower molecular weight, presumably proteolytically processed, forms. McMUC1 was similar in structure to other insect intestinal mucins (IIM) and was highly glycosylated. The expression of both proteins was restricted to the larval midgut. Lower molecular weight proteins that may represent non- and partially glycosylated forms of McMUC1 were also recognized by an anti-McMUC1 antiserum. These were preferentially degraded upon ingestion of M. configurata multi-capsid nucleopolyhedrovirus by larvae, possibly by a viral-encoded metalloprotease. A molecular model of PM structure is presented featuring the interaction of McPM1 with chitin inter-fibril junctions and McMUC1 with the extended chains in the internodal regions. The potential for interaction between PM proteins via intermolecular disulfide bond formation and through association of CBD with N-linked glycans is discussed.     

Chromodomain protein Swi6-mediated role of DNA polymerase alpha in establishment of silencing in fission Yeast

Although DNA replication has been thought to play an important role in the silencing of mating type loci in Saccharomyces cerevisiae, recent studies indicate that silencing can be decoupled from replication. In Schizosaccharomyces pombe, mating type silencing is brought about by the trans-acting proteins, namely Swi6, Clr1-Clr4, and Rhp6, in cooperation with the cis-acting silencers. The latter contain an autonomous replication sequence, suggesting that DNA replication may be critical for silencing in S. pombe. To investigate the connection between DNA replication and silencing in S. pombe, we analyzed several temperature-sensitive mutants of DNA polymerase alpha. We find that one such mutant, swi7H4, exhibits silencing defects at mat, centromere, and telomere loci. This effect is independent of the checkpoint and replication defects of the mutant. Interestingly, the extent of the silencing defect in the swi7H4 mutant at the silent mat2 locus is further enhanced in absence of the cis-acting, centromere-proximal silencer. The chromodomain protein Swi6, which is required for silencing and is localized to mat and other heterochromatin loci, interacts with DNA polymerase alpha in vivo and in vitro in wild type cells. However, it does not interact with the mutant pol alpha and is delocalized away from the silent mat loci in the mutant. Our results demonstrate a role of DNA polymerase alpha in the establishment of silencing. We propose a recruitment model for the coupling of DNA replication with the establishment of silencing by the chromodomain protein Swi6, which may be applicable to higher eukaryotes.     

Characterization of toxin from cheilanthes fern and its effect on lymphocyte proliferation and DNA fragmentation

Thin layer chromatography of aqueous extract of whole Cheilanthesfarinosa fern indicated the presence of ptaquiloside or ptaquiloside like compound, coinciding Rf values with that of Pterosin B standard. HPLC analysis revealed the presence of 26.3 mg/kg ptaquiloside. In vitro studies of the aqueous extract on lymphocyte culture revealed a correlation between stimulative indices and concentration of aqueous extract. Stimulation in lymphocyte proliferation was in order of bracken > cheilanthes > ConA> ptaquiloside standard. On incubation of lymphocyte with aqueous extract of ferns, no DNA damage was observed in isolated DNA.     

Structure-guided design of peptidic ligand for human prostate specific antigen.

Prostate specific antigen (PSA) is a member of kallikrein family having serine protease-like activity and acts as a prognostic marker of prostate carcinoma. Various studies have been performed on inhibition of PSA and such targeting requires the identification of highly selective peptide inhibitors. PSA was purified from human seminal plasma by rapid and efficient methods, and binding studies for various peptides were carried out by fluorescence spectroscopy and SPR. The 'S' of PSA is predominated by hydrophobic residues, and hence many hydrophobic peptides were used to determine their binding affinity to PSA by fluorescence spectroscopy. We observed that LLFW, FFKW, and KFW binds strongly to PSA, among them LLFW showed strong binding. SPR also showed strong binding affinity of PSA toward peptides with hydrophobic and basic residues. Among the peptides used, FWYS showed dramatic increase in binding affinity (10(-10) M). The peptides analyzed for binding studies, suggests that peptide with Trp residue along with basic or hydrophobic amino acids may be useful for designing specific inhibitors for PSA. The strong affinities of designed peptides for PSA can be a valuable tool for designing therapeutic agents for prostate carcinomas.     

