Studies on the product of antigenic recognition. I. Formation of the product in recognition of alloantigens.

A product of antigenic recognition (PAR) was produced whenever receptors for alloantigens from T lymphocytes or a principle present in T-cell dependent alloantisera interacted with alloantigen. With two forms of the PAR assay (direct and indirect) the mechanisms underlying these interactions have been analyzed. For the interaction of T-lymphocyte receptors with alloantigen measured with direct PAR assays, the following conclusion emerged: upon confrontation with alloantigen, receptors (if not already present in secreted form) had first to be released from T-cell membranes. Shed T-cell receptors interacted with alloantigens by solubilizing them. Both processes could be prevented by fixing cells with formaldehyde. Release of T-cell receptors was temperature-dependent, solubilization of alloantigens was not. Because in mixed cell cultures receptors had first to be shed, this process was considerably slower and, in concordance with temperature dependence of receptor release, took place only at 37 degrees C. Titration of T lymphocytes with 'bound' receptors by the direct PAR test revealed that in the presence of excess alloantigen 10(2) T cells sufficed to give measurable responses. Supernates of parental strain lymphocytes containing numerous T-cell receptor specificities could be depleted of one of them. Alloantisera raised in presence of T helper cells ('T alloantisera') contained a principle capable of recognizing alloantigens, alloantisera incited in the absence of T helpers ('B alloantisera') did not. The recognizing principle appeared to be IgG. Like T-cell receptors, it was capable of solubilizing alloantigens form target cell membranes. B alloantisera lacked this capacity and their alloantigen-recognizing moiety was found to be monomeric IgM. The mode of interaction of this IgM with alloantigen most likely consisted in fixation to and shielding of antigen.     

Approximating the product spectrum and product concentrations in continuous photochemical reactions.

Several approximations to a common photochemical rate law, in which the rate is proportional to the fraction of light absorbed by the reactant chromophore, have been developed to permit the product spectrum to be determined from a sequence of spectra during irradiation that exhibit isosbestic points. The methods were tested on the photolysis of [Cr(NH(3))(6)](3+) and [Cr(en)(3)](3+) (en = ethylenediamine) in water, and [Fe(Et(2)dtc)(3)], tris(diethyldithiocarbamato)iron(III), in CHCl(3).     

Studies on the product of antigenic recognition. III. Nature of PAR.

The product of antigenic recognition (PAR) is an antigen-antibody complex with granulotactic activity. The following results support his conclusion. PAR generated by cell-bound or shed T-cell RS or the recognizing structure of T alloantisera and alloantigen can be destroyed by heating at 56 degrees C for 30 min, because of inactivation of a heat-labile component supplied by alloantigen. Recognizing partners of these complexes are heat-stable. They form PAR anew when alloantigen is added or upon confrontation with anti-RS serum. They are also capable of inducing anti-RS sera. PAR preparations composed of these recognizing structures and antibodies to them are heat-inactivated at much higher temperatures, because both partners of the complex are relatively heat-stable. Heat-labile PAR complexes (recognizing structures and alloantigen) pass anti-mouse immunoglobulin immunosorbent, but are retained specifically by alloantiserum (via alloantigen of the complex) or by anti-RS serum (via RS of the complex) bound to the column.     

Identification of the ftsA gene product.

A nonsense mutation was identified in the essential cell division gene ftsA of Escherichia coli. A gamma-transducing phage was isolated which complemented this mutation. This phage programmed the synthesis of four bacterial proteins in UV-irradiated cells. By substituting the nonsense mutation for the ftsA+ allele in this transducing phage and comparing the proteins programmed by it in UV-treated Su+ and Su- cells, the product of the ftsA gene was identified as a protein with a molecular weight of 50,000.     

Studies on yellow fever vaccine. II--Stability of the reconstituted product

This work evaluated the stability of diluted yellow fever vaccine in order to determine conditions that maintain the minimum of 3 log10 of 17D virus per human dose as required by WHO. The vaccines were held at 0 degrees C or at 37 degrees C and were diluted either with distilled water, with 0.15 M saline or with 0.15 M PBS at pH 5.5, 7.2 and 8.0. In a next step, stabilizer substances such as gelatin and peptone were added to the vaccines. Dilution of the vaccines in distilled water maintained the virus titre for up to three hours at 37 degrees C and this diluent has been adopted for routine use in Brazil.     

Identification and amplification of the E. coli phr gene product.

We have constructed a series of multicopy plasmids that complement mutations in the phr gene of Escherichia coli. By subcloning into a tac plasmid vector we obtained a phr plasmid that upon induction overproduces two proteins of Mr's 49,000 and 20,000. Tn1000 insertions into the phr gene caused the disappearance of the 49,000 dalton protein, thus demonstrating this protein to be the phr gene product, DNA photolyase. The photolyase encoded by the phr gene makes up about 15% of total cellular proteins after induction of cells carrying a tac-phr plasmid. This protein binds specifically to UV (254 nm) irradiated DNA and upon exposure to near UV (300-500 nm) illumination repairs the UV damage and dissociates from DNA.     

