Determination of cyclooxygenase products and prostaglandin metabolites using high-pressure liquid chromatography and radioimmunoassay.

A method is described for measurement of the cyclooxygenase products, thromboxane,prostacyclin, and prostaglandins (PG), and several prostaglandin metabolites. The procedure involves separation of the compounds by high-pressure liquid chromatography combined with identification and estimation by serologic analysis. These combined procedures have been used to identify and estimate five such products, PGE₂, PGE₁ PGF2α, PGF_1α, and 6-keto-PGF_1α, in the culture fluids of dog kidney cells stimulated by a tumor-promoting phorbol diester. The prostaglandin metabolites, 13,14-dihydro-15-keto-PGE₂, 13,14-dihydro-15-keto-PF2_2α, 13,14-dihydro-PGE₂, and 13,14-dihydro-PGF_2α, were not found in these culture fluids.     

Differential effects of sulfhydryl reagents on saxitoxin and tetrodotoxin block of voltage-dependent Na channels.

We have probed a cysteine residue that confers resistance to tetrodotoxin (TTX) block in heart Na channels, with membrane-impermeant, cysteine-specific, methanethiosulfonate (MTS) analogs. Covalent addition of a positively charged group to the cysteinyl sulfhydryl reduced pore conductance by 87%. The effect was selectively prevented by treatment with TTX, but not saxitoxin (STX). Addition of a negatively charged group selectively inhibited STX block without affecting TTX block. These results agree with models that place an exposed cysteinyl sulfhydryl in the TTX site adjacent to the mouth of the pore, but do not support the contention that STX and TTX are interchangeable. The surprising differences between the two toxins are consistent with the hypothesis that the toxin-receptor complex can assume different conformations when STX or TTX bound.     

Molecular Cloning of the Wild-Type and Mutant thyA Gene from Shigella flexneri Y

The thyA gene which codes for thymidylate synthase has been cloned and sequenced from the wild-type Shigella flexneri Y strain SH4 and a thyA mutant TSF21 after amplifying the gene by polymerase chain reaction (PCR). The nucleotide sequence revealed 98% homology to the E. coli K-12 thyA gene. The sequence of the wild-type thyA gene of Shigella flexneri Y was identical with that of the thyA mutant except that the residue T at position 345 was replaced by residue A in the thyA mutant. This change would cause a predicted amino acid substitution of leucine at position 44 in the polypeptide product of the wild type by glutamine in the mutant. Thus, Leu⁴⁴ may be critical in enzymatic activity of the thyA gene product thymidylate synthase.     

Improvement of terrestrial groundwater sampling method affects microbial community analysis

Groundwater sampling is a critical step in subsurface microbial ecology. Here, we compared two different sampling methods: commonly used disposable bailers (unimproved sampler) and an improved sampler, the latter of which was devised to minimize exposure to the aerobic atmosphere. Microbial community analysis using the 16S rRNA and methyl coenzyme-M reductase (mcrA) genes in the lignite seam groundwater revealed that the archaeal communities in samples obtained by the improved sampler were dominated by hydrogenotrophic methanogen Methanobacterium. These results suggested that the improved sampler would be more favorable for obtaining methanogenic archaeal community than the unimproved one, and that the sampling method affected the microbial community analysis in the investigated subterranean lignite seams.     

Complete Mitochondrial Genome of Great Frigatebird (Fregata minor): Phylogenetic Position and Gene Rearrangement

Biochemical Genetics - The complete mitogenome sequence of the Great Frigatebird, Fregata minor was sequenced for the first time in this study. The mitogenome (16,899 bp) comprises of 13...     

Frequency Distribution of F-Latencies (DFL) has Physiological Significance and Gives Distribution of Conduction Velocity (DCV) of Motor Nerve Fibres With Implications for Diagnosis

On electrical stimulation of a peripheral motor nerve, a delayed and reduced F-response is obtained, which is known to occur due to random backfiring of a few percent of the motor nerve fibres at the spinal end after antidromic conduction. F-latencies obtained from multiple stimulations vary in latency, size and shape because of this randomness. We hypothesised that, being a random process, recruitment of fibres for F-response would depend on the distribution of conduction velocity (DCV) for motor nerve fibres directly, and therefore, a frequency distribution of F-latencies (DFL) from such multiple F-responses would be an approximate mirror image of DCV, latency being inversely proportional to velocity. First, obtaining DFL from many human subjects, we have shown that this is a reproducible parameter for a nerve trunk of a subject, and hence reveals a new physiological phenomenon. DFL has a single peaked distribution, which is also expected for the DCV of a normal healthy motor nerve. To validate its hypothesised relationship to DCV further, DFLs were obtained from both median nerves of patients with unilateral carpal tunnel syndrome (CTS). The patterns of DFL from both sides remained almost the same except for a delay shift equal to that in between the two M-responses, which lends support to this hypothesis. DFL, and DCV as its suggested mirror image, appear to change systematically with certain known disorders such as cervical spondylosis, even at a subclinical stage, which needs further study. This also indicates that DFL may become a new and improved investigative diagnostic tool in neurophysiology.     

Differential expression of intestinal membrane transporters in cholera patients

Vibrio cholerae causes the cholera disease through secretion of cholera toxin (CT), resulting in severe diarrhoea by modulation of membrane transporters in the intestinal epithelium. Genes encoding membrane-spanning transporters identified as being differentially expressed during cholera disease in a microarray screening were studied by real-time PCR, immunohistochemistry and in a CaCo-2 cell model. Two amino acid transporters, SLC7A11 and SLC6A14, were upregulated in acute cholera patients compared to convalescence. Five other transporters were downregulated; aquaporin 10, SLC6A4, TRPM6, SLC23A1 and SLC30A4, which have specificity for water, serotonin (5-HT), magnesium, vitamin C and zinc, respectively. The majority of these changes appear to be attempts of the host to counteract the secretory response. Our results also support the concept that epithelial cells are involved in 5-HT signalling during acute cholera.     

Morphology, cytoskeletal organization, and myosin dynamics of mouse embryonic fibroblasts cultured on nanofibrillar surfaces

Growth of cells in tissue culture is generally performed on two-dimensional (2D) surfaces composed of polystyrene or glass. Recent work, however, has shown that such 2D cultures are incomplete and do not adequately represent the physical characteristics of native extracellular matrix (ECM)/basement membrane (BM), namely dimensionality, compliance, fibrillarity, and porosity. In the current study, a three-dimensional (3D) nanofibrillar surface composed of electrospun polyamide nanofibers was utilized to mimic the topology and physical structure of ECM/BM. Additional chemical cues were incorporated into the nanofibrillar matrix by coating the surfaces with fibronectin, collagen I, or laminin-1. Results from the current study show an enhanced response of primary mouse embryonic fibroblasts (MEFs) to culture on nanofibrillar surfaces with more dramatic changes in cell spreading and reorganization of the cytoskeleton than previously observed for established cell lines. In addition, the cells cultured on nanofibrillar and 2D surfaces exhibited differential responses to the specific ECM/BM coatings. The localization and activity of myosin II-B for MEFs cultured on nanofibers was also compared. A dynamic redistribution of myosin II-B was observed within membrane protrusions. This was previously described for cells associated with nanofibers composed of collagen I but not for cells attached to 2D surfaces coated with monomeric collagen. These results provide further evidence that nanofibrillar surfaces offer a significantly different environment for cells than 2D substrates.