Complex regulation of simple sugar transport in insulin-responsive cells.

Facilitated sugar entry into mammalian cells is catalysed by multiple isoforms of the glucose transporter and regulated by hormonal stimuli, nutritional status and oncogenesis. A large reserve of latent glucose transport capacity must be maintained by muscle and adipose cells that are sensitive to insulin, the primary activator of sugar uptake after feeding. Intracellular sequestration of sugar transporters accounts for a large part of this latent capacity, but new findings suggest that there is also reversible suppression of intrinsic catalytic activity of those glucose transporters residing at the cell surface. The mechanism of this suppression appears to be occlusion or disruption of the exofacial sugar-binding sites on the glucose-transporter proteins.     

Stereospecificity of leukotriene B4 and structure-function relationships for chemotaxis of human neutrophils

The chemotactic activity of leukotriene B4 (5S, 12R Dihydroxy 6, 14 cis, 8, 10 trans eicosatetraenoic acid) (LTB4) was examined by using a sensitive Boyden-chamber assay. The activity of LTB4 was compared to other biosynthetic stereoisomers: 5S, 12R Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (6-trans LTB4); 5S, 12S Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (12-epi-6-trans LTB4), 5S, 12S DiHETE; the metabolic product 20-Hydroxy LTB4 (20-OH LTB4); methylated LTB4 (Methyl-LTB4), and the related monoHETE 5-HETE and 12-HETE. The compounds were purified by several steps of reverse phase and straight phase HPLC. The LTB4 exhibits measurable chemotactic activity at 10(-9) M with maximal activity at 10(-7) M and an ED50 of 10(-8) M. The LTB4 isomers and monoHETE were less chemotactic than previously reported. The monoHETE (5-HETE and 12-HETE), the isomer 12-epi-6-trans LTB4, and 5S, 12S DiHETE fail to attract neutrophils at levels between 10(-6) and 10(-5) M. If these compounds are chemotactic, then activity is at least four orders of magnitude less than that of LTB4. The isomer 6-trans LTB4 at 10(-6) to 10(-5) M induced chemotaxis with an extrapolated ED50 value of 10(-5) M, indicating that a trans for cis change in configuration at position 6 reduces the chemotactic activity of LTB4 by 1000-fold. Conversely, the metabolic product 20-OH LTB4 is at least as active as the native compound LTB4. Methylation of the carboxyl group of LTB4 reduces its chemotactic activity by two orders of magnitude. These results indicate a high degree of stereospecificity for the LTB4 receptor with strict dependence on hydroxyl group, and triene configuration and considerable dependence on the carboxyl group. Modification at the aliphatic omega end of the LTB4 molecule has a minimal effect on function, suggesting that the hydrophobicity of this portion of the molecule is not important for optimal activity. Furthermore, we propose that metabolic products of LTB4 may be of greater importance than LTB4 as physiologic inflammatory mediators in vivo.     

Compensation of respiratory alkalosis induced after acclimation to simulated altitude

Conscious intact rats previously acclimated for 3 wk to barometric pressure of 370-380 Torr (3WHx) were made alkalotic for 3 h by a decrease in inspired O2 fraction from 0.10 to 0.075 at ambient barometric pressure (730-740 Torr). Controls were normoxic littermates (Nx) in which inspired O2 fraction was lowered from approximately 0.21 to 0.10 for 3 h. Arterial PCO2 decreased progressively and similarly in both groups (65-70% of control at 15 min). Initially, arterial pH increased less in 3WHx (0.09 +/- 0.004 vs. 0.15 +/- 0.008). As hypocapnia continued, delta[HCO3-]/delta pH (mmol.l-1.pH) became more negative in Nx, from -15.2 +/- 2.5 at 15 min to -37.0 +/- 2.9 at 3 h, indicating nonrespiratory compensation of alkalosis. In 3WHx, delta[HCO3-]/delta pH did not change during alkalosis. Cumulative renal excretion of base (mueq/100 g) during alkalosis increased by 73.2 +/- 11.1 in Nx and 25.4 +/- 7.3 in 3WHx. This difference was mainly due to a larger increase in HCO3- excretion in Nx. The data suggest that the smaller compensation of hypocapnic alkalosis in 3WHx is partly due to the smaller increase in renal base excretion. Because base availability limits renal base excretion, the smaller renal response of 3WHx may be secondary to the low plasma HCO3- concentration that accompanies altitude acclimation.     

Natural variants of the human papillomavirus type 16 E6 protein differ in their abilities to alter keratinocyte differentiation and to induce p53 degradation.

Three naturally occurring variant human papillomavirus type 16 (HPV-16) E6 proteins, which contained amino acid substitutions predominantly near the N terminus, exhibited significant differences in their abilities to abrogate keratinocyte differentiation in response to serum and calcium and to induce the degradation of p53 in vitro. One variant surpassed the reference E6 protein in its ability to abrogate keratinocyte differentiation responses, whereas another showed a reduction in this activity. Interestingly, the biological activities of the HPV-16 E6 proteins and their abilities to induce p53 degradation in vitro were directly correlated. These results demonstrate that naturally occurring variants of HPV-16 differ in biological and biochemical properties which might result in differences in pathogenicity.     

