Detection of polymerase chain reaction-amplified malarial DNA in infected blood and individual mosquitoes

Chelex treatment of Plasmodium falciparum and P. berghei infected tissues, in lieu of organic extraction, was followed directly by polymerase chain reaction amplification of primed circumsporozoite gene sequences. The amplified DNA products were detected in stained gels and hybridization blots of extracts from individual infected mosquitoes and dissected mosquito tissues as well as small volumes of infected blood. Parasite development, within the mosquito midgut and salivary gland, was also monitored as a function of time post infectious blood meal. The temporal presence of amplifiable circumsporozoite gene sequences in the infected mosquito midgut lumen, midgut endothelium, and salivary glands corresponded directly to the visual identification of ookinetes, oocysts, and salivary gland sporozoites, respectively.     

Biochemical and molecular studies on the resistance mechanisms in tea [<Emphasis Type="Italic">Camellia sinensis</Emphasis> (L.) O. Kuntze] against blister blight disease

Tea (Camellia sinensis) plantations are exposed to biotic and abiotic stresses. Among the biotic factors, blister blight (BB), caused by Exobasidium vexans, affects the quality and quantity of the product and demands high fungicide application. A long term solution for disease resistance would require the knowledge of the basic molecular and biochemical changes occurring in plant as an attempt to resist the pathogen and limit the spread of the disease which can further help in developing resistant cultivars using biotechnological tools. Thus, gene expression studies using the cDNA based suppressive subtractive hybridization library, characterization of genes for pathogenesis related (PR) proteins [chitinase (CsCHIT), glucanase (CsGLUC), phenylalanine ammonia lyase (CsPAL)] and genes in flavonoid pathway were accessed in the BB resistant and susceptible cultivars, SA6 and TES34, respectively. Further, biochemical analysis of PR and antioxidant enzymes (POX, APX, SOD) involved in BB resistance have been carried out to investigate the potential molecular and biochemical changes. Various stages of pathogen development had varied impact on PR protein, flavonoid pathway and anti-oxidative enzymes and indicates the possible role of reactive oxygen species, lignins, flavonoids, anthocyanins and other synthesized compounds in acting as antimicrobial/antifungal agents in tea cultivars.     

Isolation and molecular characterization of mRNA transport mutants in Schizosaccharomyces pombe.

Nucleocytoplasmic transport of mRNA is essential for eukaryotic gene expression. However, how mRNA is exported from the nucleus is mostly unknown. To elucidate the mechanisms of mRNA transport, we took a genetic approach to identify genes, the products of which play a role in that process. From about 1000 temperature -sensitive (ts- or cs-) mutants, we identified five ts- mutants that are defective in poly(A)+ RNA transport by using a situ hybridization with an oligo(dT)50 as a probe. These mutants accumulate poly(A)+ RNA in the nuclei when shifted to a nonpermissive temperature. All five mutations are tightly linked to the ts- growth defects, are recessive, and fall into four different groups designated as ptr 1-4 (poly(A)+ RNA transport). Interestingly, each group of mutants has a differential localization pattern of poly(A)+ RNA in the nuclei at the nonpermissive temperature, suggesting that they have defects at different steps of the mRNA transport pathway. Localization of a nucleoplasmin-green fluorescent protein fusion suggests that ptr2 and ptr3 have defects also in nuclear protein import. Among the isolated mutants, only ptr2 showed a defect in pre-mRNA splicing. We cloned the ptr2+ and ptr3+ genes and found that they encode Schizosaccharomyces pombe homologues of the mammalian RCC1, a guanine nucleotide exchange factor for RAN/TC4, and the ubiquitin-activating enzyme E1 involved in ubiquitin conjugation, respectively. The ptr3+ gene is essential for cell viability, and Ptr3p tagged with green fluorescent protein was localized in both the nucleus and the cytoplasm. This is the first report suggesting that the ubiquitin system plays a role in mRNA export.     

A C-terminal domain of HIV-1 accessory protein Vpr is involved in penetration, mitochondrial dysfunction and apoptosis of human CD4+ lymphocytes

We have previously shown that expression of HIV-1 vpr in yeast results in cell growth arrest and structural defects, and identified a C-terminal domain of Vpr as being responsible for these effects in yeast.1 In this report we show that recombinant Vpr and C-terminal peptides of Vpr containing the conserved sequence HFRIGCRHSRIG caused permeabilization of CD4+ T lymphocytes, a dramatic reduction of mitochondrial membrane potential and finally cell death. Vpr and Vpr peptides containing the conserved sequence rapidly penetrated cells, co-localized with the DNA, and caused increased granularity and formation of dense apoptotic bodies. The above results suggest that Vpr treated cells undergo apoptosis and this was confirmed by demonstration of DNA fragmentation by the highly sensitive TUNEL assay. Our results, together with the demonstration of extracellular Vpr in HIV infected individuals,2,3 suggest the possibility that extracellular Vpr could contribute to the apoptotic death and depletion of bystander cells in lymphoid tissues4,5 during HIV infection.     

