Cell type-specific proteasomal processing of HIV-1 Gag-p24 results in an altered epitope repertoire

Proteasomes are critical for the processing of antigens for presentation through the major histocompatibility complex (MHC) class I pathway. HIV-1 Gag protein is a component of several experimental HIV-1 vaccines. Therefore, understanding the processing of HIV-1 Gag protein and the resulting epitope repertoire is essential. Purified proteasomes from mature dendritic cells (DC) and activated CD4(+) T cells from the same volunteer were used to cleave full-length Gag-p24 protein, and the resulting peptide fragments were identified by mass spectrometry. Distinct proteasomal degradation patterns and peptide fragments were unique to either mature DC or activated CD4(+) T cells. Almost half of the peptides generated were cell type specific. Two additional differences were observed in the peptides identified from the two cell types. These were in the HLA-B35-Px epitope and the HLA-B27-KK10 epitope. These epitopes have been linked to HIV-1 disease progression. Our results suggest that the source of generation of precursor MHC class I epitopes may be a critical factor for the induction of relevant epitope-specific cytotoxic T cells.     

Galanin regulation of LH release in male rats

The present study examines the role of cerebroventricular administered (IIIrd ventricle) galanin on LHRH and LH release in adult and immature male rats. In both age groups, galanin stimulated LHRH synthesis and release from the hypothalamus, leading to a higher release of pituitary LH which in turn increased plasma LH levels. Galantide, a galanin receptor blocker, on the other hand, drastically reduced hypothalamic LHRH and plasma LH while increasing pituitary LH. In vitro incubation of anterior pituitary cells with galanin followed by LHRH resulted in increased release of pituitary LH but not by galanin alone. Galantide exhibited no such effect either alone or with LHRH. These results indicate that galanin is an important regulator for both hypothalamic LHRH and hypophysial LH and its role is independent of age in the case of male rats.     

Investigations of �� Initiator Protein-mediated Interaction between Replication Origins �� and �� of the Plasmid R6K

A typical plasmid replicon of Escherichia coli, such as ori γ of R6K, contains tandem iterons (iterated initiator protein binding sites), an AT-rich region that melts upon initiator-iteron interaction, two binding sites for the bacterial initiator protein DnaA, and a binding site for the DNA-bending protein IHF. R6K also contains two structurally atypical origins called α and β that are located on either side of γ and contain a single and a half-iteron, respectively. Individually, these sites do not bind to initiator protein π but access it by DNA looping-mediated interaction with the seven π-bound γ iterons. The π protein exists in 2 interconvertible forms: inert dimers and active monomers. Initiator dimers generally function as negative regulators of replication by promoting iteron pairing (“handcuffing”) between pairs of replicons that turn off both origins. Contrary to this existing paradigm, here we show that both the dimeric and the monomeric π are necessary for ori α-driven plasmid maintenance. Furthermore, efficient looping interaction between α and γ or between 2 γ iterons in vitro also required both forms of π. Why does α-γ iteron pairing promote α activation rather than repression? We show that a weak, transitory α-γ interaction at the iteron pairs was essential for α-driven plasmid maintenance. Swapping the α iteron with one of γ without changing the original sequence context that caused enhanced looping in vitro caused a significant inhibition of α-mediated plasmid maintenance. Therefore, the affinity of α iteron for π-bound γ and not the sequence context determined whether the origin was activated or repressed.     

Somatic embryogenesis and shoot regeneration from intact seedlings of Phaseolus acutifolius A., P. aureus (L.) Wilczek,P. coccineus L., and P. wrightii L.

