A gene locus affecting tolerance to BGG in mice.

A genetic analysis was made of the ease of tolerance induction to bovine γ-globulin (BGG) in DBA/2, BALB/c, F₁ and backcross generation mice. Like parental DBA/2 mice, the F₁ generation of BALB/c × DBA/2 becomes tolerant when treated with 2 mg BGG. A backcross of this F₁ to DBA/2 parents produced mice that all became tolerant to this dose of BGG. A backcross of F₁ mice to BALB/c parents produced 50% offspring tolerized by the same dose of BGG and 50% resistant to tolerance induction.The data suggest a single autosomal locus affecting tolerance induction. Data presented elsewhere suggest that the locus affects macrophage function. We propose that this locus be called tolerance (symbol Tol-l) and the two alleles be (Tol-l^a (DBA/2 type) and Tol-l^b (BALB/c type) with Tol-l^a being dominant.     

Reaction of human lymphocytes with aggregated IgG columns. Effect on antibody-dependent cytotoxicity and EAC rosette formation.

The origin and life span of long-lived small lymphocytes in the bone marrow of mice have been evaluated by the use of radioautography, scintillation counting, and anti-theta serum. Thymus-deprived BALB/C mice and nude mice had a smaller percentage of long-lived lymphocytes in bone marrow and in thoracic duct lymph than sham-operated or normal littermates. Furthermore, the long-lived lymphocytes in the marrow of nudes have more varied—but generally shorter—life spans than long-lived lymphocytes from mice having a thymus. In thoracic duct lymph of nude mice a more homogeneous long-lived population—according to life span—was found.It was concluded that both long-lived T cells and long-lived B cells are normal residents in the bone marrow. Furthermore, it was concluded that cells of variable life spans comprise the B lymphocyte population: short-lived cells with life spans of 3–5 days and long-lived lymphocytes with life spans of weeks to months.     

A mutant of Anabaena sp. CA with oxygen-sensitive nitrogenase activity.

Studies on the O₂ protection mechanism for nitrogenase in a mutant (PM10) of $\text{Anabaena}$ sp. CA indicated that the ability to protect nitrogenase from O₂ was functionally impaired. Growth rates of PM10 were substantially improved when cells were cultured under microaerobic conditions. Nitrogenase activity was totally inhibited by exposure to O₂ for 30 min; partial restoration of activity was attained when cell suspensions were subsequently made microaerobic. Experiments in which induction of nitrogenase activity was followed indicated that the synthesis of the O₂ protection mechanism was temporally separated from synthesis of heterocysts and nitrogenase.     

New automated fluorometric peptide microassay for carnosine in mouse olfactory bulb

A procedure for assaying peptides at the picomole level in tissue extracts has been developed and used to measure the dipeptide carnosine in mouse olfactory bulb. In this procedure the tissue extract is reacted with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF), and the resultant fluorophors are resolved on a high performance reverse-phase column. Quantitation is performed in a filter fluorometer equipped with a flow cell. Carnosine was found to be present at a level of 1.93 ± 0.44 nmol/mg of tissue (mean + SD of 11 samples), in agreement with previous findings by other methods.     

Cell-associated immunity to measles (rubeola): The demonstration of in vitro lymphocyte tritiated thymidine incorporation in response to measles complement fixation antigen

Lack of a reliable in vitro assay for lymphocyte responsiveness to measles (rubeola) has hampered our understanding of the cell-associated response in diseases caused by, or related to, the measles virus. We report a reliable and reproducible system for demonstrating specific lymphocyte incorporation of ³H-thymidine in response to measles complement fixation antigen (CFA). Seventeen patients with positive histories of measles as children demonstrated a dose-response curve that varied between individuals but was constant for each individual. Kinetic data disclosed maximal responsiveness on day 7, and viral inactivation experiments disclosed that live virus was neither necessary for nor inhibitory to the reaction. The implications of this assay in terms of our understanding of the cell-associated response to measles virus in clinical measles and SSPE are discussed. The concept is explored that membrane-associated antigen is crucial in demonstrating the host's cellular immune response to viruses that can grow by cell-to-contiguous cell spread.     

Induction of booster antibody formation without specific antigenic drive.

Injection of antigen into the rabbit eye leads to the development of an immunogenic uveitis and local immunologic memory, with extensive intraocular formation of immunoglobulins. During both primary and booster responses within the eye, only a small proportion of Ig-forming cells contain antibody specific for the inciting antigen. Preliminary evidence suggests that most of the antibody formed is specific for antigens within the prior extraocular experience of the host. It is postulated that the inflammatory reaction is accompanied by the local release of lymphokines capable of attracting a representative sample of B cells from the blood, and of nonspecifically activating them to booster antibody formation without specific antigenic drive.     

