Prolongation of insulin-induced activation of mitogen-activated protein kinases ERK 1/2 and phosphatidylinositol 3-kinase by vanadyl sulfate, a protein tyrosine phosphatase inhibitor

Vanadium salts such as vanadyl sulfate (VS), potent inhibitors of protein tyrosine phosphatases, have been shown to mimic, augment, and prolong insulin's action. However, the molecular mechanism of responses to these salts is not clear. In the present studies, we examined if VS-induced effects on insulin action are associated with enhancement or augmentation in the activation state of key components of the insulin signaling pathway. Treatment of insulin receptor-overexpressing cells with insulin or VS resulted in a time-dependent transient increase in phosphorylation and activation of extracellular signal-regulated kinases 1 and 2 (ERK 1/2) that peaked at about 5 min, then declined rapidly to about baseline within 30 min. However, when the cells were treated with VS before stimulation with insulin, sustained ERK 1/2 phosphorylation and activation were observed well beyond 60 min. VS treatment also prolonged the insulin-stimulated activation of phosphatidylinositol 3-kinase (PI3-K), which was associated with sustained interaction between insulin receptor substrate-1 (IRS-1) and the p(85 alpha) subunit of phosphatidylinositol 3-kinase (PI3-K) in response to insulin. These data indicate that prolongation of insulin-stimulated ERK 1/2 and PI3-K activation by VS is due to a more stable complex formation of IRS-1 with the p(85 alpha) subunit which may, in turn, be responsible for its ability to enhance and extend the biological effects of insulin.     

Phenotypic characteristics and population genetics of Enterococcus faecalis cultured from patients in Tehran during 2000-2001

Conventional bacteriology techniques were used to identify enterococci isolates cultured from patients at different hospitals in Tehran during 2000-2001. The identification was confirmed using species-specific PCR targeting the D-alanyl-D-alanine ligase gene. A total of 59 isolates of Enterococcus faecalis were identified. The rates of resistance to different antibiotics were in the following order: penicillin 84%, ciprofloxacin 42%, high-level gentamicin 30%, nitrofurantoin 14%, imipenem 4%, and chloramphenicol 2%. Resistance to ampicillin was found to be rare among the Iranian isolates of E. faecalis. Multi-locus enzyme electrophoresis was then used to analyze the strains. Forty-five electrophoretic types were obtained when 10 enzyme loci were screened. Although the collection of bacterial isolates was limited in time and location, considerable heterogeneity was found. Analysis of strains for linkage disequilibrium demonstrated that the studied population is not clonal, since the index of association was not significantly different from zero (Ia = 0.0296). Enterococcus faecalis isolates recovered from patients in Tehran were genetically diverse and seemed to possess a high potential for genetic recombinations, though none were resistant to vancomycin.     

Y-chromosomal DNA variation in Pakistan

Eighteen binary polymorphisms and 16 multiallelic, short-tandem-repeat (STR) loci from the nonrecombining portion of the human Y chromosome were typed in 718 male subjects belonging to 12 ethnic groups of Pakistan. These identified 11 stable haplogroups and 503 combination binary marker/STR haplotypes. Haplogroup frequencies were generally similar to those in neighboring geographical areas, and the Pakistani populations speaking a language isolate (the Burushos), a Dravidian language (the Brahui), or a Sino-Tibetan language (the Balti) resembled the Indo-European-speaking majority. Nevertheless, median-joining networks of haplotypes revealed considerable substructuring of Y variation within Pakistan, with many populations showing distinct clusters of haplotypes. These patterns can be accounted for by a common pool of Y lineages, with substantial isolation between populations and drift in the smaller ones. Few comparative genetic or historical data are available for most populations, but the results can be compared with oral traditions about origins. The Y data support the well-established origin of the Parsis in Iran, the suggested descent of the Hazaras from Genghis Khan's army, and the origin of the Negroid Makrani in Africa, but do not support traditions of Tibetan, Syrian, Greek, or Jewish origins for other populations.     