A rapid, one-step procedure has been developed for inducing direct organogenesis and somatic embryogenesis in cultures of Phaseolus coccineus L., P. acutifolius A., P. aureus L. [Vigna radiata L. Wilczek] and P. wrightii L. Development of somatic embryos and shoot buds occurred within 6–8 weeks of culture from intact seedlings raised on MS (Murashige and Skoog 1962) medium supplemented with N⁶-benzylaminopurine (BAP). Shoot buds or embryoids originated from subepidermal tissue of the regions adjacent to the shoot apex, hypocotyl and cotyledonary axils. While P. acutifolius and P. aureus were regenerated via shoot formation and P. wrightii by somatic embryogenesis, both embryogenesis and shoot regeneration were observed in P. coccineus. Relatively higher levels of BAP, 50–80 M, were found to be optimal for inducing regeneration while lower concentrations were ineffective. About 40–70 shoots and 70–250 somatic embryos were produced per responding seedling. Regenerated shoots and somatic embryos developed into whole plants on a basal medium or the one supplemented with 1 M naphthaleneacetic acid.     

Resistance in certain maize cultivars to first and third-instar Chilo partellus larvae

The resistance/susceptibility levels of ten maize (Zea mays L.) cultivars to neonates and 3rd-instar larvae of Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae) were measured in the field and screenhouse experiments. The maize cultivars tested were: Inbred A, Mp 704, V-37, Population 10, Hybrid 511, Katumani Composite B, ER-29 SVR, Poza Rica 7832, ICZ2-CM, and MMV 400. Most cultivars were found susceptible to 3rd-instar larvae based on the incidence of dead heart symptoms, foliar feeding and stem tunnelling caused by stem borers in both the field and screenhouse experiments. However, the maize cultivars V-37, MMV 400 and Poza Rica 7832 sustained significantly lesser damage than the other cultivars when infested with neonates or 3rd instar larvae.The screenhouse experiments conducted with five maize cultivars, i.e. Inbred A, Mp 704, V-37, ER-29 SVR, and Poza Rica 7832 showed that neonates fed less on the resistant than the susceptible maize cultivars but feeding by the 3rd instars was equally high on all the maize cultivars except Mp 704 on which the larvae fed less than the others. In the cultivars infested with neonates, the mean period for 50% moth emergence P₅₀ was longest on Mp 704 followed by Poza Rica 7832, ER-29 SVR, V-37 and Inbred A. In the cultivars infested with 3rd-instars, P₅₀ was longest for Poza Rica 7832 followed equally by the remaining cultivars.On the basis of the evidence presented we here conclude that neonates of C. partellus are sensitive to the growth inhibiting effects of the resistant maize cultivars Mp 704, V-37, and Poza Rica 7832 but the older larvae are insensitive to these effects of Mp 704 and V-37 but not of Poza Rica 7832.     

Anionic phospholipids regulate native and expressed epithelial sodium channel (ENaC).

Using patch clamp techniques, we found that the epithelial sodium channel (ENaC) activity in the apical membrane of A6 distal nephron cells showed a sudden rundown beginning at 4 min after forming the inside-out configuration. This sudden rundown was prevented by addition of anionic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP(2)), phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), and phosphatidylserine (PS) to the "cytoplasmic" bath. Conversely, chelation of endogenous PIP(2) with anti-PIP(2) antibody, hydrolysis of PIP(2) with either exogenous phospholipase C (PLC) or activation of endogenous PLC by extracellular ATP, or application of the positively charged molecule, poly-L-lysine, accelerated channel rundown. However, neutral phosphatidylcholine had no effect on ENaC activity. By two-electrode voltage clamp recordings, we demonstrated that PIP(2) and PIP(3) significantly increased amiloride-sensitive current in Xenopus oocytes injected with cRNAs of rat alpha-, beta-, and gamma-ENaC. However, PIP(2) and PIP(3) did not affect surface expression of ENaC, indicating that PIP(2) and PIP(3) regulate ENaC at the level of the inner plasma membrane through a mechanism that is independent of ENaC trafficking. These data suggest that anionic phospholipids may mediate the regulation of ENaC by PLC- or phosphoinositide 3-kinase-coupled receptors.     