Juglone–ascorbic acid synergy inhibits metastasis and induces apoptotic cell death in poorly differentiated thyroid carcinoma by perturbing SOD and catalase activities

Anaplastic thyroid carcinoma (ATC) requires more innovative approaches as the current regimes for therapy are inadequate, also most anticancer drugs cause general suppression of physiological functions. However, therapy with limited nontarget tissue damage is desirable. In the present study, we show prooxidant ability of ascorbic acid, which enhances cytotoxicity induced by juglone. We decipher that juglone–ascorbate combination induces reactive oxygen species‐mediated apoptosis leading to cell death in ARO cell line originated from ATC. This combination also affects enzyme activity of catalase, glutathione reductase, and superoxide dismutase destabilizing redox balance in cell and thereby making juglone effective at a lower dose. We also show that juglone–ascorbate combination suppresses cell migration, invasion, and expression of tumor‐promoting, and angiogenic genes in ARO cell line, thereby disrupting epithelial–mesenchymal transition ability of the cells. Overall, we show that ascorbic acid increases cytotoxic potency of juglone through redox cycling when used in synergy.     

Identification,mapping, and marker development of stem rust resistance genes in durum wheat ‘Lebsock’

Wheat production in many wheat-growing regions is vulnerable to stem rust, caused by Puccinia graminis f. sp. tritici (Pgt). Several previous studies showed that most of the durum cultivars adapted to the upper Great Plains in the USA have good resistance to the major Pgt pathotypes, including the Ug99 race group. To identify the stem rust resistance (Sr) genes in the durum cultivar ‘Lebsock’, a tetraploid doubled haploid (DH) population derived from a cross between Lebsock and Triticum turgidum ssp. carthlicum PI 94749 was screened with the Pgt races TTKSK, TRTTF, and TTTTF. The stem rust data and the genotypic data previously developed were used to identify quantitative trait loci (QTL) associated with resistance. We identified one QTL each on chromosome arms 4AL, 6AS, 6AL, and 2BL. Based on marker and race-specification analysis, we postulated that the QTL on 4AL, 6AS, 6AL, and 2BL correspond to Sr7a, Sr8155B1, Sr13, and likely Sr9e, respectively. The results indicated that most of the US durum germplasm adapted to the upper Great Plains likely harbors the four major Sr genes characterized in this study. Among these genes, Sr8155B1 was recently identified and shown to be unique in that it conferred susceptibility to TTKSK but resistance to variant race TTKST. Two, three, and one thermal asymmetric reverse PCR (STARP) markers were developed for Sr7a, Sr8155-B1, and Sr13, respectively. Knowledge of the Sr genes in durum germplasm and the new STARP markers will be useful to pyramid and deploy multiple Sr genes in future durum and wheat cultivars.     

The start of the birch pollen season in Finnish Lapland: separating non‐local from local birch pollen and the implication for allergy sufferers

Determining the start of the birch pollen season requires the reliable separation of non‐local from locally produced birch pollen. The research was undertaken close to the latitudinal birch tree line at the Kevo Subarctic Research Institute (69°45′N 27°01′E) in northern Finland. By comparing phenological and aerobiological observations, the proportion of birch pollen present in the air before local anthesis commences can be delimited. We coupled this with data of pollen deposition monitored by means of a modified Tauber trap. The dominant birch species at Kevo is the mountain birch Betula pubescens ssp. czerepanovii, whereas B. pubescens ssp. pubescens is very rare, hence we consider the proportion of the southerly B. pubescens‐type pollen deposited in the pollen trap to be non‐local in origin.

We did not observe any trend towards an earlier start of the phenologically observed mountain birch anthesis at Kevo as predicted from work elsewhere. Moreover, the fixed 2.5% threshold method for determining the birch pollen season proved not to be applicable since in many years this threshold was reached before the end of continuous snow cover. The results indicate that in some years non‐local birch pollen contributes considerably to the allergen load in Lapland with up to 57% of the total birch pollen sum being recorded before the day on which local anthesis commenced, and up to 70% of the annual birch pollen deposited being of the southerly birch type.     

