Evidence that type II 5'-deiodinase is not a selenoprotein.

Brain type II 5'-iodothyronine deiodinase and liver type I 5'-iodothyronine deiodinase activities are decreased in rats fed a Se(2+)-deficient diet suggesting that both enzymes are Se(2+)-dependent proteins. Since serum thyroxine (T4) concentrations are twice normal in the Se(2+)-deficient animals, it is unclear whether the Se2+ deficiency or the increased circulating T4 account for the decrease in the brain enzyme. In order to separate these two possibilities, the effects of Se2+ on 5'-deiodinase in glial cells (type II) and LLC-PK1 cells (type I) were examined. LLC-PK1 and glial cells were grown in serum-free defined medium containing 0, 1 pM, 10 nM, and 40 nM Se2+ for 3-5 days or in medium containing 75Se2+ for 24 h. Deiodinase isozymes were determined by measuring catalytic activity and by quantification of the BrAc[125I]T4 affinity-labeled substrate binding subunits. Se2+ deficiency was confirmed by measuring the activity of the selenoprotein, glutathione peroxidase. Se2+ caused a concentration-dependent increase in glutathione peroxidase activity in both cell types, as well as in the type I enzyme, but had no effect on the type II enzyme. LLC-PK1 cells contained multiple 75Se(2+)-labeled proteins including the 27-kDa substrate binding subunit of the type I 5'-deiodinase. Glial cells contained seven 75Se(2+)-labeled proteins ranging in size from 12 to 62 kDa, none of which corresponded to the type II substrate binding subunit. these data show that, unlike the type I enzyme, the type II enzyme does not contain a selenocysteine or selenomethionine, further emphasizing the differences between these two isozymes.     

The Effect of Cyclohexyladenosine on the Penischemic Increases of Hippocampal Glutamate and Glycine in the Rabbit

We investigated the ability of N6-cyclohexyladenosine (CHA), a potent and selective agonist of the adenosine A1 receptor, to attenuate elevations of levels of extracellular hippocampal glutamate and glycine that result from episodes of transient global cerebral ischemia (TGCI). A total of 30 New Zealand white rabbits were randomly assigned to receive 0 (n = 5), 0.1 (n = 8), 1.0 (n = 6), 10 (n = 6), or 100 (n = 5) microM CHA. The drug was dissolved in artificial CSF (vehicle) and administered via a microdialysis probe placed stereotactically into the dorsal hippocampus. A second microdialysis probe placed into the contralateral hippocampus of each animal was perfused with vehicle alone. Ten minutes of TGCI was induced by neck tourniquet inflation and deliberate hypotension from 0 to 10 min. Microdialysis samples were collected as follows: every 20 min preischemia (at -80, -60, -40, -20, and 0 min); every 5 min during ischemia and in the immediate reperfusion period (at 5, 10, 15, and 20 min); and every 20 min for the remainder of the reperfusion period (at 40, 60, and 80 min). Samples were then analyzed for their concentration of glutamate and glycine by HPLC. Following 10 min of ischemia, glutamate levels increased to a peak of 3.28 +/- 0.55 times baseline and returned to preischemic levels by 40 min, i.e., during reperfusion. Glycine concentrations increased to 5.41 +/- 0.91 times over baseline and remained elevated for the duration of the study.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationships between chromosome segregation, cell shape and temperature in Escherichia coli.

The partitioning of chromosomes into daughter cells during the division of Escherichia coli is non-random. As a result, the chromosome containing the older template DNA strand has a higher probability of segregating toward the old cell pole than toward the new cell pole. The numerical value of this probability is a function of the incubation temperature. It is shown here that a recent model for explaining the physiological basis for non-random chromosome segregation also explains the temperature dependence of the segregation process.     

Immunoregulation in cancer-bearing hosts. Down-regulation of gene expression and cytotoxic function in CD8+ T cells.

The causes of the decreased immune responsiveness in tumor-bearing hosts are incompletely understood. The impact of a decreased immune response in cancer patients on the clinical response in immunotherapy trials has not been evaluated. The present report demonstrates a marked decrease in the therapeutic efficacy of adoptively transferred T lymphocytes obtained from murine hosts bearing tumor for greater than 30 days [late tumor-bearing mice (TBM)] as compared with normal mice and mice bearing tumor for less than 21 days (early TBM). In vitro analysis of the functions of the T lymphocytes from late TBM showed an apparently normal proliferative response to anti-CD3 and IL-2 with adequate lymphokine production from CD4+ cells, but a significant decrease in the cytotoxic function of CD8+ cells. The decreased cytotoxicity was not because of cell-mediated suppression. The expression of granzyme B mRNA was significantly delayed and decreased in magnitude in CD8+ cells from late TBM. Culture supernatants from two unrelated tumor cell lines were able to inhibit the cytotoxic activity of normal CD8+ cells in vitro. The tumor-derived suppressive factor is not transforming growth factor-beta (TGF-beta), but it has not been further characterized. The data suggest that one potential mechanism responsible for immunologic defects in patients with large tumor burdens is a tumor-induced defect that compromises the function of CD8+ effector T cells.     

