Nonuniform size distribution of nascent globin peptides, evidence for pause localization sites, and a cotranslational protein-folding model

Examination of nascent globin peptides accumulatingin vitro during globin synthesis in rabbit reticulocyte lysates was carried out. A view was supported that nonrandom distribution of codons with different usage frequencies in mRNA may determine the messenger's translation kinetics. Regions of reduced translation of - and -globin polypeptide chains were localized, and the cotranslational protein-folding model suggested previously was substantiated. An active conjunction of synthesis and folding of proteins was proposed as one of the main destinations of a translation nonuniformity.     

Cirrhosis with atypia. A potential pitfall in the interpretation of liver aspirates

Needle aspirations of the liver yielding highly atypical hepatocytes present a diagnostic challenge, with the differential diagnosis lying between hepatocellular carcinoma and benign reactive atypia. A case of a healing liver abscess in a patient with cirrhosis, mistakenly diagnosed as an hepatocellular carcinoma, is presented. Criteria for the avoidance of false-positive diagnoses of hepatocellular carcinoma on needle aspirates are presented, and the concept of "liver cell dysplasia" as a cytodiagnostic entity is discussed.     

Design and synthesis of metabolically stable atrial natriuretic factor analogs. Amino- and carboxy-terminal stabilization

Two analogs of rat atrial natriuretic factor, rANF7-28-NH2 and [Mpr7,Ala20,D-Arg27]rANF7-27-NH2, were prepared by the solid-phase method. These peptides had 2-fold and 7-fold less affinity, respectively, than rANF1-28 in binding to membranes prepared from cultured aortic smooth muscle cells, and both peptides were 5-fold less potent than rANF1-28 in relaxing serotonin-contracted rabbit aortic rings. rANF7-28-NH2 was rapidly degraded by rat kidney homogenates but [Mpr7,Ala20,D-Arg27]rANF7-27-NH2 had enhanced stability against rat kidney homogenate degradation. However, this in vitro stability did not translate into an extended duration of action in vivo.     

Calcium dependence during single-cycle catalysis of the sarcoplasmic reticulum ATPase

We have investigated the kinetic and thermodynamic properties of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum under conditions that result in a single transport cycle. Simultaneous addition of ATP and EGTA to sarcoplasmic reticulum vesicles, preincubated with calcium, resulted in a transient of intermediate species. In the presence of saturating Ca2+ levels, total E-P species reached a maximum of 2.3 nmol/mg at 100 ms, followed by a monoexponential decay with kobs = 3.6 s-1. The data are interpreted in terms of Ca2+ sequestration, either by occlusion as Ca2+ in the phosphorylated enzyme or chelation by EGTA. Maximum Ca2+ uptake was 8.3 nmol/mg with the release of 4.4 nmol/mg Pi. The ratio of Ca2+ uptake to Pi release approached 1.9 over a wide [Ca2+] range. Equilibrium Ca2+ binding, in the absence of ATP, showed a K0.5 of 0.88 microM with a Hill coefficient of 1.9. The Ca2+ concentration dependence of Ca2+ uptake during single-cycle catalysis showed a 10-fold enhanced affinity (K0.5 = 0.06 microM) and was noncooperative (nH = 0.9). Quench with excess EGTA (greater than 2 mM) decreased Ca2+ uptake to 1 nmol/mg, indicating an "off" rate of Ca2+ from high affinity sites that exceeds 100 s-1. The ATP concentration dependence for a single-cycle catalysis showed an apparent K0.5 of 1.1 microM, similar to that for ATP equilibrium binding. It is proposed that enzyme phosphorylation proceeds only following binding of a second calcium ion to externally oriented sites whose intrinsic affinity is in the same range as the calcium dependence of a single-cycle turnover.     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. IV. Recognition and activation by cloned glycoproteins gB and gD

Results of studies in mice and clinical observations in man indicate that T cell-mediated immunity is important in resistance to herpes simplex virus (HSV) infections. This study was undertaken to elucidate the viral antigen specificity of human HSV-immune T cells. Purified HSV-1 glycoproteins gB-1 and gD-1, cloned and expressed in mammalian cells, were found to stimulate proliferation of, and interleukin 2 (IL 2) production by, peripheral blood lymphocytes (PBL) of HSV seropositive individuals, indicating the presence of memory T cells to gB-1 and gD-1 in individuals with serologic evidence of immunity to HSV. Second, T cell clones, generated by stimulation of PBL with HSV-1, were found to recognize gB-1 or gD-1, as evidenced by the ability of the clones to proliferate in response to stimulation with gB-1 or gD-1 in the absence of exogenous IL 2. Third, HSV-specific T cell clones, lytic for HSV-1 or both HSV-1- and HSV-2-infected autologous target cells, were generated after stimulation of PBL with purified cloned gB-1 or gD-1. Our findings, that human HSV-specific T cells can recognize and be activated by HSV subunit antigens gB-1 or gD-1, imply that these glycoproteins play a role in human T cell-mediated immunity to HSV and support the contention that a gB-1 or gD-1 subunit vaccine may be protective in man.     

