Hepatic glucose metabolism during intraduodenal glucose infusion: impact of infection

We previously reported that infection decreases hepatic glucose uptake when glucose is given as a constant peripheral glucose infusion (8 mg. kg(-1) x min(-1)). This impairment persisted despite greater hyperinsulinemia in the infected group. In a normal setting, hepatic glucose uptake can be further enhanced if glucose is given gastrointestinally. Thus the aim of this study was to determine whether hepatic glucose uptake is impaired during an infection when glucose is given gastrointestinally. Thirty-six hours before study, a sham (SH, n = 7) or Escherichia coli-containing (2 x 10(9) organisms/kg; INF; n = 7) fibrin clot was placed in the peritoneal cavity of chronically catheterized dogs. After the 36 h, a glucose bolus (150 mg/kg) followed by a continuous infusion (8 mg. kg(-1). min(-1)) of glucose was given intraduodenally to conscious dogs for 240 min. Tracer ([3-(3)H]glucose and [U-(14)C]glucose) and arterial-venous difference techniques were used to assess hepatic and intestinal glucose metabolism. Infection increased hepatic blood flow (35 +/- 5 vs. 47+/-3 ml x g(-1) x min(-1); SH vs. INF) and basal glucose rate of appearance (2.1+/-0.2 vs. 3.3+/-0.1 mg x kg(-1) x min(-1)). Arterial insulin concentrations increased similarly in SH and INF during the last hour of glucose infusion (38+/-8 vs. 46+/-20 microU/ml), and arterial glucagon concentrations fell (62+/-14 to 30+/-3 vs. 624+/-191 to 208+/-97 pg/ml). Net intestinal glucose absorption was decreased in INF, attenuating the increase in blood glucose caused by the glucose load. Despite this, net hepatic glucose uptake (1.6+/-0.8 vs. 2.4+/- 0.9 mg x kg(-1) x min(-1); SH vs. INF) and consequently tracer-determined glycogen synthesis (1.3+/-0.3 vs. 1.0+/-0.3 mg. kg(-1) x min(-1)) were similar between groups. In summary, infection impairs net glucose absorption, but not net hepatic glucose uptake or glycogen deposition, when glucose is given intraduodenally.     

Acute ascorbate supplementation alone or combined with arginase inhibition augments reflex cutaneous vasodilation in aged human skin

Full expression of reflex cutaneous vasodilation (VD) is dependent on nitric oxide (NO) and is attenuated in older humans. NO may be decreased by an age-related increase in reactive oxygen species or a decrease in L-arginine availability via upregulated arginase. The purpose of this study was to determine the effect of acute antioxidant supplementation alone and combined with arginase inhibition on reflex VD in aged skin. Eleven young (Y; 22 +/- 1 yr) and 10 older (O; 68 +/- 1 yr) human subjects were instrumented with four intradermal microdialysis (MD) fibers. MD sites were control (Co), NO synthase inhibited (NOS-I), L-ascorbate supplemented (Asc), and Asc + arginase-inhibited (Asc + A-I). After baseline measurements, subjects underwent whole body heating to increase oral temperature (T(or)) by 0.8 degrees C. Red blood cell flux was measured by using laser-Doppler flowmetry, and cutaneous vascular conductance (CVC) was calculated (CVC = flux/mean arterial pressure) and normalized to maximal (CVC(max)). VD during heating was attenuated in O (Y: 37 +/- 3 vs. O: 28 +/- 3% CVC(max); P < 0.05). NOS-I decreased VD in both groups compared with Co (Y: 20 +/- 4; O: 15 +/- 2% CVC(max); P < 0.05 vs. Co within group). Asc and Asc + A-I increased VD beyond Co in O (Asc: 35 +/- 4% CVC(max); Asc + A-I: 41 +/- 3% CVC(max); P < 0.001) but not in Y (Asc: 36 +/- 3% CVC(max); Asc + A-I: 40 +/- 5% CVC(max); P > 0.05). Combined Asc + A-I resulted in a greater increase in VD than Asc alone in O (P = 0.001). Acute Asc supplementation increased reflex VD in aged skin. Asc combined with arginase inhibition resulted in a further increase in VD above Asc alone, effectively restoring CVC to the level of young subjects.     

Mapping the anthrax protective antigen binding site on the lethal and edema factors.

