Polymerization of deoxyhemoglobin CHarlem (β6 Glu → Val, β73 Asp → Asn): The effect of β73 asparagine on the gelation and crystallization of hemoglobin

The kinetics of aggregation and the solubility of deoxy Hb2 C_Harlem (α₂β₂ 6 Val, 73 Asn) in concentrated phosphate buffers were studied in comparison with those of deoxy Hb S and deoxy Hb A. Deoxy Hb C_Harlem aggregated with a clear exhibition of a delay time. The length of the delay and aggregation times and the degree of the aggregation depended upon the initial hemoglobin concentration.The initial hemoglobin concentration required for the aggregation of deoxy Hb C_Harlem was approximately 200% of its solubility, a value much higher than that required for the aggregation of deoxy Hb S (120%). With the same hemoglobin concentration, the delay time for the aggregation of deoxy Hb C_Harlem was approximately 100 times longer than that of deoxy Hb S. The logarithmic plotting of the delay time versus hemoglobin concentration in 1.8 m-phosphate buffer (pH 7.4) showed linear lines with a slope (n) of 4.0 for deoxy Hb C_Harlem. In contrast to the results for the aggregation of deoxy Hb S, n values for deoxy Hb C_Harlem were unchanged with phosphate concentrations varying from 1.2 m to 2.0 m. The solubilities of deoxy Hb S and deoxy Hb C_Harlem were increased exponentially by lowering the pH of the medium, with the increase being more conspicuous for Hb C_Harlem. The gels (or aggregates) of Hb C_Harlem were converted to crystals at a rate much faster than were those of Hb A and Hb S. The kinetics for gelation and crystallization of deoxy Hb C_Harlem can be explained by the following scheme, where nuclei G and nuclei C are formed before gelation and crystallization, respectively. Monomenc deoxy Hb

The hemoglobin concentration required for the crystallization of deoxy Hb C_Harlem was about ten times lower than that required for deoxy Hb A. The solubility of deoxy Hb C_Harlem after aggregation was about twice that of deoxy Hb S, suggesting that the substitution of Asn for Asp at the β73 residue inhibits the formation of nuclei G and accelerates the formation of nuclei C.     

Amber mutations in the structural gene for RNA polymerase sigma factor of Escherichia coli

Summary Amber mutants of Escherichia coli K-12 affected in the structural gene (rpoD) for th subunit of RNA polymerase have been obtained from a strain harboring a temperature-sensitive amber suppressor (supF-Ts6) which is active only at low temperatures. These mutants grow normally at low temperature (30°C) but do not grow at high temperature (42°C) due to the inability to synthesize factor. In one mutant studied in detail (rpoD40), the rate of -factor synthesis at 30°C is about half that of the wild type and is decreased to 10%–15% within 1 h of incubation at 42°C. The synthesis of core polymerase subunits or bulk protein is virtually unaffected at least for 2 h. The defect of the mutant in synthesis and growth at high temperature can be suppressed by any of the amber suppressors tested (supD, supE or supF). RNA-polymerase holoenzymes prepared from the mutant cells carrying each of the suppressors (grown at 42°C) exhibit different thermostabilities attributable to alterations in the factor. The reduced synthesis in the mutant is accompanied by the synthesis of polypeptide tentatively identified as amber fragment. These results as well as the genetic mapping data indicate that the amber mutation (rpoD40) resides within the structural gene for the factor and directly affects synthesis upon inactivation of the suppressor at high temperature.     

Establishment of Autoantibody-Producing Cell Lines from Peripheral Blood Lymphocytes of Patients with Systemic Lupus Erythematosus

Autoantibody-producing B cell lines were established from peripheral blood lymphocytes of patients with systemic lupus erythematosus. Peripheral blood lymphocytes obtained from five of seven patients were successfully transformed by Epstein-Barr virus. Two of four established B lymphoblastoid cell lines examined in this study produced anti-nuclear factor antibodies and one of them produced anti-single-stranded DNA and anti-double-stranded DNA antibodies. These results indicate that B cell clones committed to self antigens are transformed by Epstein-Barr virus and continue to produce autoantibodies. In order to establish a monoclonal autoantibody-producing B cell line, the cells were cloned by a limiting dilution method. The data suggest that it is possible to establish a monoclonal autoantibody-producing B cell line by the combination of transformation of B cells by Epstein-Barr virus and extensive cloning.     