Gas Chromatography- Mass Spectrometry Based Metabolomic Approach for Optimization and Toxicity Evaluation of Earthworm Sub-Lethal Responses to Carbofuran

Despite recent advances in understanding mechanism of toxicity, the development of biomarkers (biochemicals that vary significantly with exposure to chemicals) for pesticides and environmental contaminants exposure is still a challenging task. Carbofuran is one of the most commonly used pesticides in agriculture and said to be most toxic carbamate pesticide. It is necessary to identify the biochemicals that can vary significantly after carbofuran exposure on earthworms which will help to assess the soil ecotoxicity. Initially, we have optimized the extraction conditions which are suitable for high-throughput gas chromatography mass spectrometry (GC-MS) based metabolomics for the tissue of earthworm, Metaphire posthuma. Upon evaluation of five different extraction solvent systems, 80% methanol was found to have good extraction efficiency based on the yields of metabolites, multivariate analysis, total number of peaks and reproducibility of metabolites. Later the toxicity evaluation was performed to characterize the tissue specific metabolomic perturbation of earthworm, Metaphire posthuma after exposure to carbofuran at three different concentration levels (0.15, 0.3 and 0.6 mg/kg of soil). Seventeen metabolites, contributing to the best classification performance of highest dose dependent carbofuran exposed earthworms from healthy controls were identified. This study suggests that GC-MS based metabolomic approach was precise and sensitive to measure the earthworm responses to carbofuran exposure in soil, and can be used as a promising tool for environmental eco-toxicological studies.     

Identification of amino acid residues involved in the binding of Huperzine A to cholinesterases.

Huperzine A, a potential agent for therapy in Alzheimer's disease and for prophylaxis of organophosphate toxicity, has recently been characterized as a reversible inhibitor of cholinesterases. To examine the specificity of this novel compound in more detail, we have examined the interaction of the 2 stereoisomers of Huperzine A with cholinesterases and site-specific mutants that detail the involvement of specific amino acid residues. Inhibition of fetal bovine serum acetylcholinesterase by (-)-Huperzine A was 35-fold more potent than (+)-Huperzine A, with KI values of 6.2 nM and 210 nM, respectively. In addition, (-)-Huperzine A was 88-fold more potent in inhibiting Torpedo acetylcholinesterase than (+)-Huperzine A, with KI values of 0.25 microM and 22 microM, respectively. Far larger KI values that did not differ between the 2 stereoisomers were observed with horse and human serum butyrylcholinesterases. Mammalian acetylcholinesterase, Torpedo acetylcholinesterase, and mammalian butyrylcholinesterase can be distinguished by the amino acid Tyr, Phe, or Ala in the 330 position, respectively. Studies with mouse acetylcholinesterase mutants, Tyr 337 (330) Phe and Tyr 337 (330) Ala yielded a difference in reactivity that closely mimicked the native enzymes. In contrast, mutation of the conserved Glu 199 residue to Gln in Torpedo acetylcholinesterase produced only a 3-fold increase in KI value for the binding of Huperzine A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and characterization of a mitochondrial glyoxalase II from Brassica juncea that is upregulated by NaCl, Zn, and ABA

A cDNA (1061 bp) Bj glyII was cloned from a mannitol induced library of Brassica juncea. It encoded a protein of 335 amino acids with a molecular weight of 36.52 kDa. The deduced amino acid sequence of the clone showed 92% and 56% identity with Pennisetum and rice glyoxalase II, respectively, and 30% identity was observed with the human glyoxalase II. Search for the identical residues revealed the presence of highly conserved THHHXDH domain which is involved in zinc binding. p-NN and pSORT analysis of this sequence revealed a N-terminal mitochondrial target peptide. The cDNA was cloned in pMAL and a fusion protein with MBP (78 kDa) was expressed in Escherichia coli. The recombinant protein was purified approximately sixfold by affinity purification on amylose column and showed its pH optima at 7.0. The K(m) was determined to be 120 microM using S-d-lactoylglutathione as substrate. The expression of Bj glyII under various abiotic stress conditions showed that it is upregulated by salinity, heavy metal stress, and ABA.