Entry of anthrax edema factor (EF) and lethal factor (LF) into the cytosol of eukaryotic cells depends on their ability to translocate across the endosomal membrane in the presence of anthrax protective antigen (PA). Here we report attributes of the N-terminal domains of EF and LF (EF(N) and LF(N), respectively) that are critical for their initial interaction with PA. We found that deletion of the first 36 residues of LF(N) had no effect on its binding to PA or its ability to be translocated. To map the binding site for PA, we used the three-dimensional structure of LF and sequence similarity between EF and LF to select positions for mutagenesis. We identified seven sites in LF(N) (Asp-182, Asp-187, Leu-188, Tyr-223, His-229, Leu-235, and Tyr-236) where mutation to Ala produced significant binding defects, with H229A and Y236A almost completely eliminating binding. Homologous mutants of EF(N) displayed nearly identical defects. Cytotoxicity assays confirmed that the LF(N) mutations impact intoxication. The seven mutation-sensitive amino acids are clustered on the surface of LF and form a small convoluted patch with both hydrophobic and hydrophilic character. We propose that this patch constitutes the recognition site for PA.     

Improved Culture Flask for Obligate Anaerobes

An improved flask system for the growth of extremely oxygen-sensitive bacteria in liquid culture is described. The improvement described utilizes an all-glass, neoprene-stoppered flask designed for growth of 50- to 1,000-ml cultures of bacteria with continuous gassing.     

Developmental and adult acclimation effects of ambient temperature on temperature regulation of mice selected for high and low levels of nest-building

Summary Genetic and environmental components of adaptation to cold inMus musculus were assessed in a study of the effects of selective breeding for behavioral temperature regulation (indexed by high and low levels of nest-building), rearing mice from birth in the cold, and cold acclimation of adult animals, on thermoregulatory traits. Mice from the eleventh selected generation of a high-nesting line maintained higher resting metabolic rates and body temperatures, while at the same time consuming less food when compared with mice from the low-nesting line (Table 1). High-nesting mice were also more discriminating in their temperature preference when placed on a thermal gradient. Thus, common genetic loci must influence a variety of energy conservation measures important for survival in the cold, including insulative nest-building, metabolic efficiency, and optimum microhabitat selection.Rearing mice at 5°C from birth until 70 days of age resulted in permanent increases in nonshivering thermogenesis, weight of interscapular brown adipose tissue, and core body temperature when compared to mice raised at 22°C (Table 1). These greater heat production capacities were accompanied by consumption of more food. Cold acclimation of adults at 5°C for 3 weeks similarly increased measures of thermogenic capacity (nonshivering thermogenesis and interscapular brown adipose tissue) as well as food consumption, when compared to the effects of warm acclimation, but differed from the effects of cold-rearing in that while resting metabolic rates were elevated, no significant differences in body temperature were found (Table 1).Sex differences were also noted for most of the thermoregulatory measures, with the lighter females scoring higher on thermal preference, resting metabolic rate, nonshivering thermogenesis, brown fat, and food consumption.In general, these results suggest that a more precise partitioning of the genetic and environmental factors which influence thermoregulatory traits in mammals could eventually result in a better understanding of the differences which exist between acclimated and acclimatized animals.     

Influenza A-specific, HLA-A2.1-restricted cytotoxic T lymphocytes from HLA-A2.1 transgenic mice recognize fragments of the M1 protein.

Previous studies have indicated that in transgenic mice expressing human class I MHC molecules, it is difficult to demonstrate a significant CTL response to a viral Ag in the context of the transgenic molecule. In this paper, a procedure is reported for the isolation of influenza-specific murine CTL restricted by the human class I molecule HLA-A2.1. The principal specificity of such CTL is for a fragment of the influenza M1 protein that has been previously shown to be immunodominant for human HLA-A2.1-restricted CTL. CTL of this specificity were also established through the use of peptide-pulsed rather than virus-infected stimulators. The dependence of murine CTL recognition upon peptide length and HLA-A2 structure was established to be similar to that previously reported for human CTL. However, the fine specificity of CTL maintained on virus-infected stimulators was somewhat different from that of CTL maintained with M1 peptide. This suggests that differences in surface density or peptide structure between peptide-pulsed and virus-infected stimulators may result in the outgrowth of T cells with different receptor structures. The immunodominance of the M1 peptide determinant in both mice and humans suggests that species-specific differences in TCR structure, Ag-processing systems, and self-tolerance are of less importance than limitations on the ability of antigenic peptides to bind to appropriate class I molecules. These results thus establish the utility of the transgenic system for the identification of human class I MHC-restricted T cell epitopes.