Envelope proteins of human T cell leukemia virus type I: characterization by antisera to synthetic peptides and identification of a natural epitope

Three peptides corresponding to selected regions of the env gene products of human T cell leukemia virus type I were synthesized by solid-phase Merrifield techniques. The sequence of peptide designated SP-65 was identical to the predicted C-terminal 12 residues of the transmembrane protein p21env, and peptide SP-74 was inferred from a region shown to be highly conserved among mammalian retroviruses. The third peptide, SP-70, was derived from a C-terminal region of the surface glycoprotein gp46. Antibodies to each peptide were raised in rabbits and were used to identify and further characterize the proteins coded by the env gene. Despite being present at very low levels in purified viral preparations, these proteins were chromatographed by reverse-phase high pressure liquid chromatography and were located by Western blot analysis of the column fractions. Anti-SP-70 recognized the surface glycoprotein (gp46) and also its C-terminal cleavage fragment (gp16). Anti-SP-65 and anti-SP-74 both reacted with the hydrophobic transmembrane protein (p21) and provided evidence that this protein does not undergo apparent C-terminal processing during viral maturation, unlike the trans-membrane protein of murine leukemia virus. As expected, anti-SP-74 also reacted with homologous proteins from other Type C and Type D viruses, confirming that peptide SP-74 corresponds to a broadly conserved region of retroviral transmembrane proteins. SP-70, which is predicted to be quite near the C terminus of the major surface glycoprotein, was also reactive with sera of HTLV-I-positive patients, indicating that this peptide corresponds to, or is part of, a native epitope recognized by the natural host.     

Acute toxoplasma infection of mice induces spleen NK cells that are cytotoxic for T. gondii in vitro

Murine spleen natural killer (NK) cells from normal and Toxoplasma-infected BALB/c mice were examined for their reactivity against RH strain tachyzoites in vitro. First, the effect of suspending medium on survival of extracellular RH tachyzoites was determined. Optimal parasite viability (by ethidium bromide-acridine orange staining) was observed when tachyzoites were incubated in phosphate-buffered saline (PBS) containing 10% horse serum (HS) for as long as 5 hr. In addition, parasite viability in PBS-HS correlated with subsequent infectivity, because freshly harvested and PBS-HS-incubated tachyzoites were equivalent in their ability to cause lethal infections in normal mice and to survive within normal mouse macrophages. Furthermore, viability and tumoricidal capacity of murine spleen NK cells incubated in PBS-HS was comparable to that of NK cells incubated in a standard cytotoxicity medium. Incubation of effector NK cells and target tachyzoites in PBS-HS in vitro revealed that spleen NK cells from 3-day Toxoplasma-infected mice had significantly greater cytotoxicity for extracellular RH tachyzoites than did control cells from uninfected mice. Moreover, Toxoplasma gondii-induced spleen NK cell toxoplasmacidal activity was significant at all effector to target cell ratios tested, and appeared to be mediated by direct contact between the host cell and the parasite. These in vitro results suggest that NK cells may be important in host defense against T. gondii.     

Monoclonal antibody that inhibits infection of HeLa and rhabdomyosarcoma cells by selected enteroviruses through receptor blockade.

BALB/c mice were immunized with HeLa cells, and their spleen cells were fused with myeloma cells to produce hybridomas. Initial screening of culture fluids from 800 fusion products in a cell protection assay against coxsackievirus B3 (CB3) and the CB3-RD virus variant yielded five presumptive monoclonal antibodies with three specificities: protection against CB3 on HeLa, protection against CB3-RD on rhabdomyosarcoma (RD) cells, and protection against both viruses on the respective cells. Only one of the monoclonal antibodies (with dual specificity) survived two subclonings and was studied in detail. The antibody was determined to have an immunoglobulin G2a isotype and protected cells by blockade of cellular receptors, since attachment of [35S]methionine-labeled CB3 was inhibited by greater than 90%. The monoclonal antibody protected HeLa cells against infection by CB1, CB3, CB5, echovirus 6, and coxsackievirus A21 and RD cells against CB1-RD, CB3-RD, and CB5-RD virus variants. The monoclonal antibody did not protect either cell type against 16 other immunotypes of picornaviruses. The monoclonal antibody produced only positive fluorescence on those cells which were protected against infection, and 125I-labeled antibody confirmed the specific binding to HeLa and RD cells. The results suggest that this monoclonal antibody possesses some of the receptor specificity of the group B coxsackieviruses.     

