Molecular characterization of the sex steroid binding protein (SBP) of plasma. Re-examination of rabbit SBP and comparison with the human, macaque and baboon proteins

Physico-chemical characterization of the sex steroid-binding protein, SBP, of rabbit plasma reveals that it is a dimer of mol. wt 85,800 composed of similar subunits of mol. wt 43,000. These data confirm our original proposal for a dimeric structure. The protein contains 9% carbohydrate, comprised of mannose, galactose, N-acetylglucosamine and sialic acid. It is devoid of N-acetylgalactosamine and fucose. The protein binds one molecule of 5 alpha-dihydrotestosterone per dimer with a Kd of 0.89 nM (12 degrees C). Comparison with the human, monkey and baboon SBPs indicates that all these proteins have the same dimeric molecular organization and exhibit microheterogeneity in SDS-PAGE and isoelectricfocusing. Rabbit SBP, however, contains less carbohydrate and has a higher polypeptide molecular weight than all the other SBPs. Spectrophotometric data also indicate that some tryptophan residues are in a different chemical environment than those in other SBPs. The observed microheterogeneity in all four SBP species is due for the most part to variable glycosylation of the subunit and variability at the amino-terminal region of the subunit. Combination of these and other phenomena will generate a significant number of isomeric forms of the SBP subunit which will then interact stoichiometrically to yield active dimeric SBP molecules. These differ slightly from each other depending upon the charge and size of the subunit comprising the dimeric structure, and will result in the observed microheterogeneity of pure SBP preparations. Based on these results along with more recent amino acid sequence data, we conclude that all four SBPs are dimers composed of identical polypeptide chains.     

Mechanism of peptidoleukotriene-induced increases in pulmonary transvascular fluid filtration

We examined the effects of leukotrienes C4 (LTC4) and D4 (LTD4) (1 microgram) on the pulmonary vascular filtration coefficient, a measure of vessel wall conductivity to water, and the alterations in pulmonary vascular resistance (PVR) in isolated-perfused guinea pig lungs. We also assessed whether LTC4 and LTD4 increased the permeability to albumin in cultured monolayers of pulmonary artery endothelial cells. In Ringer-perfused and blood-perfused lungs, LTC4 resulted in increases in pulmonary arterial pressure (Ppa) and the pulmonary capillary pressure (Pcap) measured as the equilibration pressure after simultaneous pulmonary arterial and venous occlusions. Pulmonary venous resistance (Rv) increased to a greater extent than arterial resistance (Ra) in both Ringer-perfused and blood-perused lungs challenged with LTC4. The greater increase in PVR in blood-perfused lungs corresponded with a greater elevation of lung effluent thromboxane B2 (TxB2) concentration. The LTC4-stimulated increase in PVR was prevented by pretreatment with meclofenamate (10(-4) M). LTD4 also induced rapid increases in Ppa and Pcap in both Ringer-perfused and blood-perfused lungs; however, Ppa decreased before stabilizing at a pressure higher than base line. The increases in Rv with LTD4 were greater than Ra. The LTD4-stimulated increases in Ra and Rv also paralleled the elevation in TxB2 concentration. As with LTC4, the increases in Ppa, Pcap, PVR, and TxB2 concentration were greater in blood-perfused than in Ringer-perfused lungs. Pretreatment with meclofenamate reduced the magnitude of the initial increase in Ppa, but did not prevent the response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preliminary clinical observations with recombinant interleukin-2 in patients with AIDS or LAS

The toxicity of recombinant Interleukin-2 (IL-2) was studied in patients with acquired immunodeficiency syndrome (AIDS) or persistent lymphadenopathy syndrome (LAS). Increasing doses of the drug from 10(3) Units/m2 to 10(6) U/m2 were given as an intravenous bolus injection. At the high-dose levels some minor effects, such as fever up to 39.5 degrees C, chills, malaise or vomiting, were observed. The administration of 10(6) U/m2 as a 4-hour infusion showed identical results. No particular alterations of laboratory parameters were found. At the high-dose level the serum concentration of neopterin, which is released from macrophages after interferon gamma stimulation, was significantly (p less than 0.001) elevated above pretreatment levels. The clinical observation of daily infusions of 10(6)/m2 for 14 days revealed the same side effects. All patients developed lymphocytosis and eosinophilia. Two patients had suffered from severe diarrhoea for several weeks presumably due to cryptosporidiosis. In both cases diarrhoea ceased under the treatment with IL-2 and did not occur in the following two months.     