Development of peptide inhibitor as a therapeutic agent against head and neck squamous cell carcinoma (HNSCC) targeting p38α MAP kinase

Background

The p38α MAP kinase pathway is involved in inflammation, cell differentiation, growth, apoptosis and production of pro-inflammatory cytokines TNF-α and IL-1β. The overproduction of these cytokines plays an important role in cancer. The aim of this work was to design a peptide inhibitor on the basis of structural information of the active site of p38α.

Methods

A tetrapeptide, VWCS as p38α inhibitor was designed on the basis of structural information of the ATP binding site by molecular modeling. The inhibition study of peptide with p38α was performed by ELISA, binding study by Surface Plasmon Resonance and anti-proliferative assays by MTT and flow cytometry.

Results

The percentage inhibition of designed VWCS against pure p38α protein and serum of HNSCC patients was 70.30 and 71.5%, respectively. The biochemical assay demonstrated the K_D and IC₅₀ of the selective peptide as 7.22 × 10^− 9 M and 20.08 nM, respectively. The VWCS as inhibitor significantly reduced viability of oral cancer KB cell line with an IC₅₀ value of 10 μM and induced apoptosis by activating Caspase 3 and 7.

Conclusions

VWCS efficiently interacted at the ATP binding pocket of p38α with high potency and can be used as a potent inhibitor in case of HNSCC.

General significance

VWCS can act as an anticancer agent as it potentially inhibits the cell growth and induces apoptosis in oral cancer cell-line in a dose as well as time dependent manner. Hence, p38α MAP kinase inhibitor can be a potential therapeutic agent for human oral cancer.     

Molecular mapping of leaf rust resistance gene Lr15 in hexaploid wheat

Leaf rust is a widespread and commonly occurring rust disease of wheat. Genetic resistance is the most economical method of reducing losses due to leaf rust. Lr15 has been shown to be present on wheat chromosome 2D and is reported to be a seedling resistance gene. However, tightly linked markers associated with Lr15 have not been reported to date. To identify molecular markers linked to Lr15, an F₂ mapping population of Thatcher × Thatcher-Lr15 was generated. Available wheat simple sequence repeat markers were utilized in parental screening and polymorphic markers were used to analyze the entire population of 221 plants. Phenotypic evaluations of the F₂-derived F₃ progenies with Puccinia triticina Eriks. pathotype 162A (93R15) confirmed the monogenic inheritance of Lr15. The linkage group representing chromosome 2DS was constructed at LOD 4.0 which revealed the closest flanking markers Xgwm4562 and Xgwm102 at a distance of 3.1 and 9.3 cM, respectively. Furthermore, utilization of these flanking markers in combination has successfully identified wheat lines with or without Lr15. These markers could potentially be useful in gene pyramiding with other genes to enhance rust resistance in wheat.     

Maintenance of Intestinal Th17 Cells and Reduced Microbial Translocation in SIV-infected Rhesus Macaques Treated with Interleukin (IL)-21

In pathogenic HIV and SIV infections of humans and rhesus macaques (RMs), preferential depletion of CD4⁺ Th17 cells correlates with mucosal immune dysfunction and disease progression. Interleukin (IL)-21 promotes differentiation of Th17 cells, long-term maintenance of functional CD8⁺ T cells, and differentiation of memory B cells and antibody-secreting plasma cells. We hypothesized that administration of IL-21 will improve mucosal function in the context of pathogenic HIV/SIV infections. To test this hypothesis, we infected 12 RMs with SIV_mac239 and at day 14 post-infection treated six of them with rhesus rIL-21-IgFc. IL-21-treatment was safe and did not increase plasma viral load or systemic immune activation. Compared to untreated animals, IL-21-treated RMs showed (i) higher expression of perforin and granzyme B in total and SIV-specific CD8⁺ T cells and (ii) higher levels of intestinal Th17 cells. Remarkably, increased levels of Th17 cells were associated with reduced levels of intestinal T cell proliferation, microbial translocation and systemic activation/inflammation in the chronic infection. In conclusion, IL-21-treatment in SIV-infected RMs improved mucosal immune function through enhanced preservation of Th17 cells. Further preclinical studies of IL-21 may be warranted to test its potential use during chronic infection in conjunction with antiretroviral therapy.