Retroperitoneal fibromatosis and acquired immunodeficiency syndrome in macaques: clinical and immunologic studies

A simian acquired immunodeficiency syndrome (SAIDS) associated with retroperitoneal fibromatosis (RF) has been observed in several species of macaque at the Washington Regional Primate Research Center. Clinical signs were recurrent diarrhea, weight loss, mesenteric lymphadenopathy, and opportunistic infections. Most affected macaques in the later stages of illness showed marked immunodeficiency. Response of peripheral blood mononuclear cells to mitogens was impaired significantly. There was sharply depressed primary and secondary antibody response to the T-cell dependent antigen, bacteriophage phi X174. Affected monkeys did not switch from IgM to IgG antibody following a secondary immunization, as did normal macaques. Twenty-four (67%) of 36 affected animals with progressive RF or deteriorated stages of illness had hypoproteinemia and hypoalbuminemia. Quantitative serum immunoglobulins of 23 cases showed that eight (35%) had hypogammaglobulinemia, six (26%) had hypergammaglobulinemia, and the remainder (39%) were within the normal range. Opportunistic infections were predominantly bacterial pathogens. Type D retrovirus appeared to be closely associated with RF-affected macaques (12/12 or 100%). The case fatality rate (including animals sacrificed after prolonged illness) was 98%. The leading cause of death was due directly to RF lesions in 43%, to enterocolitis in 36%, septicemia in 12%, amyloidosis in 5%, and malignant lymphoma (2%). Clinical, immunologic and pathologic changes reveal an acquired immunodeficiency syndrome that has many similarities to human AIDS. SAIDS and RF may be a useful model for studying human AIDS.     

Allometry in the placoderm Bothriolepis canadensis and its significance to antiarch evolution

A set of 18 measurements of the dermal armour of Bothriolepis canadensis Whiteaves (Placodermi, Anti-archa) is analysed with respect to allometric growth patterns. The strongest allometric patterns were found for the orbital fenestra and premedian plate of the head-shield. and the anterior median dorsal plate of the trunk-shield. These are all areas of the greatest importance in antiarch phylogeny and imply a role for ontogenetic effects such as paedomorphosis in the evolution of antiarchs. It is suggested that this is partly a result of the severe constraints on growth in a closed box such as the armour of placoderms, and may be generally true of such arrangements.     

Sapwood area ofPinus contorta stands as a function of mean size and density

Summary An indirect test of the relationship between leaf area and the combination of mean size and density is made in stands of lodgepole pine (Pinus contorta Dougl.). Total sapwood cross-sectional area of these stands is a function of the product of density and mean diameter raised to an exponent of about 1.6. Results from other studies, representing four species, suggest that this relationship between sapwood area and the combination of mean size and density may be general. The implications of the relationship are discussed in the context of evapotranspiration, competition and self-thinning.Utah Agricultural Experiment Station Journal Paper No. 3072     

Expression and secretion of biologically active human atrial natriuretic peptide in Saccharomyces cerevisiae

A hybrid gene was constructed containing a fusion between the DNA sequences encoding the secretory precursor of the yeast mating pheromone alpha-factor and a synthetic sequence encoding a biologically active 24-amino acid carboxyl-terminal portion of the human atrial natriuretic peptide (hANP) precursor. Transformation of Saccharomyces cerevisiae with the hybrid gene resulted in the yeast cells secreting biologically active hANP into the extracellular medium. The secreted hANP was purified and found to be accurately processed at the junction in the chimeric alpha-factor/hANP protein, producing the desired mature hANP amino terminus. The secreted product was also folded correctly with respect to the single disulfide bond. However, the carboxyl terminus of the secreted hANP material was heterogeneous such that the major form lacked the last two amino acids of the peptide while the minor form was the full length material. The observed processing at the carboxyl terminus of the secreted hANP may reflect a normal processing event involved in alpha-factor peptide maturation.     