Binding of C-reactive protein to nucleated cells leads to complement activation without cytolysis

C-reactive protein (CRP) is an acute-phase reactant that is found bound to cells at sites of inflammation. We have passively sensitized HEp-2 cells for CRP binding and examined the effect of this treatment on complement activation and cell lysis. When cells were treated with protamine sulfate and CRP and were incubated with normal human serum in a 4-hr 51Cr-release assay, no significant lysis was noted. In contrast, HEp-2 cells treated with antibody and normal human serum were lysed. The consumption of complement components in normal human serum after incubation with cells treated with protamine and CRP was measured by hemolytic assays. CRP-treated cells consumed over 80% of C1, C4, and C2 and about 40% of C3 present. No significant consumption of C5 through C9 components was observed. Cells treated with antibody and complement showed consumption of C1 through C9. Cells were also sensitized for CRP binding by using diazophenylphosphocholine. This treatment also led to CRP binding and activation of the early classical pathway (C1, C4, C2, and to a lesser extent C3). The components of the membrane attack complex (C5 through C9) were not activated. Both a mouse monoclonal IgM and a human IgG antibody to phosphocholine activated the entire classical pathway. These results indicate that CRP activation of the classical complement pathway is restricted to the early part of the pathway. In the absence of activation of the membrane attack complex, complement-mediated cell lysis cannot occur.     

Absorbance and fluorescence properties of 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate bound to coupled and uncoupled Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum

Preincubation of skeletal muscle sarcoplasmic reticulum vesicles in the presence of the calcium chelator, [ethylenebis(oxyethylenenitrilo)tetraacetic acid] (EGTA), irreversibly uncouples calcium transport from ATP hydrolysis. Uncoupling cannot be explained by increased membrane permeability, but is associated with decreased capacity of the Ca2+-ATPase to bind noncatalytic, tightly bound ATP and ADP (Berman, M. C. (1982) Biochim. Biophys. Acta 694, 95-121). The effects of EGTA-induced uncoupling on absorbance and fluorescence properties of the bound ATP analog, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), have been studied under static and turnover conditions. Binding of 4.5-4.9 nmol of TNP-ATP/mg, as determined by absorbance difference titration, was relatively unaffected in the uncoupled state. TNP-ATP, bound to coupled vesicles during turnover, showed 6-8-fold enhanced fluorescence and a shift in the difference absorbance maximum from 510 to 493 nm, indicating increased hydrophobicity of the noncatalytic site. Turnover-dependent fluorescence enhancement was diminished by 60-70% in the uncoupled state, while the absorbance maximum wavelength shift was abolished. These data, correlating changes in the environment of the noncatalytic or regulatory nucleotide binding site on the Ca2+-ATPase with coupling activity, indicate that uncoupling is an intramolecular process, involving a ligand binding site on the ATPase, and that exclusion of H2O from the site occupied by noncatalytic nucleotides, during at least part of the catalytic cycle, is an event associated with energy transduction.     

Isolation and properties of the soluble c-type cytochromes of the dinoflagellate Peridinium cinctum

Four soluble cytochromes of the c type were isolated from the freshwater dinoflagellate Peridinium cinctum collected from Lake Kinneret, Israel. Cytochrome c with alpha-band maximum at 550 nm in the reduced state had a molecular mass of 10,200 Da, pI 7.4, and Em of 278 m V. This cytochrome was active in the respiratory chain of beef heart Keilin-Hartree particles. Cytochrome c-553 had a molecular mass of 13,200 Da, pI 4.9, and Em of 384 m V, and was active in light induced electron transport of Euglena gracilis chloroplast fragments. Cytochrome c-554 had a molecular mass of 13,500 Da, pI 4.4, and Em of 326 m V. This cytochrome was inactive in light induced electron transport but competed with cytochrome c-552 of Euglena in the assay. The acidic cytochrome c-557 was present in very small quantities. The properties of the soluble c-type cytochromes of P. cinctum are compatible with the classification of dinoflagellates as primitive eucaryotes.     

Amygdala kindled seizure stage is related to altered benzodiazepine binding site density

Benzodiazepine receptor binding was examined in rats at 3 stages of amygdaloid kindling (i.e., initial afterdischarge, Stage 3 and Stage 5) immediately or 24 hr after seizure. 3H-diazepam binding site density (Bmax) was significantly increased 24 hr after Stage 3 and Stage 5 kindled seizures in the hippocampus but not in the amygdala. There were no significant differences in the dissociation constants (KD) between kindled and control rats at any time point examined for either brain region. These results demonstrate that changes in benzodiazepine binding are observed with partial kindled seizures (i.e., Stage 3), indicating that generalized seizures are not prerequisite to increased benzodiazepine receptor site density.     

Dependence of phytoplankton assimilation quotients on light and nitrogen source: implications for oceanic primary productivity

Photosynthetic production of oxygen by phytoplankton assemblagedominated by Peridinium in Lake Kinneret, Israel, generallyexceeds the molar equivalent rate of carbon assimilation. Carbonassimilation occurs only if oxygenic photosynthesis exceedsa light-dependent threshold. Assimilation quotients (mol C molO₂^–1) are a variable function of irradiance, and typicallyonly about one-half of the photoreductant produced during oxygenicphotosynthesis is used for reduction of carbon dioxide. Mostof the residual oxygenic photoreductant probably is used forlight-dependent reduction of nitrate, which competes with carbondioxide for oxygenic photoreductant. Nitrate is an importantsource of nitrogen for this algal assemblage, and light-dependentnitrate reduction probably is much larger than carbon dioxidereduction at lowest irradiances in the euphotic zone. Oxygenproduction also may be much larger than carbon assimilationat low light levels in other environments where oxidized formsof nitrogen are important nitrogenous nutrients for phytoplankton,as in the lower euphotic zone of the sea, where low rates ofcarbon assimilation by phytoplankton have been thought to beinconsistent with the amount of oxygen that accumulates duringsummer.