Compartment-ablation studies of GLUT4 distribution in adipocytes: evidence for multiple intracellular pools

The available data suggest that GLUT4 does populate the recycling endosomal system to some extent, but that a large proportion of the intracellular GLUT4 resides in a compartment that is devoid of transferrin receptors and may have properties more akin to specialized secretory vesicles. The study of the nature and biogenesis of this compartment will provide important insight into the mechanism by which insulin stimulates glucose transport. Further study of the role of the synaptobrevins in these distinct subcellular compartments will probably shed further light on the mechanism by which insulin stimulates GLUT4 translocation.     

OXIDATION OF HYDROCARBONS BY HEMIN1

A model system for cytochrome P-450 comprised of hemin, thiosalicylic acid, and solvent (acetone and water), oxidized cyclohemne and n-hexane at pH 2.7. Oxidation of cyclohexane yielded more cyclohexanol than cyclohexanone under mild reaction conditions, whereas the oxidation of n-hexane generated more 3- and 2-hexanones than 3- and 2-hemnols. Oxygen was a limiting factor in the reaction, and increased oxygen pressures increased the ratio of cyclohexanone to cyclohexanol formed. Lag time for product formation decreased in a linear fashion with an increase in temperature. In addition, cyclohexanone decreased relative to cyclohexanol as the temperature was increased. Antioxidants increased the lag time for product formation, but had little effect on the final quantity or ratios of the oxidative products generated. 1,3 Diphenylisobenzafiran had no effect on the lag time, whereas Tiron and catechol increased the lag time. In contrast to aliphatic thiols, aromatic thiols were capable of driving the oxidative reaction. The model hemin system was approximately 6% as active as natural cytochrome P-450. Hemin immobilized on glass beads effectively catalyzed the oxidation of cyclohexane.     

Stepwise transplantation of an active site loop between heat-labile enterotoxins LT-II and LT-I and characterization of the obtained hybrid toxins

Members of the cholera toxin family, including Escherichia coli heat- labile enterotoxins LT-I and LT-II, catalyze the covalent modification of intracellular proteins by transfer of ADP-ribose from NAD to a specific arginine of the target protein. The ADP-ribosylating activity of these toxins is located in the A-subunit, for which LT-I and LT-II share a 63% sequence identity. The flexible loop in LT-I, ranging from residue 47 to 56, closes over the active site cleft. Previous studies have shown that point mutations in this loop have dramatic effects on the activity of LT-I. Yet, in LT-II the sequence of the equivalent loop differs at four positions from LT-I. Therefore five mutants of the active site loop were created by a stepwise replacement of the loop sequence in LT-I with virtually all the corresponding residues in LT- II. Since we discovered that LT-II had no activity versus the artificial substrate diethylamino-benzylidine-aminoguanidine (DEABAG) while LT-I does, our active site mutants most likely probe the NAD binding, not the arginine binding region of the active site. The five hybrid toxins obtained (Q49A, F52N, V53T, Q49V/F52N and Q49V/F52N/V53T) show (i) great differences in holotoxin assembly efficiency; (ii) decreased cytotoxicity in Chinese hamster ovary cells; and (iii) increased in vitro enzymatic activity compared with wild type LT-I. Specifically, the three mutants containing the F52N substitution display a greater Vmax for NAD than wild type LT-I. The enzymatic activity of the V53T mutant is significantly higher than that of wild type LT-I. Apparently this subtle variation at position 53 is beneficial, in contrast to several other substitutions at position 53 which previously had been shown to be deleterious for activity. The most striking result of this study is that the active site loop of LT- I, despite great sensitivity for point mutations, can essentially be replaced by the active site loop of LT-II, yielding an active 'hybrid enzyme' as well as 'hybrid toxin'.      

Prevalence and numbers of Salmonella and Campylobacter spp. on raw,whole chickens in relation to sampling methods

Salmonella and Campylobacter continue to be major foodborne pathogens and raw poultry is considered to be an important source of these bacteria. In this study, the prevalence and numbers of Salmonella and Campylobacter spp. in relation to isolation/sampling methods were determined in 241 whole raw chickens purchased from retail outlets in England during the winters of 1998/1999 (101 chickens) and 1999/2000 (140 chickens). The packaging of the 140 chickens was also examined for the presence of the above pathogens. The prevalence and numbers of enterococci were examined in 21 of the 101 chickens. In total, Salmonella and Campylobacter spp. were present in 25% and 83% of the chickens, respectively. Salmonella were isolated from a sample representing both the inside and outside of the packaging in 19% of the chickens, while the corresponding figure for Campylobacter spp. was 56%. Both of these pathogens were isolated from the outside of the packaging in 6% of the chickens. Salmonella was more frequently isolated from samples containing chicken skin in comparison with those containing carcass-rinse fluid only. Two chickens (0.8%) were positive for Salmonella by direct enumeration methods with contamination levels of log10 3.8 and 4.5 colony forming units (cfu) per carcass, respectively. The most prevalent serotypes were S. Hadar, S. Enteritidis and S. Indiana and two different serotypes were identified in 5/20 salmonella-positive chickens. Resistance to at least one antibiotic was found in 70% of the strains, 46% were multiresistant (resistant to > or = four drugs) and 52% showed a lowered susceptibility to ciprofloxacin. The likelihood of isolating Campylobacter spp. from neck-skin, carcass-rinse or carcass-rinse plus whole skin samples was similar, Campylobacter spp. were found in higher levels in carcass-rinse or carcass-rinse plus whole skin samples than in neck-skin. The log10 cfu of Campylobacter spp. were 2.70-4.99 in 18% of the chickens and 5.00-6.99 in 20%. Campylobacter isolates (425) comprised Campylobacter jejuni (98%) and C. coli (2%) and 98 different sero/phagetypes of these two species were identified. Resistance to at least one antibiotic was found in 73% of the strains and 13% were multiresistant. Thirteen percent of the strains showed lowered susceptibility to ciprofloxacin, while 4.9% were resistant to erythromycin. Vancomycin-resistant enterococci (VRE), able to grow on agar containing 15 mg l(-1) vancomycin (VRE15), were present in 19 chickens. The log10 cfu of VRE15 was 2.90-3.99 in 10 chickens and between 4.00 and 4.99 in two chickens. The data presented here contribute to risk assessment and highlight the need to continue to emphasise the safe handling of raw retail poultry.     