Stimulation of facilitated [3H]uridine transport by thyroid hormone in GH1 cells. Evidence for regulation by the thyroid hormone nuclear receptor

We have previously shown that 3,5,3'-triiodo-L-thyronine (L-T3) stimulates cell growth and a 4- to 8-fold increase in growth hormone mRNA in GH1 cells. These effects appear to be mediated by a thyroid hormone nuclear receptor with an equilibrium dissociation constant for L-T3 of 0.2 nM and an abundance of about 10,000 receptors per cell nucleus. In this report, we show that L-T3 exerts a pleiotypic effect on GH1 cells to rapidly (within 2 h) stimulate [3H]uridine uptake to a maximal value of 2.5- to 3-fold after 24 h. This results from an increase in the number of functional uridine "transport sites" as shown by studies documenting an increase in the apparent Vmax with no change in the Km, 17 microM. Although the labeling of the cellular uridine pool and pools of all phosphorylated uridine derivatives was increased by L-T3, there was no change in the relative amounts of the individual pools in cells incubated with or without hormone. The intracellular concentration of [3H]uridine was estimated to be similar to that of the medium, suggesting that facilitated transport mediates [3H]uridine uptake. That this increase in [3H]uridine transport was nuclear receptor-mediated is supported by the excellent correspondence of the L-T3 dose-response curve for [3H]uridine uptake and that for L-T3 binding to receptor. Finally, inhibition of protein synthesis by cycloheximide and RNA synthesis by actinomycin D demonstrated that the L-T3 effect required continuing protein and RNA synthesis. These results are consistent with an effect of the L-T3-nuclear receptor complex to increase uridine uptake in GH1 cells by altering the expression of gene(s) essential for the transport process.     

Isolation of a human stromal cell strain secreting hemopoietic growth factors

A diploid fibroblastoid cell strain, termed "ST-1," has been established from a long-term liquid culture of human fetal liver cells. ST-1 cells are nonphagocytic, nonspecific esterase negative and do not possess factor VIII-related antigen but stain with antibodies specific for fibronectin and type I collagen. The ST-1 cells produce nondialyzable hemopoietic growth factors capable of stimulating the development of erythroid bursts, mixed granulocyte-macrophage colonies, pure granulocyte colonies, and pure macrophage colonies. These factors are active on both human fetal liver and human adult bone marrow progenitors. When liquid cultures of human fetal liver hemopoietic progenitors are established with a preformed monolayer of ST-1 cells, the yields of nonadherent cells, erythroid progenitors, and myeloid progenitors are greatly increased. These studies demonstrate that the fibroblastoid ST-1 cells support hemopoiesis in vitro and may be a critical element in the stromal microenviroment in vivo.     

豚鼠冰水游泳应激后血浆、肾上腺髓质和脑内组织的亮—脑啡肽含量变化

豚鼠冰水游泳应激3min后,用放射免疫法测定血浆、肾上腺髓质和脑内三个脑区组织的亮—脑啡肽(Leu-enkephalin,LEK,)含量和血浆的血管紧张肽Ⅱ(Angiotensin Ⅱ,ATⅡ)浓度变化。结果表明:在冰水中游泳应激组豚鼠的血浆、肾上腺髓质、下丘脑和纹状体组织的LEK含量和血浆的ATⅡ浓度,均较对照组豚鼠明显升高(P值均<0.01)。提示:豚鼠在冰水中紧张、剧烈游泳后,可能激活了中枢神经和周围组织内的脑啡肽能神经元和血管紧张肽原酶—血管紧张肽Ⅱ系统,从而促进LEK和ATⅡ的释放增加。