The human T cell antigen gp39, a member of the TNF gene family, is a ligand for the CD40 receptor: expression of a soluble form of gp39 with B cell co-stimulatory activity.

Signals delivered to B cells via CD40 can synergize with those provided by other B cell surface receptors to induce B cell proliferation and antibody class switching as well as modulate cytokine production and cell adhesion. Recently, it has been shown that the ligand for CD40 is a cell surface protein of approximately 39 kDa expressed by activated T cells, gp39. Here we report on the isolation and characterization of a cDNA clone encoding human gp39, a type II membrane protein with homology to TNF, and the construction and characterization of a soluble recombinant form of gp39. COS cell transfectants expressing gp39 synergized with either anti-CD20 mAb or PMA to drive strong B cell proliferation and alone were able to drive B cells to proliferate weakly. In all cases the B cell proliferation induced by gp39-expressing COS cells was reduced to background levels by the addition of soluble CD40. Unlike gp39-expressing COS cells, recombinant soluble gp39 was not mitogenic alone and required co-stimulation to drive B cell proliferation. These results suggest that B cells require a second signal besides gp39-CD40 to drive proliferation and that soluble gp39 alone in a non-membrane bound form is able to provide co-stimulatory signals to B cells.     

Stability of solvent formation in Clostridium acetobutylicum during repeated subculturing

Summary Clostridium acetobutylicum ATCC 824 was submitted to repeated subculturing at 24-hour intervals for 218 days. The organism retained its ability to form solvents, although the fermentation slowly became increasingly acidogenic during the first 200 days. Except for the initial spore inoculum, the cultures were not subjected to heat shocking between the serial transfers. When the inoculum volume was doubled from 3.3% to 6.7% after 200 days of subculturing, the product formation pattern quickly shifted back from acids to primarily butanol. Acetone production also resumed after being undetectable for more than 50 days. The relative formation of acetate and ethanol remained nearly constant throughout the experiments, while the formation of butyrate mirrored that of butanol.     

Glycerol catabolite regulation of d-cycloserine production in Streptomyces garyphalus

A fluorescent pigment was isolated from the culture fluid of Methanobacterium thermoautotrophicum strain H. This pigment was shown to be 7,8-didemethyl-8-hydroxy-5-deazariboflavin by various spectroscopic and chromatographic techniques. This compound was previously described as the FO acid hydrolysis fragment of coenzyme F₄₂₀. On the basis of the time of appearance of the pigment in the course of fermentation, it is suggested that this substance may be an over-produced biosynthetic precursor of F₄₂₀.     

Electron microscopic observations on iron-labelled secondary lysosomes in renal tubules in mice

Summary Labelling of renal tubule cytosomes with electron dense iron granules can be attained by daily intramuscular injections to mice of an iron sorbitol citric-acid compound in a total of approximately 50 mg Fe⁺⁺⁺/100 g of body weight. The labelled cytosomes correspond to secondary lysosomes and represent heterolysosomes or ambilysosomes. The evidence suggests that tubule and lysosome function are undisturbed by the labelling procedure. The use of this method for fine structural studies of the interaction between secondary lysosomes and other cytoplasmic organelles and elements is indicated.Microbodies do not incorporate administered Fe⁺⁺⁺. The morphological observations support the opinion that these bodies are formed in specialized portions of the smooth surfaced endoplasmic reticulum of proximal tubule cells.Supported in part by grants from the Swedish Medical Research Council (Projects No. K 67-12x-1006-2, B 67-12x-1006-02K, K 68-12x-1006-03, and B 69-12x-1006-04A). The assistance of Miss Silwa Mengarelli and Miss Britt-Marie Pettersson is gratefully acknowledged.     

Relative positions of some epitopes on carcinoembryonic antigen

Summary To investigate whether anti-(carcinoembryonic antigen) monoclonal antibodies (mAb) react with single or repeated epitopes, sandwich radioimmunoassays in homologous and heterologous combinations were performed. Four mAb (I-27, I-47, II-17 and to some degree II-16) gave homologous binding while two mAb (I-38S1 and II-10) did not. Taken together with previous immunoprecipitation studies we conclude that all these mAb except II-10 react with repeated epitopes. The relative positions of the epitopes recognized by these mAb and of three additional mAb (II-6, II-7 and CB-CEA-1) were investigated using a plate antibody competition test with enzyme-labelled carcinoembryonic antigen (CEA). mAb I-38S1, II-6, II-7, II-10, II-16 and CB-CEA-1 were mutually cross-reactive, and were classified as belonging to one epitope group. mAb I-27 and I-47 fell outside this group and did not interfere with the binding of CEA conjugate to mAb II-17 either. They therefore represent a second epitope group. mAb II-17 showed no interference with the binding of CEA to any of the other mAb and must therefore represent a third epitope group. The slopes of the plate antibody competition curves were used for calculation of a correlation matrix, which in turn was used to depict the relative positions of the epitopes recognized by the mAb in the large group.     