Dissimilar phloem loading in leaves with symplasmic or apoplasmic minor-vein configurations

Plant species were selected on the basis of abundant or no symplasmic continuity between sieveelement-companion-cell (SE-CC) complexes and adjacent cells in the minor veins. Symplasmic continuity and discontinuity are denoted, respectively, as symplasmic and apoplasmic minor-vein configurations. Discs of predarkened leaves from which the lower epidermis had been removed, were exposed to ¹⁴CO₂. After 2 h of subsequent incubation, phloem loading in control discs and discs treated with p-chloromercuribenzenesulfonic acid (PCMBS) was recorded by autoradiography. Phloem loading was strongly suppressed by PCMBS in minor veins with symplasmically isolated SE-CC complexes (Centaurea, Impatiens, Ligularia, Pelargonium, Pisum, Symphytum). No significant inhibition of phloem loading by PCMBS was observed in minor veins containing sieve elements with abundant symplasmic connections (Epilobium, Fuchsia, Hydrangea, Oenothera, Origanum, Stachys). Phloem loading in minor veins with both types of SE-CC complex (Acanthus) had apoplasmic features. The results provide strong evidence for coincidence between the mode of phloem loading and the minor-vein configuration. The widespread occurrence of a symplasmic mode of phloem loading is postulated.Abbreviations PCMBS p-chloromercuribenzenesulfonic acid - SE-CC complex sieve-element-companion-cell complex     

Tissue plasminogen activator has an O-linked fucose attached to threonine-61 in the epidermal growth factor domain

An unusual type of glycosylation has been observed for tissue plasminogen activator (t-PA). The monosaccharide fucose is glycosidically linked to threonine-61 in the epidermal growth factor region of t-PA. The presence of O-linked fucose was demonstrated by carbohydrate analysis and mass spectrometry of tryptic and chymotryptic peptides that contain this site. The susceptibility of the fucose residue to alpha-fucosidase indicated that it was in the alpha-anomeric configuration. Fucosylation of threonine-61 was observed in t-PA isolated from the Bowes melanoma cell line and from recombinant expression systems using Chinese hamster ovary or human embryonic kidney cells. Fucosylation of the homologous residue in prourokinase has also been reported recently. Our results indicate that this novel type of glycosylation may be common to the epidermal growth factor domains found in coagulation and fibrinolytic proteins and, therefore, suggest that the modification may have functional significance.     

Isolation of bovine Y-derived sequence: potential use in embryo sexing.

To obtain bovine Y-derived probes, we have constructed a bovine plasmid library enriched for Y-specific DNA sequences by the deletion enrichment method. The resulting clones were analyzed by hybridization to Southern blots of male and female genomic DNA. From 200 clones tested, two (BC1.2 and BC1.34) were entirely male specific, six gave a male-female differential hybridization pattern, and the remaining reacted similarly with male and female DNA. Interspecies somatic cell hybrid studies and chromosomal in situ hybridization confirmed that the BC1.2 sequence was derived from the Y chromosome. This 54-bp fragment is present at about 2000-2500 copies in the bovine male genome. No polymorphism was revealed with any of the restriction enzymes used, suggesting enzyme site conservation within blocks of repeats. Evolutionary study has shown that the BC1.2 sequence is conserved within Bos and Bison genera and remains male specific. The male specificity and repeated nature of the BC1.2 sequence have enabled us to use it as a molecular probe for sex determination on small numbers of cells by in situ hybridization.     

Roberts' — SC phocomelia syndrome with cytogenetic findings

Summary This paper reports the physical and cytogenetic findings in an eight-year-old severely mentally retarded female child with the following features: tetraphocomelia; weight, lenght, and head circumference below the third percentile; microcephaly with prominent frontal bones; hypertelorism; shallow orbits; prominent eyes; bilateral corneal opacities; micrognathia; hypoplastic alae nasi; small, low set ears; short neck; sparse silvery blond hair; severe flexion deformities of both knees and wrist joints; a cardiac murmur. Cytogenetic studies revealed premature centromere separation.