Application of electrospray ionization mass spectrometry to study the hydrophobic interaction between the epsilon and theta subunits of DNA polymerase III

The interactions between the N-terminal domain of the epsilon (epsilon186) and theta subunits of DNA polymerase III of Escherichia coli were investigated using electrospray ionization mass spectrometry. The epsilon186-theta complex was stable in 9 M ammonium actetate (pH 8), suggesting that hydrophobic interactions have a predominant contribution to the stability of the complex. Addition of primary alkanols to epsilon186-theta in 0.1 M ammonium acetate (pH 8), led to dissociation of the complex, as observed in the mass spectrometer. The concentrations of methanol, ethanol, and 1-propanol required to dissociate 50% of the complex were 8.9 M, 4.8 M, and 1.7 M, respectively. Closer scrutiny of the effect of alkanols on epsilon186, theta, and epsilon186-theta showed that epsilon186 formed soluble aggregates prior to precipitation, and that the association of epsilon186 with theta stabilized epsilon186. In-source collision-induced dissociation experiments and other results suggested that the epsilon186-theta complex dissociated in the mass spectrometer, and that the stability (with respect to dissociation) of the complex in vacuo was dependent on the solution from which it was sampled.     

Structural and functional consequences of missense mutations in exon 5 of the lipoprotein lipase gene

Missense mutations in exon 5 of the LPL gene are the most common reported cause of LPL deficiency. Exon 5 is also the region with the strongest homology to pancreatic and hepatic lipase, and is conserved in LPL from different species. Mutant LPL proteins from post-heparin plasma from patients homozygous for missense mutations at amino acid positions 176, 188, 194, 205, and 207, and from COS cells transiently transfected with the corresponding cDNAs were quantified and characterized, in an attempt to determine which aspect of enzyme function was affected by each specific mutation. All but one of the mutant proteins were present, mainly as partially denatured LPL monomer, rendering further detailed assessment of their catalytic activity, affinity to heparin, and binding to lipoprotein particles difficult. However, the fresh unstable Gly(188)-->Glu LPL and the stable Ile(194)-->Thr LPL, although in native conformation, did not express lipase activity. It is proposed that many of the exon 5 mutant proteins are unable to achieve or maintain native dimer conformation, and that the Ile(194)-->Thr substitution interferes with access of lipid substrate to the catalytic pocket. These results stress the importance of conformational evaluation of mutant LPL. Absence of catalytic activity does not necessarily imply that the substituted amino acid plays a specific direct role in catalysis.     

Discordant protein and mRNA expression in lung adenocarcinomas

The relationship between gene expression measured at the mRNA level and the corresponding protein level is not well characterized in human cancer. In this study, we compared mRNA and protein expression for a cohort of genes in the same lung adenocarcinomas. The abundance of 165 protein spots representing 98 individual genes was analyzed in 76 lung adenocarcinomas and nine non-neoplastic lung tissues using two-dimensional polyacrylamide gel electrophoresis. Specific polypeptides were identified using matrix-assisted laser desorption/ionization mass spectrometry. For the same 85 samples, mRNA levels were determined using oligonucleotide microarrays, allowing a comparative analysis of mRNA and protein expression among the 165 protein spots. Twenty-eight of the 165 protein spots (17%) or 21 of 98 genes (21.4%) had a statistically significant correlation between protein and mRNA expression (r > 0.2445; p < 0.05); however, among all 165 proteins the correlation coefficient values (r) ranged from -0.467 to 0.442. Correlation coefficient values were not related to protein abundance. Further, no significant correlation between mRNA and protein expression was found (r = -0.025) if the average levels of mRNA or protein among all samples were applied across the 165 protein spots (98 genes). The mRNA/protein correlation coefficient also varied among proteins with multiple isoforms, indicating potentially separate isoform-specific mechanisms for the regulation of protein abundance. Among the 21 genes with a significant correlation between mRNA and protein, five genes differed significantly between stage I and stage III lung adenocarcinomas. Using a quantitative analysis of mRNA and protein expression within the same lung adenocarcinomas, we showed that only a subset of the proteins exhibited a significant correlation with mRNA abundance.     

Conception rates of dairy cows following early not-pregnant diagnosis by ultrasonography and subsequent treatments with shortened Ovsynch protocol

Our objective was to determine the feasibility of prompt reinsemination of dairy cows when diagnosed not pregnant 27-29 days after first-service timed AI (TAI). We assumed that a first-wave dominant follicle was present at that time that would ovulate in response to GnRH once precocious luteal regression was induced after administration of PGF(2alpha). Cows that had not been detected in estrus and reinseminated by Days 27-29 after a first-service TAI were diagnosed not pregnant by ultrasonography. Nonpregnant cows from three herds were assigned randomly to receive either no further treatment until reinsemination (controls; n=189); 25mg i.m. of PGF(2alpha) and then reinsemination according to detected estrus (81 of 108) or at 72-80h after PGF(2alpha) treatment (PGF) in the absence of estrus (27 of 108); or 25mg i.m. of PGF(2alpha) followed by 100 microg i.m. of GnRH 48h later (PGF+GnRH) and then reinsemination after detection of estrus (9 of 160) or at 16-20h after GnRH (151 of 160). Blood samples were collected at the time of the not-pregnant diagnosis and again 48h later. Concentrations of progesterone before treatment with PGF(2alpha) were elevated (<1ng/ml) in 61% of the cows when PGF(2alpha) was administered and 81% of the cows given PGF(2alpha) had low (<1ng/ml) concentrations of progesterone 48h after PGF(2alpha). Treated cows were re-inseminated earlier (P<0.01; 31+/-1days) after first-service TAI than controls (55+/-1days). Conception rates after treatment were not different among treatments: PGF (22%), PGF+GnRH (23%), and control (23%). Average intervals from calving to conception were 22-23 days less (P<0.001) in treated cows than in controls. We concluded that treating nonpregnant cows with PGF(2alpha) on Days 27-29 after insemination produced acceptable conception rates when inseminations were made after detected estrus or when TAI was used after GnRH treatment. Further, both treatments reduced days between first-service TAI and second inseminations, and days from calving to conception.     

Hydrocarbon bioremediation potential of an unimpacted Kuwaiti oil-field environment

Seasonal variations in the hydrocarbon-degrading potential of soil samples from an unimpacted site in the Kuwaiti Burgan oil field environment were studied under mesophilic conditions. Hydrocarbon-degrading microorganisms occurred but varied all-year-round, and their numbers ranged from 1.3 x 10(7) to 9.3 x 10(7) CFU g(-1) dry soil, while hydrocarbon-degrading fungi ranged from 3.0 x 10(4) - 3.8 x 10(5) CFU g(-1) dry soil, depending on the sampling period. These hydrocarbon-degraders also comprised variable but generally high proportions of the total aerobic heterotrophic organisms (2 to > 98%) for bacteria and lower levels (7-9%) for fungi. The crude oil-degrading capacity of the oil-degrading populations (bacteria and fungi) ranged from 80-95% of the hexane-extractable fractions. Differential inhibition studies carried out on soil samples showed that bacteria were the greater contributors to hydrocarbon degradation (79-92%) than fungi. Pure hydrocarbon substrates, hexadecane and phenanthrene, were degraded to near completion after a 28-day incubation by both the bacterial and fungal portions of the soil flora.