Cloning and expression of ovine placental lactogen

Ovine placental lactogen (oPL) is active in a wide range of GH and PRL assays, a property that it shares with human GH (hGH). In addition, oPL is one of a small number of hormones that bind the human GH receptor with high affinity. In order to compare the sequence of oPL to the sequences of other members of the GH family, full-length cDNA clones have been isolated. These clones predict that the full sequence of oPL contains 198 amino acids preceded by a 38 amino acid signal sequence. The mature oPL sequence includes six cysteine and two tryptophan residues and shows substantially more identity to bovine PL (67%) and oPL (49%) than to mouse (31%) or human (25%) PL or to oGH (28%) or (26%) hGH. Like the natural hormone, oPL expressed in mammalian tissue cells binds with high affinity to a soluble form of the recombinant hGH receptor. Thus, oPL binds to the human receptor in spite of having a sequence that is considerably divergent from hGH. Interestingly, the sequence of oPL differs from hGH at most of the amino acids recently found by mutagenesis studies to be important residues in the binding of hGH to the human receptor.     

Measurement of hydrogen peroxide in plasma and blood

Measurement of the oxygen metabolite hydrogen peroxide (H2O2) in biological fluids such as plasma could be of interest because it might indicate participation of toxic oxygen species in tissue injury. Recently several reports claimed to measure H2O2 using spectrophotometric and high pressure liquid chromatographic (HPLC) techniques that utilize oxidation of a substrate to a product by a peroxidase. In such a system it is crucial to perform two control experiments to verify whether the measured substance is H2O2. The specificity of the assay for H2O2 should be checked with catalase, and the degradation of H2O2 or inhibition of the assay system by the sample should be checked by determining the recovery of exogenously added H2O2. We performed both types of controls for HPLC and spectrophotometric determinations of H2O2 in plasma and blood. Our results indicate that contrary to previous reports in the literature the measured substance(s) in plasma or blood is not H2O2. Moreover, quantitative measurements of H2O2 in plasma or blood by HPLC was unreliable due to the irreversible binding of H2O2 to the column surface.     

Free radical reduction in the human epidermis

The human epidermis presents the first line of defense against invading free radicals. Therefore, the surface of the skin must be equipped to deal with both the penetration of ultra-violet light as well as the neutralization of reactive photochemical products such as superoxide anion radical, hydrogen peroxide and especially hydroxyl radicals. Consequently, the human epidermis contains a variety of anti-oxidants to reduce oxygen radicals and hydrogen peroxide. The photochemical production of hydroxyl radicals, from both extracellular and intracellular hydrogen peroxide, is of special significance to the integrity of cells in the human epidermis. Recently, both biochemical and clinical studies on the healthy human population, and on patients with pigmentation disorders, suggested a connection between free radical defense by plasma membrane associated thioredoxin reductase and melanin biosynthesis. This research provided the first evidence for a direct relationship between free radical concentration and pigmentation. Furthermore, this system has been shown to be regulated by both extracellular and intracellular calcium concentrations. Clinical studies show depigmentation disorders vitiligo and tyrosinase positive albinism (Hermansky-Pudlak syndrome) appear to have defective calcium uptake systems influencing both free radical defense and melanin biosynthesis.     

The viral envelope gene is involved in macrophage tropism of a human immunodeficiency virus type 1 strain isolated from brain tissue.

Human immunodeficiency virus type 1 (HIV-1) strains isolated from the central nervous system (CNS) may represent a subgroup that displays a host cell tropism different from those isolated from peripheral blood and lymph nodes. One CNS-derived isolate, HIV-1SF128A, which can be propagated efficiently in primary macrophage culture but not in any T-cell lines, was molecularly cloned and characterized. Recombinant viruses between HIV-1SF128A and the peripheral blood isolate HIV-1SF2 were generated in order to map the viral gene(s) responsible for the macrophage tropism. The env gene sequences of the two isolates are about 91.1% homologous, with variations scattered mainly in the hypervariable regions of gp120. Recombinant viruses that have acquired the HIV-1SF128A env gene display HIV-1SF128A tropism for macrophages. Furthermore, the gp120 variable domains, V1, V2, V4, and V5, the CD4-binding domain, and the gp41 fusion domain are not directly involved in determining macrophage tropism.     