Efficient recovery of recombinant proteins using membrane-based immunoaffinity chromatography (MIC)

A systematic approach to the design and development of membrane-based immunoaffinity systems for the purification of recombinant proteins is presented. The preparation and characterization of immunoaffinity membranes are described. The immunoaffinity purification process for recombinant interferon-alpha2a is used as a model system to determine the operational parameters in membrane-based immunoaffinity chromatography. The high volumetric throughput of membranes, combined with the typically fastbinding kinetics of antigen-antibody interactions, enable the purification of recombinant proteins from dilute feed stream in less time, using less antibody than conventional systems. Three recombinant proteins, human interferon-alpha2a, interleukin-2, and interleukin-2 receptor, have been purified efficiently employing membrane-based immunoaffinity chromatography. Overall, membrane-based immunoaffinity chromatography is shown to be a viable and scalable method, ideal for the industrial-scale production of recombinant proteins. (c) 1992 John Wiley & Sons, Inc.     

Influence of verticillium wilt resistant and susceptible potato genotypes on populations of antagonistic rhizosphere and rhizoplane bacteria and free nitrogen fixers

Summary Verticillium wilt resistant A 66 107-51 and susceptible Russet Burbank potatoes differentially influenced populations of rhizosphere and rhizoplane bacteria. Although differences in total bacteria between the two genotypes were not significantly different, selective differences were evident. These included increases in bacteria antagonistic in vitro toward Verticillium dahliae strain RB 5, and bacteria capable of fixing nitrogen that were more commonly associated with the wilt-resistant potato. Bacteria that were antagonistic to V. dahliae were predominantly Bacillus spp. Other antagonists were species of Pseudomonas, Gluconobacter, Flavobacterium, and Streptomyces. Nitrogen-fixing bacteria were Azotobacter and Azomonas spp. The suppression of Verticillium wilt in Russet Burbank during the growing season following the planting of A 66 107-51 may, in part, be explained by the above findings.     

Probabilistic methods of identifying genes in prokaryotic genomes: connections to the HMM theory

In this paper, we review developments in probabilistic methods of gene recognition in prokaryotic genomes with the emphasis on connections to the general theory of hidden Markov models (HMM). We show that the Bayesian method implemented in GeneMark, a frequently used gene-finding tool, can be augmented and reintroduced as a rigorous forward-backward (FB) algorithm for local posterior decoding described in the HMM theory. Another earlier developed method, prokaryotic GeneMark.hmm, uses a modification of the Viterbi algorithm for HMM with duration to identify the most likely global path through hidden functional states given the DNA sequence. GeneMark and GeneMark.hmm programs are worth using in concert for analysing prokaryotic DNA sequences that arguably do not follow any exact mathematical model. The new extension of GeneMark using the FB algorithm was implemented in the software program GeneMark.fba. Given the DNA sequence, this program determines an a posteriori probability for each nucleotide to belong to coding or non-coding region. Also, for any open reading frame (ORF), it assigns a score defined as a probabilistic measure of all paths through hidden states that traverse the ORF as a coding region. The prediction accuracy of GeneMark.fba determined in our tests was compared favourably to the accuracy of the initial (standard) GeneMark program. Comparison to the prokaryotic GeneMark.hmm has also demonstrated a certain, yet species-specific, degree of improvement in raw gene detection, ie detection of correct reading frame (and stop codon). The accuracy of exact gene prediction, which is concerned about precise prediction of gene start (which in a prokaryotic genome unambiguously defines the reading frame and stop codon, thus, the whole protein product), still remains more accurate in GeneMarkS, which uses more elaborate HMM to specifically address this task.     

Conformational transitions induced by in vitro macromolecular crowding lead to the amyloidogenesis of buffalo heart cystatin

The biological cells and extracellular matrix exhibit a highly crowded environment, called as macromolecular crowding. Crowding significantly influences protein structure and may lead to its aggregation. In the present study, buffalo heart cystatin (BHC), after purification from buffalo heart tissue, has been used as a model protein for studying effect of macromolecular crowding in the presence of high concentrations of bovine serum albumin (BSA), poly‐ethylene glycol‐1000 (PEG‐1000), and poly‐ethylene glycol‐4000 (PEG‐4000). Cystatins are thiol protease inhibitors and found to be involved in various important physiological processes. Functional inactivation of BHC was observed upon crowding, which varied as a function of concentration and molecular weight of crowding agents as well as incubation time. Structural changes of BHC at tertiary and secondary level were detected with the help of fluorescence and CD spectroscopy. CD analysis showed changes of α‐helix to β‐sheet, which could be due to aggregation. The ANS‐fluorescence study suggested the unfolding and presence of some partially folded intermediates. Increase in ThT‐fluorescence and absorption of Congo red spectra with red shift, confirmed the amyloid type aggregation of BHC in the presence of various crowding agents. Finally, electron microscopy provided the physical evidence about the formation of amyloid fibrils. Results suggested that among the various crowding agents used, amyloidogenesis of BHC was maximal in case of BSA followed by PEG‐4000 and least for PEG‐1000. The present work makes an important contribution in crowding mediated protein aggregation, which can have implications of potential interest. Copyright © 2015 John Wiley & Sons, Ltd.