Comparison of arginase activity in red blood cells of lower mammals, primates, and man: evolution to high activity in primates.

Arginase activity in red blood cells (RBC) of various mammalian species including man was determined. In nonprimate species, the activity generally fell below the level of detectability of the assay: less than 1.0 mumol urea/g hemoglobin per hr. Activities in higher nonhuman primates were equal to or of the same order of magnitude as those in man (approximately 950 mumol/g hemoglobin per hr). RBC arginase deficiency with normal liver arginase activity has been shown to segregate as an autosomal codominant trait in Macaca fascicularis established and bred in captivity. This study confirms the presence of this polymorphism in wild populations trapped in several geographic areas and demonstrates the absence of immunologically cross-reactive material in the RBC of RBC arginase-deficient animals. These data when taken together suggest that the expression of arginase in RBC is the result of a regulatory alteration, has evolved under positive selective pressure, and is not an example of the vestigial persistence of an arcane function. The expression of arginase in the RBC results in a marked drop in the arginine content of these cells.     

Pulmonary microcirculatory responses to leukotrienes B4, C4 and D4 in sheep

The pulmonary microvascular responses to leukotrienes B₄, C₄ and D₄ (total dosage of 4 μg/kg i.v.) were examined in acutely-prepared halothane anesthetized and awake sheep prepared with lung lymp fistulas. In anesthetized as well as unanesthetized sheep, LTB₄ caused a marked and transient decrease in the circulating leukocyte count. Pulmonary transvascular protein clearance (pulmonary lymph flow x lymph-to-plasma protein concentration ratio) increased transiently in awake sheep, suggesting a small increase in pulmonary vascular permeability. The mean pulmonary artery pressure (P ) also increased. In the acutely-prepared sheep, the LTB₄-induced pulmonary hemodynamic and lymph flow responses were damped. Leukotriene C₄ increased P to a greater extent in awake sheep than in anesthetized sheep, but did not significantly affect the pulmonary lymph flow rate (Q̇_lym) and lmph-to-plasma protein concentration (L/P) ration in either group. LTD₄ increased P and Q̇_lymp in both acute and awake sheep; Q̇_lym increased without a significant change in the L/P ratio. The LTD₄-induced rise in P occurred in association with an increase in plasma thromboxane B₂ (Txb₂) cocentration. The relativity small increase in Q̇_lym with LTD₄ suggests that the increase in the transvascular fluid filtration rate is the result of a rise in the pulmonary capillary hydrostatic pressure. In conclusion, LTB₄ induces a marked neutropenia, pulmonary hypertension, and may transiently increase lung vascular permeability. Both LTC₄ and LTD₄ cause a similar degree of pulmonary hypertension in awake sheep, but had different lymph flow responses which may be due to pulmonary vasoconstriction at different sites, i.e. greather pre-capillary constriction with LTC₄ because Q̇_lym did not change and greater post-capillary constriction with LTD₄ because Q̇ increased with the same rise in P .     

Microvascular albumin permeability in isolated perfused lung: effects of EDTA

We examined the effects of decreases in perfusate concentrations of calcium and magnesium on the pulmonary vascular permeability in the isolated perfused rabbit lung. The albumin permeability-surface area product (PS) and the albumin reflection coefficient (sigma) were determined in the same lung using 125I- and 131I-labeled albumin tracers. Decreases in vascular Ca2+ and Mg2+ concentrations were induced by adding ethylenediaminetetraacetic acid (EDTA) to the perfusate. Decreases in the concentration of these cations resulted in an increase in the PS from a control value of 1.18 +/- 0.13 X 10(-3) to 7.69 +/- 0.75 X 10(-3) cm3 X min-1 X g wet lung wt-1 and a decrease in the sigma from 0.96 +/- 0.01 to 0.74 +/- 0.02. The decrease in sigma suggests an increase in the calculated equivalent pore radius from 44 to 63 A. The results indicate that Ca2+ and Mg2+ play a role in the maintenance of normal pulmonary vascular permeability to proteins.     