The effects of psychological stress on leukocyte subset distribution in humans: evidence of immune activation

This study aimed to investigate which abilities are measured by the Austin Maze. One hundred and eight university students were administered a battery of eight neuropsychological tests including, the Austin Maze, the Tower of London, the Wisconsin Card Sort Test, Block Design, the Visual Spatial Learning Test, Digit Span Backwards, the Brown-Peterson Task and the Wide Range Achievement Test of Reading. Results indicated that visuospatial ability and memory both significantly contributed to performance on the Austin Maze, but differed in the degree to which they explained the performance depending on which measure of maze performance was employed. It appears that visuospatial ability is measured in early trials of the Austin Maze when individuals are orienting themselves to the path. In later trials individuals must call upon visuospatial memory to consolidate the details of the path. Executive function and working memory were not found to be significantly implicated in performance on the Austin Maze.     

Specificity of the SH2 domains of SHP-1 in the interaction with the immunoreceptor tyrosine-based inhibitory motif-bearing receptor gp49B

Inhibitory receptors on hemopoietic cells critically regulate cellular function. Despite their expression on a variety of cell types, these inhibitory receptors signal through a common mechanism involving tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM), which engages Src homology 2 (SH2) domain-containing cytoplasmic tyrosine or inositol phosphatases. In this study, we have investigated the proximal signal-transduction pathway of an ITIM-bearing receptor, gp49B, a member of a newly described family of murine NK and mast cell receptors. We demonstrate that the tyrosine residues within the ITIMs are phosphorylated and serve for the association and activation of the cytoplasmic tyrosine phosphatase SHP-1. Furthermore, we demonstrate a physiologic association between gp49B and SHP-1 by coimmunoprecipitation studies from NK cells. To address the mechanism of binding between gp49B and SHP-1, binding studies involving glutathione S-transferase SHP-1 mutants were performed. Utilizing the tandem SH2 domains of SHP-1, we show that either SH2 domain can interact with phosphorylated gp49B. Full-length SHP-1, with an inactivated amino SH2 domain, also retained gp49B binding. However, binding to gp49B was disrupted by inactivation of the carboxyl SH2 domain of full-length SHP-1, suggesting that in the presence of the phosphatase domain, the carboxyl SH2 domain is required for the recruitment of phosphorylated gp49B. Thus, gp49B signaling involves SHP-1, and this association is dependent on tyrosine phosphorylation of the gp49B ITIMs, and an intact SHP-1 carboxyl SH2 domain.     

Responses of neurons in the nucleus of the basal optic root to translational and rotational flowfields

Wild-type Arabidopsis plants, the starch-deficient mutant TL46, and the near-starchless mutant TL25 were evaluated by noninvasive in situ methods for their capacity for net CO2 assimilation, true rates of photosynthetic O2 evolution (determined from chlorophyll fluorescence measurements of photosystem II), partitioning of photosynthate into sucrose and starch, and plant growth. Compared with wild-type plants, the starch mutants showed reduced photosynthetic capacity, with the largest reduction occurring in mutant TL25 subjected to high light and increased CO2 partial pressure. The extent of stimulation of CO2 assimilation by increasing CO2 or by reducing O2 partial pressure was significantly less for the starch mutants than for wild-type plants. Under high light and moderate to high levels of CO2, the rates of CO2 assimilation and O2 evolution and the percentage inhibition of photosynthesis by low O2 were higher for the wild type than for the mutants. The relative rates of 14CO2 incorporation into starch under high light and high CO2 followed the patterns of photosynthetic capacity, with TL46 showing 31% to 40% of the starch-labeling rates of the wild type and TL25 showing less than 14% incorporation. Overall, there were significant correlations between the rates of starch synthesis and CO2 assimilation and between the rates of starch synthesis and cumulative leaf area. These results indicate that leaf starch plays an important role as a transient reserve, the synthesis of which can ameliorate any potential reduction in photosynthesis caused by feedback regulation.