Complexity in organic evolution

The present state of knowledge of the formation of the Compounds I of peroxidases and catalases is discussed in terms of the restrictions which must be placed upon a valid mechanism. It is likely that all Compounds I contain one oxygen atom bound to the heme-iron as in the Compound I of chloroperoxidase. Thus the formation of Compound I, obtained after molecular hydrogen peroxide and the enzyme diffuse together, involves a minimum of two bond ruptures and the formation of two new bonds. Yet this amazing reaction proceeds with an activation energy equal to or less than that for the fluidity of water. This result can only be accounted for by including at least one reversible step. Since Compound I formation requires the formation of an “inner sphere” complex, the presence or absence of water in the sixth co-ordination position of the heme-iron is of crucial importance. A comparison of the rates of ligand binding with the rate of Compound I formation indicate that the inner sphere complex leading to Compound I formation is formed by an excellent nucleophile, probably the peroxide anion, formed by a proton transfer from hydrogen peroxide. This proton cannot equilibrate with the bulk solvent. A proton derived from the active site would appear to be added to the hydroxide ion which permits a molecule of water to depart upon oxygen atom addition (or substitution) to (or at) the heme-iron. It is tentatively suggested that Compound I of catalase has a single active site per subunit molecule and that Compound I of peroxidase normally has two reactive sites.     

A near-infrared method for studying hydration changes in aqueous solution: Illustration with protease reactions and protein denaturation

Proteins undergoing protease reactions, heat denaturation, or interactions with sodium dodecyl sulfate (SDS) were used to demonstrate the effectiveness of a near-infrared method for the quantitative study of changes in hydration or water binding during such processes. The spectra of different proteins showed that the liberation of $\text{COO}{}^{\mathrm{?}}\text{and NH}{}_3{}^{\mathrm{+}}$ groups during a protease reaction is associated with a large increase in hydration and excluded volume. On the basis of experiments with model compounds, other spectral changes, including development of continuum absorbance between 1.55 and 1.85 μm and a band with a peak near 2.1 μm, were also attributed to the liberation of these groups. After heat denaturation or in the presence of SDS, the rate of proteolytic hydrolysis was markedly increased, consistent with the view that some preliminary denaturation is necessary for protease activity. The validity of the hydration changes calculated for protease reactions was supported by model studies with l-lysine, and with poly-l-lysine before and after hydrolysis. The near-infrared spectrum of the protein substrate with no added protease was largely unaffected by heat treatment alone, indicating that the hydration as such was not changed to a large extent by the structural modifications of denaturation. In contrast to the protease reaction, the interactions between SDS and the proteins resulted in a decrease in hydration. Results of this paper are compared with those obtained from other methods. Some unique advantages of the near-infrared method for the study of hydration changes during reactions in aqueous solution are described.     

Uptake, retention, and efflux of Ca2+ by mitochondrial preparations from skeletal muscle

Functionally intact mitochondria, substantially free of contamination, were isolated from rabbit gastrocnemius muscle after protease digestion and their Ca2+-handling properties examined. When judged by their capacity to retain large Ca2+ loads and the magnitude of basal and Na+-stimulated Ca2+ effluxes, the most suitable isolation method was digestion of finely minced muscle in buffered isoosmotic KCl with low levels (0.4 mg/g) of trypsin or the bacterial protease nagarse, followed by differential centrifugation. Polytron disruption of skeletal muscle in both sucrose- and KCl-based media released mitochondria deficient in cytochrome c. Use of the divalent ion chelator EDTA rather than EGTA in the isolation medium sharply reduced Ca2+-dependent respiratory control and tolerance of the mitochondria to Ca2+ loads, probably by removing Mg2+ essential to membrane integrity. ADP-dependent respiratory control was not altered in mitochondria prepared in an EDTA-containing isolation medium. Purification of mitochondria on a Percoll density gradient did not improve their Ca2+-handling ability despite removal of minor contaminants. Mitochondria prepared by the protease method could accumulate micromole loads of Ca2+/mg while maintaining a low basal Ca2+ efflux. Addition of BSA to the assay medium slightly improved Ca2+ retention but was not essential either during isolation or assay. Ca2+-dependent state 3 respiration was maximal at pH 6.5-7.0 while respiratory control and Ca2+/O were optimal at pH 7.0-7.5. Neither Pi nor oxaloacetate induced Ca2+ release from loaded mitochondria when monitored for 30 min after ruthenium red addition. Na+-stimulated Ca2+ efflux had sigmoidal kinetics with a Hill coefficient of 3. Since skeletal muscle mitochondria can be isolated and assayed in simple media, functional deficiencies of mitochondria from diseased muscle are unlikely to be masked.     

Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system

The relative quantities of 26 known transfer RNAs of Escherichia coli have been measured previously (Ikemura, 1981). Based on this relative abundance, the usage of cognate codons in E. coli genes as well as in transposon and coliphage genes was examined. A strong positive correlation between tRNA content and the occurrence of respective codons was found for most E. coli genes that had been sequenced, although the correlation was less significant for transposon and phage genes. The dependence of the usage of isoaccepting tRNA, in E. coli genes encoding abundant proteins, on tRNA content was especially noticeable and was greater than that expected from the proportional relationship between the two variables, i.e. these genes selectively use codons corresponding to major tRNAs but almost completely avoid using codons of minor tRNAs. Therefore, codon choice in E. coli genes was considered to be largely constrained by tRNA availability and possibly by translational efficiency. Based on the content of isoaccepting tRNA and the nature of codon-anticodon interaction, it was then possible to predict for most amino acids the order of preference among synonymous codons. The synonymous codon predicted in this way to be the most preferred codon was thought to be optimized for the E. coli translational system and designated as the “Optimal codon”. E. coli genes encoding abundant protein species use the optimal codons selectively, and other E. coli genes, such as amino acid synthesizing genes, use optimal and “non-optimal” codons to a roughly equal degree. The finding that the frequency of usage of optimal codons is closely correlated with the production levels of individual genes was discussed from an evolutionary viewpoint.     

A study of the reaction of 2-phenyl-1,3,2-benzodiazaborolidine with benzoyl chloride

Reaction of 2-phenyl-1,3,2-benzodiazaborolidine (2) with benzoyl chloride afforded N,N′-dibenzoyl-o-phenylenediamine (8) regardless of the amount of the acid chloride employed. Depending on the work-up procedure, the 2-phenylboron moiety was isolated as either phenylboronic acid or phenylboronic acid anhydride. In the absence of added bases, the reaction was observed to be susceptible to solvent assistance by ethers, presumably by coordination with the empty p-orbital of boron. In the presence of added amines, the extent of benzoylation was altered considerably. With pyridine as a co-reagent, small amounts of 1,3-dibenzoyl-2-phenyl-1,3,2,-benzodiazaborolidine mono- or dihydrocholoride were isolated though this compound readily underwent hydrolysis or oxidation upon standing. Interaction of 2 with sodium hydride in tetrahydrofuran caused ionization of only one of the two hydrogens bound to nitrogen even upon extended reflux. Benzoylation of the heterocycle under these latter conditions again afforded only the dibenzoylated compound 8.     

Purification and characterization of protein synthesis initiation factors IF1, IF2, and IF3 from Escherichia coli.

A simple procedure is described for the purification in high yields of protein synthesis initiation factors IF1, IF2, and IF3 from Escherichia coli strain MRE 600. IF2 was separated from IF1 and IF3 by ammonium sulfate fractionation and was purified by column chromatography on phosphocellulose and diethylaminoethyl (DEAE) Sephadex. IF1 and IF3 were separated by phosphocellulose column chromatography. IF1 was purified by molecular sieve chromatography, and IF3 by phosphocellulose column chromatography in urea buffer. Each factor was analyzed by sodium dodecyl sulfate or urea polyacrylamide gel electrophoresis and was greater than 98% pure. Only one form of IF1 and IF3 was found, with molecular weights of 8,500 and 22,500, respectively. Two forms of IF2 were isolated: IF2a with a molecular weight of 118,000 and IF2b with a molecular weight of 90,000. The amino acid composition of each factor was determined, and their stimulation in a variety of assays for initiation of protein synthesis is reported.     

Structural changes during the transformation of bacteriophage T4 polyheads: II. Evidence that a conformational change in the major capsid protein precedes the expansion

The shell of the bacteriophage T4 prehead is transformed after the maturation cleavages from a fragile to a highly chemically resistant structure. A “cleaved but anchored” shell, in which the capsid protein has been cleaved but expansion to the mature structure has not yet occurred, is thought to be an intermediate in the transformation. We have compared native, trypsinized, temperature-sensitive mutant, and cleaved but anchored polyheads for differences and similarities in their capsomeres. Our results show that the altered capsomeres of the cleaved but anchored state must be attributed to a conformational change in the subunits, and not simply to the loss of the amino-terminal peptide by proteolysis.     

Determination of protein synthesis initiation factor levels in crude lysates of Escherichia coli by a sensitive radioimmune assay.