Integrated product formation and recovery.

As continues to be demonstrated, the in situ recovery of selected products froma bioreactor can have a significant positive impact on production. The strategies that are focused on here are: aqueous two-phase biocatalysis; non-aqueous biocatalysis; and membrane-enhanced biocatalysis. Additional fundamental understanding of molecular partitioning and biocatalytic activity in these environments will facilitate the rational selection of the components involved in these processing strategies.     

Studies on the mode of sugar-phosphate product inhibition of brain hexokinase.

Hexokinase I (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.1), a key regulatory glycolytic enzyme in certain tissues, is known to be markedly inhibited under physiological conditions. The action of the primary inhibitory effector, glucose-6-P, is reversed by inorganic orthophosphate (P_i). A molecular model for inhibition and deinhibition of hexokinase was recently proposed [Ellison, W. R., Lueck, J. D., and Fromm, H. J. (1975) J. Biol. Chem.250, 1864–1871]. One of the central assumptions of this model is that glucose-6-P is a normal product inhibitor of hexokinase. It has long been suggested that glucose-6-P is an allosteric inhibitor of hexokinase, whereas other sugar-phosphate products such as mannose-6-P are normal product inhibitors. In this report we investigated the kinetic mechanism of hexokinase action with mannose as substrate and mannose-6-P as an inhibitor. The data obtained show that there are no qualitative differences between glucose and mannose as substrates and glucose-6-P and mannose-6-P as inhibitors. Binding experiments indicate that glucose-6-P and mannose-6-P are competitive binding ligands with hexokinase I. Furthermore, the activation pattern observed with Pi and glucose-6-P inhibited hexokinase is also found with the mannose-6-P inhibited phosphotransferase. These findings suggest that the mechanism of inhibition of glucose-6-P and mannose-6-P represents a difference in degree rather than a difference in kind. An explanation of the results in terms of a stereochemical model is presented.     

Purification of the E. coli dnaA gene product.

The product of the dnaA gene of Escherichia coli was isolated in a highly enriched form. The purification product binds specifically to DNA containing the E. coli chromosomal origin of replication, oriC.     

Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation.

To prevent the loss of raw material in ethanol production by anaerobic yeast cultures, glycerol formation has to be reduced. In theory, this may be done by providing the yeast with amino acids, since the de novo cell synthesis of amino acids from glucose and ammonia gives rise to a surplus of NADH, which has to be reoxidized by the formation of glycerol. An industrial strain of Saccharomyces cerevisiae was cultivated in batch cultures with different nitrogen sources, i.e., ammonium salt, glutamic acid, and a mixture of amino acids, with 20 g of glucose per liter as the carbon and energy source. The effects of the nitrogen source on metabolite formation, growth, and cell composition were measured. The glycerol yields obtained with glutamic acid (0.17 mol/mol of glucose) or with the mixture of amino acids (0.10 mol/mol) as a nitrogen source were clearly lower than those for ammonium-grown cultures (0.21 mol/mol). In addition, the ethanol yield increased for growth on both glutamic acid (by 9%) and the mixture of amino acids (by 14%). Glutamic acid has a large influence on the formation of products; the production of, for example, alpha-ketoglutaric acid, succinic acid, and acetic acid, increased compared with their production with the other nitrogen sources. Cultures grown on amino acids have a higher specific growth rate (0.52 h-1) than cultures of both ammonium-grown (0.45 h-1) and glutamic acid-grown (0.33 h-1) cells. Although the product yields differed, similar compositions of the cells were attained. The NADH produced in the amino acid, RNA, and extracellular metabolite syntheses was calculated together with the corresponding glycerol formation. The lower-range values of the theoretically calculated yields of glycerol were in good agreement with the experimental yields, which may indicate that the regulation of metabolism succeeds in the most efficient balancing of the redox potential.     

Marginal product pricing in the ecosystem.

Surface membrane biosynthesis and turnover is reviewed focusing mainly on the fate of cell surface constituents after they terminated their sojourn as part of a functional cell structure. The different experimental approaches to study this problem are described and original data are presented on the turnover of surface membrane constituents of chicken embryo cells in culture. It is proposed that as a consequence of surface membrane turnover, certain surface macromolecules are continuously shed from cells. The size and charge of these molecules was found to be identical to molecules released from cells by mild trypsin treatment. The term shedding is proposed for this process which is assumed to occur both in vitro and in vivo. Many systems in which shedding of cell surface constituents is clearly demonstrated or can be tentatively suggested are described. The biological significance of cell surface carbohydrate containing macromolecules and the possible role of these shed cellular entities is discussed.     

Molecular amplification and purification of the E. coli recC gene product.

