Proteomic Analysis of the Human Anterior Pituitary Gland.

The pituitary function is regulated by a complex system involving the hypothalamus and biological networks within the pituitary. Although the hormones secreted from the pituitary have been well studied, comprehensive analyses of the pituitary proteome are limited. Pituitary proteomics is a field of postgenomic research that is crucial to understand human health and pituitary diseases. In this context, we report here a systematic proteomic profiling of human anterior pituitary gland (adenohypophysis) using high-resolution Fourier transform mass spectrometry. A total of 2164 proteins were identified in this study, of which 105 proteins were identified for the first time compared with high-throughput proteomic-based studies from human pituitary glands. In addition, we identified 480 proteins with secretory potential and 187 N-terminally acetylated proteins. These are the first region-specific data that could serve as a vital resource for further investigations on the physiological role of the human anterior pituitary glands and the proteins secreted by them. We anticipate that the identification of previously unknown proteins in the present study will accelerate biomedical research to decipher their role in functioning of the human anterior pituitary gland and associated human diseases.

Endoribonuclease type II toxin-antitoxin systems: functional or selfish?

Most bacterial genomes have multiple type II toxin-antitoxin systems (TAs) that encode two proteins which are referred to as a toxin and an antitoxin. Toxins inhibit a cellular process, while the interaction of the antitoxin with the toxin attenuates the toxin's activity. Endoribonuclease-encoding TAs cleave RNA in a sequence-dependent fashion, resulting in translational inhibition. To account for their prevalence and retention by bacterial genomes, TAs are credited with clinically significant phenomena, such as bacterial programmed cell death, persistence, biofilms and anti-addiction to plasmids. However, the programmed cell death and persistence hypotheses have been challenged because of conceptual, methodological and/or strain issues. In an alternative view, chromosomal TAs seem to be retained by virtue of addiction at two levels: via a poison-antidote combination (TA proteins) and via transcriptional reprogramming of the downstream core gene (due to integration). Any perturbation in the chromosomal TA operons could cause fitness loss due to polar effects on the downstream genes and hence be detrimental under natural conditions. The endoribonucleases encoding chromosomal TAs are most likely selfish DNA as they are retained by bacterial genomes, even though TAs do not confer a direct advantage via the TA proteins. TAs are likely used by various replicons as 'genetic arms' that allow the maintenance of themselves and associated genetic elements. TAs seem to be the 'selfish arms' that make the best use of the 'arms race' between bacterial genomes and plasmids.

Horizontal gene transfer of chromosomal Type II toxin-antitoxin systems of Escherichia coli.

Type II toxin-antitoxin systems (TAs) are small autoregulated bicistronic operons that encode a toxin protein with the potential to inhibit metabolic processes and an antitoxin protein to neutralize the toxin. Most of the bacterial genomes encode multiple TAs. However, the diversity and accumulation of TAs on bacterial genomes and its physiological implications are highly debated. Here we provide evidence that Escherichia coli chromosomal TAs (encoding RNase toxins) are 'acquired' DNA likely originated from heterologous DNA and are the smallest known autoregulated operons with the potential for horizontal propagation. Sequence analyses revealed that integration of TAs into the bacterial genome is unique and contributes to variations in the coding and/or regulatory regions of flanking host genome sequences. Plasmids and genomes encoding identical TAs of natural isolates are mutually exclusive. Chromosomal TAs might play significant roles in the evolution and ecology of bacteria by contributing to host genome variation and by moderation of plasmid maintenance.

Streptomycin affinity depends on 13 amino acids forming a loop in homology modelled ribosomal S12 protein (rpsL gene) of Lysinibacillus sphaericus DSLS5 associated with marine sponge (Tedania anhelans).

Streptomycin, an antibiotic used against microbial infections, inhibits the protein synthesis by binding to ribosomal protein S12, encoded by rpsL12 gene, and associated mutations cause streptomycin resistance. A streptomycin resistant, Lysinibacillus sphaericus DSLS5 (MIC >300 µg/mL for streptomycin), was isolated from a marine sponge (Tedania anhelans). The characterisation of rpsL12 gene showed a region having similarity to long terminal repeat sequences of murine lukemia virus which added 13 amino acids for loop formation in RpsL12; in addition, a K56R mutation which corresponds to K43R mutation present in streptomycin-resistant Escherichia coli is also present. The RpsL12 protein was modelled and compared with that of Lysinibacillus boronitolerans, Escherichia coli and Mycobacterium tuberculosis. The modelled proteins docked with streptomycin indicate compound had less affinity. The effect of loop on streptomycin resistance was analysed by constructing three different models of RpsL12 by, (i) removing both loop and mutation, (ii) removing the loop alone while retaining the mutation and (iii) without mutation having loop. The results showed that the presence of loop causes streptomycin resistance (decreases the affinity), and it further enhanced in the presence of mutation at 56th codon. Further study will help in understanding the evolution of streptomycin resistance in organisms.

mazEF-mediated programmed cell death in bacteria: "what is this?".