On the phosphate linkages and the structure of a disaccharide unit of the type-specific polysaccharide of pneumococcus type XIX

The structure of the capsular polysaccharide (S-XIX) of Pneumococcus Type XIX, which contains residues of d-glucose, l-rhamnose, 2-acetamido-2-deoxy- d-mannose, and phosphate, has been investigated by acid hydrolysis, treatment with acid phosphatase, mass spectrometry, and ¹³C-n.m.r. spectroscopy. Phosphoric esters in S-XIX were largely resistant to hydrolysis (4M HCl, 100°, 3 h). With M or 2M HCl at 100° for 3 h, 4-O-(2-amino-2-deoxy-β-d-mannopyranosyl)-d-glucose 4′-phosphate was liberated. More-drastic hydrolysis of S-XIX gave 2-amino-2-deoxy-d-mannose 3-, 4-, and 6-phosphates, and 4-O-(2-amino-2-deoxy-d-mannopyranosyl)-d-glucose and its 4′-phosphate.     

Volatile emissions of plant tissue cultures

Summary The low-molecular-weight volatiles released by a variety of plant tissue cultures were examined by gas chromatography. Callus cultures invariably produced carbon dioxide, ethylene, acetaldehyde and ethanol. In cultures with developed shoots, ethanol was absent and acetaldehyde was detected only rarely.     

A new assay system of phospholipid exchange activities using concanavalin a in the separation of donor and acceptor liposomes

A new assay system of phospholipid exchange activities is described. The exchange activities were quantitated by measuring the stimulation of phospholipid transfer between two separate populations of liposomes, which contained, as the major constituents, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, and cholesterol in molar ratios of 6: 2: 1: 1: 5. One population of the liposomes was made reactive to concanavalin A by the incorporation of 1.8 mol% $\text{α-}\text{d}\text{-mannosyl}\text{-(1 → 3)-α-}\text{d}\text{- mannosyl}\text{-sn-}\text{1,2-diglyceride}$ from Micrococcus lysodeikticus. The concanavalin A-reactive liposomes, a phospholipid donor, were doubly labelled with [6-³H]galactosylglucosyl ceramide and that class of ³²P-labelled phospholipids whose exchange was being measured. The ³H-labelled glycolipid served as a non-exchangeable reference marker. The other population of the liposomes, a phospholipid acceptor, was concanavalin A nonreactive. These two populations of liposomes were incubated with the cytosol protein of rat liver in a total volume of 0.2 ml.After the incubation, two different procedures were used to separate the two liposomal populations. In one procedure concanavalin A was added to agglutinate the reactive liposomes; the flocculated lectin-liposome complex was separated from the non-reactive liposomes by brief centrifugation. In the other procedure the reactive liposomes were trapped by binding to concanavalin A covalently coupled to Sepharose 2B; the complex was separated from the nonreactive liposomes by filtration through a filter paper under suction. In both assay procedures the amount of phospholipid transferred from the donor to the acceptor liposomes was calculated from the decrease of ³²P/³H ratio of the concanavalin A-reactive liposomes during the incubation. By the assay system it is possible to determine phosphatidylcholine and phosphatidylinositol exchange activities in 100 μg of rat liver cytosol protein.     

Methylmercury: Effects on electrical properties of squid axon membranes

At low concentrations (25–100 μM) methylmercury chloride caused a steady increase in the threshold for excitation and on eventual block of action potentials without changing the resting membrane potential in squid giant axons. In the axons exposed to 25 μM methylmercury chloride, peak transient and steady-state conductances were decreased by 58.8 ± 5.1% and 35.9 ± 4.3% (mean ± SEM, 4 axons), respectively and leakage conductance increased to about five times of the control value. Higher concentrations of methylmercury chloride decreased the resting membrane potential. A concentration of 0.5 mM depolarizing the nerve membrane by 16 ± 2 mV (mean ± SEM, 3 axons) in 40 minutes. These changes in ionic conductances and membrane potential were irreversible on washing the axon with drug-free sea water.     

An endo-(1→6-β-D-glucanase from Mucor hiemalis

An endo-(1→6)-β-D-glucanase (EC 3.2.1), isolated from the culture filtrate of Mucor hiemalis, was purified by ammonium sulphate fractionation and gel filtration. The homogeneity of the enzyme was confirmed by disc electrophoresis. The enzyme had a wide range of temperature and pH stability, high substrate specificity, and an action pattern of the endo-type.