Biochemical characteristics and partial amino acid sequence of the receptor-like human B cell and carcinoma antigen CDw40

The Ag CDw40 (p50, Bp50) is a phosphoprotein expressed on the surface of both B lymphocytes and on certain malignant cell types of nonhemopoietic origin. Antibodies to this Ag have been shown to act as a potent co-mitogen for B cells. In order to elucidate the function of this Ag, we have now investigated some of its biochemical characteristics as well as the relationship of B cell derived CDw40 to that derived from urinary bladder carcinoma (transitional cell carcinoma, TCC) cells. CDw40 from normal B cells or from the Burkitt lymphoma line Raji showed a characteristic pattern of three bands when analyzed by SDS-PAGE and Western blotting: a main band of 47 kDa, a degradation product of 43 kDa, and a dimer of 85 kDa. The dimer was disrupted by reduction with 2-ME but was reformed spontaneously from the purified monomers under nonreducing conditions. CDw40 from two bladder cancer cell lines gave a similar pattern but formed little or no dimer. Thirty amino acids of the amino terminal end of CDw40 from Raji and 22 amino acids of that from TCC cells (HU549) were sequenced. The sequences were unusually rich in cysteines and differed only in that the cysteine in position 6 in Raji CDw40 had been replaced by glutamine in HU549. In addition there were two conservative changes in positions 15 and 19. Taken together these results show that CDw40 derived from B cells or from TCC cells are the same or closely related molecules. Comparisons of the amino acid sequence and biochemical characteristics of CDw40 with proteins having receptor functions indicated a close structural resemblance of CDw40 to the nerve growth factor-receptor.     

The presence of two serine racemases in Streptomyces garyphalus,a d-cycloserine producer

Two serine racemases (I and II) were isolated from Streptomyces garyphalus. Serine racemase I (molecular weight 93,000) was purified to a single band in an analytical electrofocusing system. Serine racemase II (molecular weight 73,000) was partially purified. Both enzymes used pyridoxal-5-phosphate as cofactor. Besides serine the enzymes utilized alanine as substrate but no other amino acid tested. The K _m values of l-alanine and l-serine for enzyme I were 111 mM and 35 mM respectively. Enzyme I was not inhibited by d-cycloserine but by hydroxylamine. Both substances inhibited enzyme II. The serine racemases may be involved in the biosynthesis of d-cycloserine in S. garyphalus.     

Gene fusion vectors based on the gene for staphylococcal protein A

Two plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusion, have been constructed. These vectors will allow fusion of any gene to the protein A gene, thus giving hybrid proteins which can be purified, in a one-step procedure, by IgG affinity chromatography. As an example of the practical use of such vectors, the protein A gene has been fused to the lacZ gene of Escherichia coli. E. coli strains containing such plasmids produce hybrid proteins with both IgG binding and β-galactosidase activities. The hybrid protein(s) can be immobilized on IgG-Sepharose by its protein A moiety with high efficiency without losing its enzymatic activity and they can be eluted from the column by competitive elution with pure protein A. The fused protein(s) also binds to IgG-coated microtiter wells which means that the in vivo product can be used as an enzyme conjugate in ELISA tests.     

Prostaglandin F2a inhibition of epinephrine stimulated cyclic AMP and progesterone production by rat corpora lutea of various ages

Epinephrine can mimic the stimulatory effects of LH in vitro on cyclic AMP (cAMP) and progesterone production by isolated rat corpora lutea. The aim of the present study was to test whether the effects of epinephrine in vitro on the rat corpus luteum, as with LH, can be inhibited by prostaglandin F_2a (PGF_2a. The stimulatory effect of epinephrine on tissue levels of cAMP in 1-day-old corpora lutea was not inhibited by PGF₂. A dose-dependent inhibition by PGF_2a (0.5–50 μM) was seen for 3-day-old corpora lutea and this inhibition could not be overcome by higher concentrations of epinephrine (0.165–165 μM). The stimulation by epinephrine on progesterone production was inhibited by PGF_2a (5 μM) in 3- and 5-day-old, but not in 1-day-old corpora lutea. Thus, PGF_2a can inhibit the stimulatory effect of epinephrine in 3- and 5-day-old corpora lutea, but not in the newly formed corpora lutea (1-day-old) and PGF_2a shows in this respect the same agedependent inhibitory pattern as in relation to LH stimulation.