New lipoxygenase-inhibiting constituents from Calligonum polygonoides

Calligonolides A (1) and B (2), two new butanolides, and a new steroidal ester, 3, have been isolated from the whole plant of Calligonum polygonoides, together with four known compounds, tetracosan-4-olide, beta-sitosterol and its glucoside, and ursolic acid. Their structures were elucidated by spectroscopic and mass-spectrometric studies. Compounds 1-3 showed moderate inhibitory potential against lipoxygenase from soybean.     

Proteomic approach for identification and characterization of novel immunostimulatory proteins from soluble antigens of Leishmania donovani promastigotes

Visceral leishmaniasis (VL) caused by Leishmania donovani is a major parasitic disease prevalent in endemic regions of Bihar in India. In the absence of good chemotherapeutic options, there is a need to develop an effective vaccine against VL which should be dependent on the generation of a T helper type 1 (Th1) immune response. We have shown that soluble proteins from promastigote of a new clinical isolate of L. donovani (2001) ranging from 68 to 97.4 kDa (F2 fraction), induce Th1 responses in the peripheral blood mononuclear cells of cured Leishmania patients and hamsters and also showed significant prophylactic potential. To understand the nature of F2 proteins, it was further characterized using 2-DE, MALDI-TOF and MALDI-TOF/TOF-MS. In all, 63 spots were cut from a CBB stained gel for analysis and data was retrieved for 52 spots. A total of 33 proteins were identified including six hypothetical/unknown proteins. Major immunostimulatory proteins were identified as elongation factor-2, p45, heat shock protein (HSP)70, HSP83, aldolase, enolase, triosephosphate isomerase, protein disulfideisomerase and calreticulin. This study substantiates the usefulness of proteomics in characterizing a complex protein fraction (F2) map of soluble L. donovani promastigote antigen identified as Th1 stimulatory for its potential as vaccine targets against VL.     

Identification and characterization of segregation distortion loci along chromosome 5B in tetraploid wheat

Segregation distortion genes are widespread in plants and animals and function by their effect on competition among gametes for preferential fertilization. In this study, we evaluated the segregation distortion of molecular markers in multiple reciprocal backcross populations derived from unique cytogenetic stocks involving the durum cultivar Langdon (LDN) and wild emmer accessions that allowed us to study the effects of chromosome 5B in isolation. No segregation distortion of female gametes was observed, but three populations developed to analyze segregation of male gametes had genomic regions containing markers with skewed segregation ratios. One region of distortion was due to preferential transmission of LDN alleles over wild emmer alleles through male gametes. Another region required the presence of LDN 5B chromosomes in the female for preferential fertilization by male gametes harboring LDN alleles indicating that the corresponding genes in the female gametes can govern genes affecting segregation distortion of male gametes. A third region of distortion was the result of preferential transmission of wild emmer alleles over LDN alleles through male gametes. These results indicate the existence of different distorter/meiotic drive elements among different genotypes and show that distortion factors along wheat chromosome 5B differ in chromosomal location as well as underlying mechanisms.     

Reactions of peptidoglycan-mimetic beta-lactams with penicillin-binding proteins in vivo and in membranes.

The membrane-bound bacterial D-alanyl- D-alanine peptidases or penicillin-binding proteins (PBPs) catalyze the final transpeptidation reaction of bacterial cell wall biosynthesis and are the targets of beta-lactam antibiotics. Rather surprisingly, the substrate specificity of these enzymes is not well understood. In this paper, we present measurements of the reactivity of typical examples of these enzymes with peptidoglycan-mimetic beta-lactams under in vivo conditions. The minimum inhibitory concentrations of beta-lactams with Escherichia coli-specific side chains were determined against E. coli cells. Analogous measurements were made with Streptococcus pneumoniae R6. The reactivity of the relevant beta-lactams with E. coli PBPs in membrane preparations was also determined. The results show that under none of the above protocols were beta-lactams with peptidoglycan-mimetic side chains more reactive than generic analogues. This suggests that in vivo, as in vitro, these enzymes do not specifically recognize elements of peptidoglycan structure local to the reaction center. Substrate recognition must thus involve extended structure.     

Minimum population size,genetic diversity,and social structure of the Asian elephant in Cat Tien National Park and its adjoining areas,Vietnam, based on molecular genetic analyses

Vietnam’s elephant population that has suffered severe declines during the past three decades is now believed to number 60–80 individuals in the wild. Cat Tien National Park is thought to be one of the key areas for the recovery of Vietnam’s elephants. We carried out a molecular genetic study of elephants in Cat Tien National Park and its adjoining areas with the objectives of estimating minimum population size, assessing genetic diversity, and obtaining insights into social organization. We obtained a minimum population size of 11 elephants based on a combination of unique nuclear microsatellite genotypes and mitochondrial haplotypes. While mitochondrial diversity based on a 600-base pair segment was high in this small sample of individuals, the six microsatellite loci examined showed low diversity and the signature of a recent population bottleneck. Along with nuclear genetic depauperation of Cat Tien’s elephants, we also report disruption of normal social organization, with different matrilines having coalesced into a single social group because of anthropogenic disturbance. The results emphasize the critical condition of this elephant population and the need for urgent conservation measures if this population is to be saved.     