The shortened replicative life span of prohibitin mutants of yeast appears to be due to defective mitochondrial segregation in old mother cells

Prohibitin proteins have been implicated in cell proliferation, aging, respiratory chain assembly and the maintenance of mitochondrial integrity. The prohibitins of Saccharomyces cerevisiae, Phb1 and Phb2, have strong sequence similarity with their human counterparts prohibitin and BAP37, making yeast a good model organism in which to study prohibitin function. Both yeast and mammalian prohibitins form high-molecular-weight complexes (Phb1/2 or prohibitin/BAP37, respectively) in the inner mitochondrial membrane. Expression of prohibitins declines with senescence, both in mammalian fibroblasts and in yeast. With a total loss of prohibitins, the replicative (budding) life span of yeast is reduced, whilst the chronological life span (the survival of stationary cells over time) is relatively unaffected. This effect of prohibitin loss on the replicative life span is still apparent in the absence of an assembled respiratory chain. It also does not reflect the production of extrachromosomal ribosomal DNA circles (ERCs), a genetic instability thought to be a major cause of replicative senescence in yeast. Examination of cells containing a mitochondrially targeted green fluorescent protein indicates this shortened life span is a reflection of defective mitochondrial segregation from the mother to the daughter in the old mother cells of phb mutant strains. Old mother phb mutant cells display highly aberrant mitochondrial morphology and, frequently, a delayed segregation of mitochondria to the daughter. They often arrest growth with their last bud strongly attached and with the mitochondria adjacent to the septum between the mother and the daughter cell.     

Effects of simulated microgravity on thyroid carcinoma cells

We aimed to investigate whether simulated microgravity on thyroid carcinoma cells could help to perform in vitro cancer studies such as antitumor drug tests more reliable and to spare animal experiments. We cultured cancer cells at 0 g to enable formation of three-dimensional multicellular tumor spheroids (MCTS), which will resemble the originating tumors. Under microgravity human follicular cells (ML-1 cell line) keep floating with-out stirring so that initial cell-cell interactions required for spheroid formation will be induced by forces due to biochemical components actually expressed on surfaces of cells, whereas gravity related push- or shear events will not influence MCTS formation. Within 12 hours of clinorotation the monolayer turned spontaneously into MCTS with remarkable features: An increase of extracellular matrix proteins and TGF-beta 1. Thyroglobulin, ft3 and ft4 secretion were markedly reduced. These data are in agreement with the observation that astronauts show low thyroid hormone levels after spaceflight.     

Frequency of CCR5 Gene 32-bp deletion in Pakistani ethnic groups

CCR5 is a G-protein-coupled chemokine receptor that is used as a co-factor by macrophage-tropic (M-tropic) isolates of human immunodeficiency virus-1 (HIV-1) to gain entry into host cells. A 32-bp deletion in the CCR5 gene (CCR5-Delta32) leads to the production of an altered gene product that prevents HIV-1 from entering the host cell. This study was carried out to determine prevalence of CCR5-Delta32 allele frequency in a large Pakistani population sample (n = 821) representing 10 ethnic groups. No individual was homozygous for the mutant allele and the frequency of the CCR5-Delta32 allele ranged from 0.62% to 3.57%. The CCR5-Delta32 allele frequency was generally lower in populations from southern Pakistan. The overall frequency of the CCR5-Delta32 allele in Pakistan was 2.31%, which is much lower than that found in European populations and similar to that in the Middle East. This is consistent with the historical records and genetic data that indicate a close genetic affinity among these populations. This study demonstrates that the Pakistani population is highly susceptible to M-tropic isolates of HIV-1 and public health measures need to be enforced with urgency if Pakistan is to avoid an HIV epidemic.     