Antisera specific for protein synthesis initiation factors IF1, IF2, and IF3 were prepared by immunizing rabbits. When crude cell lysates are analyzed by double immunodiffusion or by immunoelectrophoresis, each antiserum forms a single precipitin line antigenically identical to its cognate factor. The antisera do not crossreact with other initiation factors or with ribosomal proteins. A radioimmune assay was developed for each initiation factor by using the specific antisera and radioactive factors prepared by reductive alkylation with [¹⁴C]formaldehyde. The assays detect as little as 10 to 30 ng of factor. Initiation factor concentrations were measured in crude Escherichin coli MRE600 extracts prepared from cells grown exponentially in a rich medium. The three initiation factors are present in approximately stoichiometric amounts and comprise about 1% of the cell protein. The molar ratio of initiation factors to ribosomes is about 0.15, which corresponds to the concentration of native ribosomal subunits.     

Crystal packing and stoichiometry of the fibre protein of adenovirus type 2

Crystals of the fibre protein of adenovirus type 2 have been grown and studied by electron microscopy and X-ray powder diffraction. The molecular packing and density of the crystals suggest that the fibre is dimeric.     

Replacement of the B protein requirement of the E. coli quinolinate synthetase system by chemically-generated iminoaspartate

Two proteins (A and B) from $\text{Escherichia}\text{,}\text{coli}$ are required for $\text{in}\text{,}\text{vitro}$ synthesis of the NAD⁺ precursor, quinolinate, from L-aspartate and dihydroxyacetone phosphate. The requirement for B protein and L-aspartate in this system can be replaced by millimolar concentrations of oxaloacetate and ammonia if they are added together. This finding supports the concept that the B protein (L-aspartate oxidase) functions to form iminoaspartate which is condensed with dihydroxyacetone phosphate by the A protein to form quinolinate.     

The synthesis of 3-nonaprenyl-4-hydroxybenzoate in rat liver mitochondria: the effect of Ca2 , Mg2+, chelators, and bacitracin on the activity of p-hydroxybenzoate-polyprenyl transferase

The formation of the first intermediate in ubiquinone-9 biosynthesis, 3-nonaprenyl-4-hydroxybenzoate (NPHB), by the enzyme p-hydroxybenzoate:polyprenyl transferase, has been studied in isolated rat liver mitochondria using solanesol pyrophosphate and p-hydroxybenzoate as the substrates. Phosphate buffer (100 mm) is inhibitory but at 20 mm inhibition is not apparent compared to other buffers at the same concentration. With various buffers at low concentration (20 mm) both EDTA and Mg²⁺ stimulate formation of NPHB while Ca²⁺ inhibits. Release of Ca²⁺ inhibition can be achieved by the addition of Mg²⁺, or EDTA, or EGTA, with EGTA being less effective than EDTA. When Mg²⁺, Ca²⁺, and EDTA are present together, a two- to threefold increase in activity of the enzyme is observed. The antibiotic bacitracin inhibits the synthesis of NPHB and the inhibition is increased when divalent cations are present. EGTA is more effective than EDTA in overcoming inhibition due to bacitracin. The possibility that these effects are partially due to alteration of mitochondrial membrane conformation as well as a direct effect on the enzyme is evaluated. The possible role of polyprenylphosphates in mitochondrial membrane function is discussed.     

Natural cytotoxicity in AKR/J mice during adjuvant induced amyloidogenesis

The induction of amyloidosis in AKR mice has previously been shown to be associated with a decrease in the incidence of spontaneous thymic leukemia (P. Ebbesen, Brit. J. Cancer29, 76, 1974). Amyloid induction with azocasein depresses the activity of the natural killer (NK) cell, a cell believed to be important in the protection against the development of malignancy. In the present studies, therefore, we examined the response of the NK cell to the induction of amyloidosis in AKR mice. Rapid and long-term depression of NK cell activity against YAC-1 tumor cells was noted following intraperitoneal administration of complete Freund's adjuvant enriched with Mycobacterium butyricum. Mixing studies suggested that active suppression did not account for the observed decrease in NK cell activity. Although some NK cell activity was noted in ascitic fluid, redirection was not felt to account for the rapid and dramatic reduction in splenic NK cell activity. Furthermore, serum from adjuvant-treated but not control mice was found to significantly inhibit NK cell activity in vitro. These studies therefore suggested a role for a serum factor in the depression of this activity. The apparent paradox of decreased NK cell activity in a setting of diminished leukemogenesis is considered in relation to previous studies describing impaired T-cell functioning after the induction of amloidosis.