MEDICAL PROGRESS: ISOTOPES IN CLINICAL AND EXPERIMENTAL MEDICINE

This is Part II of an article in two parts. Part I appeared in the July issue of California Medicine, and with it were the list of references for the entire article and the Table 1 referred to in the following text.     

Subpopulation analysis of human granular lymphocytes: associations with age, gender and cytotoxic activity

Levels of human blood granular lymphocyte subpopulations were enumerated in 105 healthy donors ranging in age from 0 to 79 years. Using double-label immunofluorescence, subpopulations of granular lymphocytes were enumerated as follows: Leu7+Leu2+, Leu7+Leu3+, Leu7+11-, Leu7+77+ and Leu7-Leu11+. The proportion of cells with granular lymphocyte morphology was determined by Giemsa staining. Natural killer (NK) cell activity against K-562 cells and lymphokine-activated killer activity against non-cultured melanoma cells were examined in parallel. Levels of total Leu7+ and Leu11+ cells increased with age (p = 0.0001) and were higher in males than females (p = 0.001). The total number of cells with granular lymphocyte morphology had an age-related increase (p = 0.001), but were not significantly higher in males than in females (p = 0.07). There was no selective increase in one granular lymphocyte subpopulation versus another since the Leu7+Leu11- (p = 0.0001), the Leu7+Leu11+ (p = 0.0001), the Leu7+Leu2+ (p = 0.0001) and the Leu7+Leu3+ (p = 0.0004) all had similar age-related increases. The one exception was the Leu7-Leu11+ (p = 0.1) granular lymphocyte subset which was low in the first decade of life but had reached maximum levels in the second decade. NK cell activity against K-562 cells was moderately increased with age (p = 0.06) with males and females exhibiting comparable activity. In contrast. lymphokine-activated killer cytotoxicity of non-cultured melanoma cells was similar in all age groups. NK cell activity was highly correlated with levels of morphologically defined granular lymphocytes (p = 0.005) and moderately with total Leu11+ cells (p = 0.06) but not with other subpopulations of granular lymphocytes.     

Perturbation of Critical Prolines in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Supports Conserved Gating Motions among Cys-loop Receptors

Gloeobacter violaceus ligand-gated ion channel (GLIC) has served as a valuable structural and functional model for the eukaryotic Cys-loop receptor superfamily. In Cys-loop and other receptors, we have previously demonstrated the crucial roles played by several conserved prolines. Here we explore the role of prolines in the gating transitions of GLIC. As conventional substitutions at some positions resulted in nonfunctional proteins, we used in vivo non-canonical amino acid mutagenesis to determine the specific structural requirements at these sites. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell electrophysiology was used to monitor channel activity. Pro-119 in the Cys-loop, Pro-198 and Pro-203 in the M1 helix, and Pro-299 in the M4 helix were sensitive to substitution, and distinct roles in receptor activity were revealed for each. In the context of the available structural data for GLIC, the behaviors of Pro-119, Pro-203, and Pro-299 mutants are consistent with earlier proline mutagenesis work. However, the Pro-198 site displays a unique phenotype that gives evidence of the importance of the region surrounding this residue for the correct functioning of GLIC.     

Unusual toxicity of riboflavin and flavin mononucleotide forCardiobacterium hominis

The chemical composition of a completely defined culture medium for the growth of strains ofCardiobacterium hominis is described.Riboflavin and flavin mononucleotide at low concentrations completely inhibit the growth response ofC. hominis. Only leucine, among a wide variety of substrates tested, prevents this toxicity over a range of riboflavin or flavin mononucleotide levels.     

Glycoside Hydrolases from a targeted Compost Metagenome, activity-screening and functional characterization

ABSTRACT: BACKGROUND: Metagenomics approaches provide access to environmental genetic diversity for biotechnology applications, enabling the discovery of new enzymes and pathways for numerous catalytic processes. Discovery of new glycoside hydrolases with improved biocatalytic properties for the efficient conversion of lignocellulosic material to biofuels is a critical challenge in the development of economically viable routes from biomass to fuels and chemicals. RESULTS: Twenty-two putative ORFs (open reading frames) were identified from a switchgrass-adapted compost community based on sequence homology to related gene families. These ORFs were expressed in E. coli and assayed for predicted activities. Seven of the ORFs were demonstrated to encode active enzymes, encompassing five classes of hemicellulases. Four enzymes were over expressed in vivo, purified to homogeneity and subjected to detailed biochemical characterization. Their pH optima ranged between 5.5 - 7.5 and they exhibit moderate thermostability up to ~60-70degreesC. CONCLUSIONS: Seven active enzymes were identified from this set of ORFs comprising five different hemicellulose activities. These enzymes have been shown to have useful properties, such as moderate thermal stability and broad pH optima, and may serve as the starting points for future protein engineering towards the goal of developing efficient enzyme cocktails for biomass degradation under diverse process conditions.     

Preliminary observations of the skeletal development in pre-flexion larvae of sablefish Anoplopoma fimbria

Ichthyological Research - Sablefish Anoplopoma fimbria support a lucrative fishery in the Gulf of Alaska, but their numbers have been declining despite a regulated fishery. Recruitment in A....