HIV promoter activity in primary antigen-specific human T lymphocytes

Human retroviruses, such as the HIV, infects human T cells, and efficient HIV replication occurs primarily in activated T cells rather than resting cells. Increased HIV production is likely caused by the activation of the retroviral promoter, and the HIV promoter may be regulated by intracellular signals induced during immune stimulation. To examine the regulation of retroviral promoter activity in normal, Ag-specific primary T lymphoblasts, a heterogeneous population of primary human T cells was transfected with either the HIV promoter or a promoter from a different retrovirus, Rous sarcoma virus (RSV) by protoplast fusion technique. Transfected T cells responded normally to Ag or mitogen stimulation, and activation of these T cells increased both the HIV and RSV promoter activity. Promoter activity was assessed by using transient expression assays after the T cells were restimulated with Ag, mitogen, or IL-2. In situ hybridization of transfected human T cells showed that 68 to 95% of activated lymphocytes expressed CAT mRNA directed by HIV or RSV. Thus, protoplast transfection of primary T cells was efficient in that the majority of cells expressed CAT message. By deletion of different regions of the HIV promoter, the enhancer region was identified as necessary for effective HIV promoter activity. In addition these deletion studies identified a region that negatively affects HIV promoter activity in primary T cells. Cotransfection of the HIV promoter with the HIV transactivator protein, tat, increases HIV promoter activity in both resting and activated primary human T cells only when the tat target sequences were present.     

Subacute and Chronic In Vivo Lithium Treatment Inhibits Agonist- and Sodium Fluoride-Stimulated Inositol Phosphate Production in Rat Cortex

We have investigated the effects of in vivo lithium treatment on cerebral inositol phospholipid metabolism. Twice-daily treatment of rats with LiCl (3 mEq/kg) for 3 or 16 days resulted in a 25-40% reduction in agonist-stimulated inositol phosphate production, compared with NaCl-treated controls, in cortical slices prelabelled with [3H]inositol. A small effect was also seen with 5-hydroxytryptamine (5-HT) 24 h after a single dose of LiCl (10 mEq/kg). Dose-response curves to carbachol and 5-HT showed that lithium treatment reduced the maximal agonist response without altering the EC50 value. This inhibition was not affected by the concentration of LiCl in the assay buffer. Stimulation of inositol phosphate formation by 10 mM NaF in membranes prepared from cortex of 3-day lithium-treated rats was also inhibited, by 35% compared with NaCl-treated controls. Lithium treatment did not alter the kinetic profile of inositol polyphosphate formation in cortical slices stimulated with carbachol. Muscarinic cholinergic and 5-HT2 bindings were unaltered by lithium, as was cortical phospholipase C activity and isoproterenol-stimulated cyclic AMP formation. [3H]Inositol labelling of phosphatidylinositol 4,5-bisphosphate was significantly enhanced by 3-day lithium treatment. The results, therefore, indicate that subacute or chronic in vivo lithium treatment reduces agonist-stimulated inositol phospholipid metabolism in cerebral cortex; this persistent inhibition appears to be at the level of G-protein-phospholipase C coupling.     

Changes in the amounts of amino acids associated with nerve endings (synaptosomes) during synaptogenesis

Changes in the amounts of proteins and amino acids in synaptosomes and whole tissue from the olfactory bulb and cerebral cortex of rats were measured during the period 5-25 days postnatal. The amount of neurotransmitter type amino acids (such as GABA, glutamate and aspartate) associated with synaptosomes obtained from 1g of brain tissue increased dramatically with the age of the animals, whereas non-transmitter type amino acids (such as serine and glutamine) showed relatively little change. The results were in harmony with an earlier cessation of synaptogenesis in the olfactory bulb than in the cerebral cortex.     

Nucleotide sequence of the Rickettsia prowazekii ATP/ADP translocase-encoding gene

The Rickettsia prowazekii ATP/ADP translocase (Tlc) gene (tlc), previously cloned in Escherichia coli was localized to a 1.6-kb chromosomal fragment. Nucleotide sequence analysis of this fragment revealed an open reading frame of 1494 bp that could encode a hydrophobic protein of 497 amino acids (aa) with an M_r of 56 668. Analysis of the deduced aa sequence revealed that it contained twelve potential membrane-spanning regions. Comparisons between the deduced aa sequence of the R. prowazekii ATP/ADP Tlc and the sequences of mitochondrial (mt) Tic revealed no detectable homologies between the rickettsial and mt sequences. The major protein synthesized in E. coli minicells containing the rickettsial gene exhibited an M_r of approx. 34000.