Population genetics and phylogenetics of DNA sequence variation at multiple loci within the Drosophila melanogaster species complex

Two regions of the genome, a 1-kbp portion of the zeste locus and a 1.1- kbp portion of the yolk protein 2 locus, were sequenced in six individuals from each of four species: Drosophila melanogaster, D. simulans, D. mauritiana, and D. sechellia. The species and strains were the same as those of a previous study of a 1.9-kbp region of the period locus. No evidence was found for recent balancing or directional selection or for the accumulation of selected differences between species. Yolk protein 2 has a high level of amino acid replacement variation and a low level of synonymous variation, while zeste has the opposite pattern. This contrast is consistent with information on gene function and patterns of codon bias. Polymorphism levels are consistent with a ranking of effective population sizes, from low to high, in the following order: D. sechellia, D. melanogaster, D.mauritiana, and D. simulans. The apparent species relationships are very similar to those suggested by the period locus study. In particular, D. simulans appears to be a large population that is still segregating variation that arose before the separation of D. mauritiana and D. sechellia. It is estimated that the separation of ancestral D. melanogaster from the other species occurred 2.5-3.4 Mya. The separations of D. sechellia and D. mauritiana from ancestral D. simulans appear to have occurred 0.58- 0.86 Mya, with D. mauritiana having diverged from ancestral D. simulans 0.1 Myr more recently than D. sechellia.      

Modifications to United States Environmental Protection Agency Methods 1622 and 1623 for Detection of Cryptosporidium Oocysts and Giardia Cysts in Water

Collaborative and in-house laboratory trials were conducted to evaluate Cryptosporidium oocyst and Giardia cyst recoveries from source and finished-water samples by utilizing the Filta-Max system and U.S. Environmental Protection Agency (EPA) methods 1622 and 1623. Collaborative trials with the Filta-Max system were conducted in accordance with manufacturer protocols for sample collection and processing. The mean oocyst recovery from seeded, filtered tap water was 48.4% ± 11.8%, while the mean cyst recovery was 57.1% ± 10.9%. Recovery percentages from raw source water samples ranged from 19.5 to 54.5% for oocysts and from 46.7 to 70.0% for cysts. When modifications were made in the elution and concentration steps to streamline the Filta-Max procedure, the mean percentages of recovery from filtered tap water were 40.2% ± 16.3% for oocysts and 49.4% ± 12.3% for cysts by the modified procedures, while matrix spike oocyst recovery percentages ranged from 2.1 to 36.5% and cyst recovery percentages ranged from 22.7 to 68.3%. Blinded matrix spike samples were analyzed quarterly as part of voluntary participation in the U.S. EPA protozoan performance evaluation program. A total of 15 blind samples were analyzed by using the Filta-Max system. The mean oocyst recovery percentages was 50.2% ± 13.8%, while the mean cyst recovery percentages was 41.2% ± 9.9%. As part of the quality assurance objectives of methods 1622 and 1623, reagent water samples were seeded with a predetermined number of Cryptosporidium oocysts and Giardia cysts. Mean recovery percentages of 45.4% ± 11.1% and 61.3% ± 3.8% were obtained for Cryptosporidium oocysts and Giardia cysts, respectively. These studies demonstrated that the Filta-Max system meets the acceptance criteria described in U.S. EPA methods 1622 and 1623.     

Discontinuation of cervical spine immobilisation in unconscious patients with trauma in intensive care units--telephone survey of practice in south and west region.

OBJECTIVE: To study how the cervical spine is assessed before discontinuation of cervical spine immobilisation in unconscious trauma patients in intensive care units. DESIGN: Telephone interview of consultants responsible for adult intensive care units. SETTING: All 25 intensive care units in the South and West region that admit victims of major trauma. MAIN OUTCOME MEASURES: The clinical and radiological basis on which the decision is made to stop cervical spine immobilisation in unconscious patients with trauma. RESULTS: In 19 units cervical spine immobilisation was stopped in unconscious patients on the basis of radiology alone, and six units combined radiology with clinical examination after the patient had regained consciousness. Sixteen units relied on a normal lateral radiological view of the cervical spine alone, five required a normal lateral and anteroposterior view, and four required a normal lateral, anteroposterior, and open mouth peg view. CONCLUSIONS: There are inconsistencies in the clinical and radiological approach to assessing the cervical spine in unconscious patients with trauma before the removal of immobilisation precautions. There is an overreliance on the lateral cervical spine view alone, which has been shown to be insensitive in this setting.     

Appearanc of cytotoxic cells within the bronchus after local infection with herpes simplex virus.

Cells from rabbit spleens, bronchial washings (BW) and bronchus-associated lymphoid tissues (BALT) were examined for their ability to lyse cells infected with herpes simplex virus (HSV). Specific lysis of HSV-infected cells was mediated by BW cells as early as 4 days after intratracheal infection of the rabbits with the virus whereas lysis by spleen cells and BALT cells was not detected until 7 or more days after infection. Lysis by spleen cells was initially detected 7 days after intraperitoneal injection of the virus but lysis by BW and BALT cells was not observed until 14 days after infection. Although spleen, BW, and BALT cells could lyse antibody-coated target cells, antibodies detectable by antibody-dependent cellular cytotoxicity could not be detected in bronchial washings until 7 or more days after infection. The data suggest that cells capable of direct cytotoxicity of virus-infected cells appear within the bronchus after local infection by the virus.