Toxin-antitoxin (TA) systems consist of a bicistronic operon, encoding a toxin and an antitoxin. They are widely distributed in the prokaryotic kingdom, often in multiple numbers. TAs are implicated in contradicting phenomena of persistence and programmed cell death (PCD) in bacteria. mazEF TA system, one of the widely distributed type II toxin-antitoxin systems, is particularly implicated in PCD of Escherichia coli. Nutrient starvation, antibiotic stress, heat shock, DNA damage and other kinds of stresses are shown to elicit mazEF-mediated-PCD. ppGpp and extracellular death factor play a central role in regulating mazEF-mediated PCD. The activation of mazEF system is achieved through inhibition of transcription or translation of mazEF loci. Upon activation, MazF cleaves RNA in a ribosome-independent fashion and subsequent processes result in cell death. It is hypothesized that PCD aids in perseverance of the population during stress; the surviving minority of the cells can scavenge the nutrients released by the dead cells, a kind of "nutritional-altruism." Issues regarding the strains, reproducibility of experimental results and ecological plausibility necessitate speculation. We review the molecular mechanisms of the activation of mazEF TA system, the consequences leading to cell death and the pros and cons of the altruism hypothesis from an ecological perspective.

Plants: a source for new antimycobacterial drugs.

Tuberculosis, also called TB, is currently a major health hazard due to multidrug-resistant forms of bacilli. Global efforts are underway to eradicate TB using new drugs with new modes of action, higher activity, and fewer side effects in combination with vaccines. For this reason, unexplored new sources and previously explored sources were examined and around 353 antimycobacterial compounds (Nat Prod Rep 2007; 24: 278-297) 7 have been previously reported. To develop drugs from these new sources, additional work is required for preclinical and clinical results. Since ancient times, different plant part extracts have been used as traditional medicines against diseases including tuberculosis. This knowledge may be useful in developing future powerful drugs. Plant natural products are again becoming important in this regard. In this review, we report 127 antimycobacterial compounds and their antimycobacterial activities. Of these, 27 compounds had a minimum inhibitory concentration of < 10 µg/mL. In some cases, the mechanism of activity has been determined. We hope that some of these compounds may eventually develop into effective new drugs against tuberculosis.

Comparative proteomics of human male and female tears by two-dimensional electrophoresis.

The proteomic profile of tear fluid is of fundamental interest in eye research. In this study we optimized the tear sample preparation method for two-dimensional (2D) analysis and determined the protein profile of tear fluid from healthy males and females. To find the most efficient method for tear sample preparation, four widely applied precipitation methods and ultrafiltration were compared. Of these, TCA precipitation & ultrafiltration resulted in efficient sample concentration and desalting. Use of a nonionic wetting agent, Tergitol NP7, in rehydration solution during isoelectric focusing improves protein separation in 2D gel electrophoresis considerably. Using this optimized method, tear protein profile was analyzed from healthy males and females. Of the thirty six tear proteins identified by LC-MS/MS, seven tear proteins were found to be significantly up regulated in the healthy female tear samples when compared to the male tear samples. These results indicate that the tear protein profile differs with respect to the sex. Mostly, the up regulated proteins are involved in the local immune defense; implying that there may be a sex difference in the ability to defend against infection at the anterior segment of the eyes of normal individuals.

Cloning of mce1 locus of Mycobacterium leprae in Mycobacterium smegmatis mc2 155 SMR5 and evaluation of expression of mce1 genes in M. smegmatis and M. leprae.

Plasmid pSET152 is a broad host range mobilizable vector which integrates into streptomyces chromosome utilizing att site and int function of slashed circleC31. Transformation of this plasmid into Mycobacterium smegmatis mc2 155 SMR5 gave stable transformants carrying the pSET152 as an integrated copy. Integration occurred at the cross over sequence 5'TTG disrupting the gatA gene (Glu-tRNA(Gln) amidotransferase subunitA), which is non-essential under conditions used. Recombinant pSET152 plasmids carrying mce1 locus of Mycobacterium leprae were used to construct M. smegmatis transformants carrying the mce1 locus in their chromosome. RT-PCR analysis revealed specific transcripts of M. leprae mce in M. smegmatis. The transcribed mRNA carried intergenic regions between genes of mce1 locus indicating that mce1 locus is an operon. Examination of M. leprae specific mRNA from lepromatous leprosy patient's biopsy showed that mce locus is transcribed as an operon in the pathogen also.