Isolation and Characterization of a Virus Inhibitor from Spinach (Spinacia oleracea L.)

A virus inhibiting protein (VI) was isolated from spinach (Spinacia oleracea L.). The VI inhibited infections of test plants with plus- and minus-strand RNA viruses. Inoculation of both local lesion and systemic hosts with TMV in the presence of varying amounts of the VI resulted in typical dose response curves for the number of local lesions or the amount of virus respectively. The lowest concentration of VI leading to a significant reduction in the number of local lesions was 0.06 μg/ml. The VI was found to inhibit local lesion formation only when applied within 2–3 h p.i. but still reduced the number of local lesions when applied up to 9 h prior to virus inoculation. The antiviral activity could be attributed to a protein of molecular weight 29,000 dalton with an isoelectric point of 10.3. Its activity was destroyed by heating for 30 min to 70°C. These characteristics resemble those of other virus inhibiting proteins described for members of the order Caryophyllales such as the Phytolacca inhibitor against which a serological relationship was obtained.     

Regulation by glucocorticoids of rat-liver phenylalanine hydroxylase in vivo

Phenylalanine hydroxylase, a liver-associated enzyme, is induced markedly by glucocorticoids in two permanent rat-hepatoma cell lines. In order to gain evidence that this phenomenon also occurs in vivo, we examined the effect of adrenalectomy and/or hormone supplementation on the levels of phenylalanine hydroxylase in the livers of adult rats: glucocorticoid administration increases, and adrenal ablation reduces, the activity of the hepatic enzyme, and the diminution occurring in the latter instance is entirely prevented by concurrent hormone replacement. These results thus corroborate earlier findings from a single experiment and are consistent with the hypothesis that adrenal corticosteroid hormones participate in modulating phenylalanine-hydroxylase levels within the diploid hepatocyte.     

Ineffective erythropoiesis in acute human P. falciparum malaria

An analysis of erythroblast cell kinetics utilizing quantitative 14C-autoradiography has been performed in five cases of acute Plasmodium falciparum malaria prior to and, in four patients, 3 or 6 days after the onset of antimalarial therapy. Associated with no or only moderate anemia were changes of erythroblast morphology, a considerable shift in the frequency of red and white blood cell precursors in the bone marrow, and a reduced rate of erythroblast proliferation. There was a marked loss of polychromatic erythroblasts, which was smaller but still detectable during the therapeutic phase. The results provide some quantitative data on the extent of "parenchymal damage" of bone marrow and stress the impact of ineffective erythropoiesis and reduced rate of erythropoietic proliferation on the emergence of anemia in Plasmodium falciparum malaria.     

Mode of interference of chlorosis-inducing herbicides with peroxisomal enzyme activities

In rye leaves ( Secale cereale L. cv. Petkus "Kustro") bleached in the presence of the chlorosis-inducing herbicides aminotriazole, haloxidine, San 6706 or difunone in white light of 54.2 W m^-2 (5000 lx), catalase activity was very low. In addition, the activities of glycolate oxidase and hydroxypyruvate reductase were strongly diminished in treatments with San 6706 and difunone. The lowering of the peroxisomal enzyme activities was observed in red, but not in blue light and did not occur after treatment with the non-bleaching pyridazinone derivative San 9785. The deficiencies of the peroxisomal enzymes did not appear to be involved in the initiation of the chlorosis. Instead they are probably produced as secondary consequences of the bleaching. Low peroxisomal enzyme activities were also obtained without herbicide treatment by growing the leaves in an atmosphere of 2% O₂ and 3% CO₂, but in this case were not accompanied by an increased sensitivity of the Chl to photooxidative bleaching. The peroxisomal enzymes reached as high activities as in untreated controls when the herbicide-treated leaves were grown at a low light intensity of 0.106 W m^-2 (10 lx). After transfer of herbicide-treated leaves grown under 0.106 W m^-2 to 306 W m^-2 (30 000 lx), catalase was strongly inactivated, even at 0°C. In treatments with San 6706 and difunone the increase of the activities of glycolate oxidase and hydroxypyruvate reductase was either stopped, remaining unchanged, or the enzymes were slightly inactivated after exposure to 306 W m^-2 (30 000 lx). The observations suggest that the inactivation of peroxisomal enzymes results from photooxidative events in the chloroplasts.