The level of recC gene expression has been analysed using Mud(bla lac) fusions to the recC promoter. The constitutive level of expression is very low and remains so even under SOS inducing conditions. The recC gene product has been amplified by harnessing the gene to the phage lambda leftward promoter in a plasmid. From cells harbouring this plasmid, RecC protein, which represented approximately 6% of the total cellular protein, was purified to electrophoretic homogeneity.     

Synthesis and conformational analysis of the insulin-like 4 gene product.

Insulin-like 4 (INSL-4) is a protein expressed in the early placenta. Its primary structure is insulin-like with reference to the distribution of cysteine residues and the single chain pro-form. Insulin-like 4 was generated by solid-phase peptide synthesis of the two chains followed by the sequential synthesis of the three disulfide bonds. Two disulfide isomers were produced, one with an insulin-like disulfide bonding pattern and the other with a reversed chain orientation. The CD spectra of the two disulfide isomers were indistinguishable without any features produced by periodic structures. In addition, the hydrodynamic properties of the two isomers were identical which implied a very open structure of the disulfide-bonded two-chain molecules. It appears that insulin-likeness cannot be defined solely on the basis of the primary structure of cDNA.     

Characterization and expression of the human rhoH12 gene product.

The rho genes constitute an evolutionarily conserved family having significant homology to the ras oncogene family. These genes have been found in Saccharomyces cerevisiae, Drosophila melanogaster, rat, and human; their 21,000-dalton products show strong conservation of structure. In humans, three classes of rho cDNA clones have been identified which differ by virtue of the presence of variable C-terminal domains: rhoH12, rhoH6, and rhoH9. The predicted 193 amino acids of human rhoH12 protein show 88% similarity with those of the human rhoH6 clone, 96.8% similarity with those of the Aplysia rho product, and 81.8% similarity with those of the yeast RHO1 protein. Rat-1 and NIH 3T3 mouse fibroblasts were transfected with clones containing the normal human rhoH12 allele as well as the variants encoding valine in place of the glycine and leucine in place of the glutamine normally found at residues 14 and 64, respectively. These replacements mirror the changes responsible for oncogenic activation of the related ras-encoded p21 proteins. These mutant rhoH12 clone alleles did not cause focus formation in monolayers or growth in soft agar. However, amplification of normal rhoH12 via cotransfection with a dihydrofolate reductase gene resulted in colonies that displayed reduced dependence on serum for growth, grew to higher saturation densities, and were tumorigenic when inoculated into nude mice. Normal p21rho protein was detected in the transfected cell lines as well as in normal cell lines by Western immunoblot and immunoprecipitation analysis with rabbit antibodies raised against the peptide corresponding to amino acids 122 to 135.     

Identification and characterization of the Myxococcus xanthus bsgA gene product.

The bsgA mutants of Myxococcus xanthus are blocked at a very early stage of the developmental program. They fail to produce fruiting bodies or to sporulate under normal conditions but can be rescued by extracellular complementation in mixtures with wild-type cells. A bsgA-lacZ gene fusion was constructed and expressed in Escherichia coli. The resulting fusion protein, which has beta-galactosidase enzyme activity, was partially purified by affinity chromatography and preparative polyacrylamide gel electrophoresis. The protein was used to immunize mice, which produced a hybridoma secreting monoclonal antibody that was specific for the bsgA gene product. The monoclonal antibody was used in Western blot (immunoblot) experiments to determine the apparent cellular location of the bsgA protein in M. xanthus and to compare the level of this protein at various times in the Myxococcus life cycle.     

Heterologous expression and characterization of the human R-ras gene product.

We directly expressed human R-ras 23,000-dalton protein (p23) cDNA in Escherichia coli under the control of the trp promoter. GTP-dependent phosphorylation of a p23 threonine 85 substitution mutant was observed. This result is in direct analogy to the autokinase activity of H-ras and K-ras threonine 59 substitution mutants. Normal p23 protein was detected in the human fibrosarcoma cell line HT1080 by immunoprecipitation with rabbit antibodies raised against an E. coli-expressed R-ras fusion protein. The R-ras p23 protein was found to be 3H labeled in the presence of [9,10(n)-3H]palmitic acid and is associated with the P100 membrane fraction of HT1080 cells. These data suggest that human R-ras p23 has biochemical properties very similar to those of the p21 products of the H-, K-, and N-ras proto-oncogenes. We constructed an R-ras minigene and engineered the expression of normal and mutant alleles from the simian virus 40 early region promoter. Normal and mutant R-ras gene products were authenticated by transient expression in COS-7 cells and immunoprecipitation. The valine 38-substituted R-ras p23 displayed reduced electrophoretic mobility. R-ras p21-like proteins, made by eliminating the first 26 R-ras codons, displayed evident mobility differences between the pro form and mature form, along with a valine 12 substitution-dependent change in electrophoretic mobility. Rat-1 fibroblasts were transfected with normal and mutant R-ras alleles and normal and activated H-ras alleles. Unlike the human T24 bladder oncogene-encoded p21, mutant R-ras alleles do not cause monolayer focus formation or growth in soft agar of rat fibroblasts.