Substrate specificities and functional characterization of a thermo-tolerant uracil DNA glycosylase (UdgB) from Mycobacterium tuberculosis

Uracil DNA glycosylases (UDGs) excise uracil from DNA and initiate the base (uracil) excision repair pathway. Ung, a highly conserved protein, is the only UDG characterized so far in mycobacteria. Here, we show that Rv1259 from Mycobacterium tuberculosis codes for a double-stranded DNA (dsDNA) specific UDG (MtuUdgB). MtuUdgB is thermo-tolerant, contains Fe-S cluster and, in addition to uracil, it excises ethenocytosine and hypoxanthine from dsDNA. MtuUdgB is product inhibited by AP-site containing dsDNA but not by uracil. While MtuUdgB excises uracil present as a single-nucleotide bulge in dsDNA, it is insensitive to inhibition by dsDNA containing AP-site in the bulge. Interestingly, in the presence of cellular factors, the uracil excision activity of MtuUdgB is enhanced, and when introduced into E. coli (ung(-)), it rescues its mutator phenotype and prevents C to T mutations in DNA. Novel features of the mechanism of action of MtuUdgB and the physiological significance of the family 5 UDG in mycobacteria have been discussed.     

In-depth analysis of the adipocyte proteome by mass spectrometry and bioinformatics

Adipocytes are central players in energy metabolism and the obesity epidemic, yet their protein composition remains largely unexplored. We investigated the adipocyte proteome by combining high accuracy, high sensitivity protein identification technology with subcellular fractionation of nuclei, mitochondria, membrane, and cytosol of 3T3-L1 adipocytes. We identified 3,287 proteins while essentially eliminating false positives, making this one of the largest high confidence proteomes reported to date. Comprehensive bioinformatics analysis revealed that the adipocyte proteome, despite its specialized role, is very complex. Comparison with microarray data showed that the mRNA abundance of detected versus non-detected proteins differed by less than 2-fold and that proteomics covered as large a proportion of the insulin signaling pathway. We used the Endeavour gene prioritization algorithm to associate a number of factors with vesicle transport in response to insulin stimulation, a key function of adipocytes. Our data and analysis can serve as a model for cellular proteomics. The adipocyte proteome is available as supplemental material and from the Max-Planck Unified Proteome database.     

Comprehensive proteomic analysis of human cervical-vaginal fluid

Cervical-vaginal fluid (CVF) is a potential rich source of biomarkers for enhancing our understanding of human parturition and pathologic conditions affecting pregnancy. In this study, we performed a comprehensive survey of the CVF proteome in pregnancy utilizing multidimensional liquid chromatography (2D-LC) coupled with mass spectrometry and gel-electrophoresis-based protein separation and identification. In total, 150 unique proteins were identified using multiple protein identification algorithms. Metabolism (32%) and immune response-related (22%) proteins are the major functional categories represented in the CVF proteome. A comparison of the CVF, serum, and amniotic fluid proteomes showed that 77 proteins are unique to CVF, while 56 and 17 CVF proteins also occur in serum and amniotic fluid, respectively. This data set provides a foundation for evaluation of these proteins as potential CVF biomarkers for noninvasive diagnosis of pregnancy-related disorders.     

Reliable detection of deamidated peptides from lens crystallin proteins using changes in reversed-phase elution times and parent ion masses

Identifying deamidated peptides using low-resolution mass spectrometry is difficult because traditional database search programs cannot accurately detect modified peptides when the mass differences are only 0.984 Da. In this study, we utilized differential reversed-phase elution behavior of deamidated and corresponding unmodified peptide forms to significantly improve deamidation detection on a low-resolution LCQ ion trap instrument. We also improved the mass measurements of unmodified and deamidated peptide forms by averaging survey scans across each chromatogram peak. Tryptic digests of a series of normal (3-day old, 2-year old, 18-year old, 35-year old, and 70-year old) and cataractous (93-year old) human lens samples were used to produce large numbers of potentially deamidated peptides. The complex peptide mixtures were separated by strong cation exchange (SCX) chromatography followed by reversed-phase (RP) chromatography. Synthetic peptides were used to show that unmodified and deamidated peptides coeluted during the SCX separation and were completely resolved with the RP conditions used. Retention time shifts (RTS) and mass differences (DeltaM) of deamidated lens peptides and their corresponding unmodified forms were manually determined for the 70-year old lens sample. These values were used to assign correct or incorrect deamidation identifications from SEQUEST searches where deamidation was specified as a variable modification. Manual validation of SEQUEST identifications from synthetic peptides, 3-day old, and 70-year old samples had an overall 42% deamidation detection accuracy. Filtering SEQUEST identifications using RTS and DeltaM constraints resulted in >93% deamidation detection accuracy. An algorithm was developed to automate this method, and 72 Crystallin deamidation sites, 18 of which were not previously reported in human lens tissue, were detected.