In situ atomic force microscopy of partially demineralized human dentin collagen fibrils

Dentin collagen fibrils were studied in situ by atomic force microscopy (AFM). New data on size distribution and the axial repeat distance of hydrated and dehydrated collagen type I fibrils are presented. Polished dentin disks from third molars were partially demineralized with citric acid, leaving proteins and the collagen matrix. At this stage collagen fibrils were not resolved by AFM, but after exposure to NaOCl(aq) for 100-240 s, and presumably due to the removal of noncollagenous proteins, individual collagen fibrils and the fibril network of dentin connected to the mineralized substrate were revealed. High-aspect-ratio silicon tips in tapping mode were used to image the soft fibril network. Hydrated fibrils showed three distinct groups of diameters: 100, 91, and 83 nm and a narrow distribution of the axial repeat distance at 67 nm. Dehydration resulted in a broad distribution of the fibril diameters between 75 and 105 nm and a division of the axial repeat distance into three groups at 67, 62, and 57 nm. Subfibrillar features (4 nm) were observed on hydrated and dehydrated fibrils. The gap depth between the thick and thin repeating segments of the fibrils varied from 3 to 7 nm. Phase mode revealed mineral particles on the transition from the gap to the overlap zone of the fibrils. This method appears to be a powerful tool for the analysis of fibrillar collagen structures in calcified tissues and may aid in understanding the differences in collagen affected by chemical treatments or by diseases.     

Containment of biogenic sulfide production in continuous up-flow packed-bed bioreactors with nitrate or nitrite

Produced water from the Coleville oil field in Saskatchewan, Canada was used to inoculate continuous up-flow packed-bed bioreactors. When 7.8 mM sulfate and 25 mM lactate were present in the in-flowing medium, H(2)S production (souring) by sulfate-reducing bacteria (SRB) was prevented by addition of 17.5 mM nitrate or 20 mM nitrite. Changing the sulfate or lactate concentration of the in-flowing medium indicated that the concentrations of nitrate or nitrite required for containment of souring decreased proportionally with a lowered concentration of the electron donor lactate, while the sulfate concentration of the medium had no effect. Microbial communities were dominated by SRB. Nitrate addition did not give rise to changes in community composition, indicating that lactate oxidation and H(2)S removal were caused by the combined action of SRB and nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB). Apparently the nitrite concentrations formed by these NR-SOB did not inhibit the SRB sufficiently to cause community shifts. In contrast, significant community shifts were observed upon direct addition of high concentrations (20 mM) of nitrite. Strains NO3A and NO2B, two newly isolated, nitrate-reducing bacteria (NRB) emerged as major community members. These were found to belong to the epsilon-division of the Proteobacteria, to be most closely related to Campylobacter lari, and to oxidize lactate with nitrate or nitrite as the electron acceptor. Thus the mechanism of microbial H(2)S removal in up-flow packed-bed bioreactors depended on whether nitrate (SRB/NR-SOB) or nitrite (SRB/NR-SOB as well as NRB) was used. However, the amount of nitrate or nitrite needed to completely remove H(2)S was dictated by the electron donor (lactate) concentration, irrespective of mechanism.     

An ultra-high throughput screening approach for an adenine transferase using fluorescence polarization

We have developed a novel assay for measuring the activity of an enzyme that transfers multiple adenine-containing groups to an acceptor protein. The assay is based on fluorescence polarization (FP) technology in a 1536-well plate format. In the assay, a long wavelength fluorescence tracer, Texas Red (Rhodamine), was covalently conjugated to adenine of the donor substrate through a C(6) spacer arm. As a result of the transfer of the adenine-containing moieties to the acceptor protein substrate, the rotational correlation time of the Texas Red conjugate increased, hence increasing the degree of fluorescence polarization. The pharmacological profile and kinetics of the enzyme measured according to the FP method were consistent with those determined previously by conventional analysis. We have successfully executed a 250,000-compound high throughput screening program based on the FP assay method. The quality and validity of the assay were verified by a variety of statistical analyses.     

580. FRITILLARIA KARELINII

Summary.  Fritillaria karelinii Fisch. ex D.Don ( Liliaceae ) is illustrated. Its relationships with other species and the position of subgenus Rhinopetalum are discussed. Instructions for its cultivation are given.