Human B cell responsiveness to B cell growth factor after activation by phorbol ester and monoclonal anti-mu antibody

The effect of phorbol ester on human B cell activation was examined. Picomolar to nanomolar concentrations of phorbol ester induced a high level of proliferation in small IgM-positive B cells isolated from peripheral blood by fluorescence-activated cell sorting. The addition of optimal doses of anti-mu antibody resulted in enhanced proliferation of phorbol ester-activated B cells. The addition of B cell growth factor (BCGF) to phorbol ester-activated B cells also resulted in a dose-dependent synergistic effect and maximal enhancement on day 3. BCGF activity could be absorbed with either phorbol ester- or anti-mu-activated B cells, but not with resting B cells, thus confirming the induction of functional BCGF receptor expression. Cell proliferation was not necessary for the induction of functional BCGF receptors. Phorbol ester was a more efficient inducer of BCGF receptor expression than was anti-mu antibody; gamma-interferon treatment had no effect. BCGF enhanced transferrin receptor expression by phorbol ester-activated B cells. The results suggest that phorbol ester-activated small B cells can be used to monitor BCGF activity, and this synergistic combination may be useful in establishing BCGF-dependent B cell clones in culture.     

Preliminary crystallographic data for cross-linked (lysine7-lysine41)-ribonuclease A

A cross-linked derivative of ribonuclease A, N^ε,N^ε′-(2,4-dinitrophenylene-1,5)-(lysine⁷-lysine⁴¹)-RNase A, has been crystallized by dialysis against 30% ($\text{v}\text{v}$) ethanol/water mixtures buffered at high pH. Single crystals belong to the orthorhombic space group P2₁2₁2₁, $\text{a = 37.2}\text{A}\text{?}\text{, b = 41.2}\text{A}\text{?}\text{, b = 41.2}\text{A}\text{?}$, with one molecule in the Crystallographic asymmetric unit.     

Water-soluble glucans from the seed of Coix lacryma-jobi var. ma-yuen

The water-soluble major polysaccharides from the seed of Coix lacryma-jobi var. ma-yuen eluted as a broad peak by gel filtration on Sepharose CL-2B. The mixture (CS-Glucan) was resolved into 7 glucans by HPLC on the column of Asahi-Pak GS-510 + GS-320. Similarities were observed between M, shown in the gel filtration profile and the elution volume in HPLC. Methylation analysis indicated that the ethanol-fractionated CS-glucan contained 4-O- and 4,6-di-O-substituted glucosyl residues. ¹H and ¹³C NMR data accorded with the results of methylation analysis, and the glycosidic linkages were shown to have an α-configuration. Thus, CS-glucan contained (1 → 4) linked α-d-glucans to which are attached glucosyl side chains at O-6 of the main chain in a similar way to amylopectin. Each purified glucan was shown to have different absorption maxima ( > 550 nm or 530 nm) in the iodine reaction. The results of the methylation analysis and of the pullulanase digestion suggest that the 550 nm-glucan has a lower branching frequency and shorter side chains than the 530 nm-glucan. Although CS-glucan was found to have weak anti-complementary activity, HPLC-purified > 550 nm-glucan was found to be more potent than the 530 nm-glucan. Thus CS-glucan is highly heterogeneous, and the glucans which form a tight complex when tested with iodine, generally tend to have considerable anti-complementary activity.     

On the Role of Stored mRNA in Protein Synthesis in Embryonic Axes of Germinating Vigna unguiculata Seeds

Polyadenylated (poly A⁺) RNAs were prepared from both dry and incubated embryonic axes of Vigna unguiculata seeds and were translated by a wheat germ translation system. Analysis with gel electrophoresis and fluorography showed that translation products of poly A⁺ RNA from dry embryonic axes were nearly the same as those from 2-hour incubated axes but somewhat different from those of 4- to 24-hour incubated axes, and that translation products remained almost unchanged between the 4- and 24-hour stages of postimbibition. The results indicate the possibility that the stored mRNA (poly A⁺ RNA from dry embryonic axes) directs the protein synthesis required for early stages of germination. This is supported by comparison of the in vitro translation products of poly A⁺ RNAs with those of polysomal RNAs. Experiments with α-amanitin, a specific inhibitor of RNA polymerase II (J. Jendrisak 1980 J Biol Chem 255: 8529-8533), suggested that the synthesis of some of the stored mRNA species is resumed as early as 4 hours after the onset of imbibition.     

N-terminal amino acid sequence of an insect neurohormone, melanization and reddish coloration hormone (MRCH): heterogeneity and sequence homology with human insulin-like growth factor II

Catecholamine release from chromaffin cells in response to carbamylcholine and high K+ is transient. Monitoring intracellular free calcium ([Ca2+]i) using quin2 demonstrated a transient rise in [Ca2+]i in response to carbamylcholine. The termination of secretion due to carbamylcholine is probably a consequence of the return of [Ca2+]i to resting levels as the nicotinic receptors desensitise. Depolarisation with 55 mM K+ led to a long-lasting rise in [Ca2+]i which persisted after the secretory response had terminated. The maintained rise in [Ca2+]i appeared to be due to continued opening of verapamil-sensitive Ca2+ channels. These results suggest that inactivation of voltage-dependent Ca2+ channels does not account for the transient nature of the secretory response in chromaffin cells.     

Identification of the active site in penicillin-binding protein 3 of Escherichia coli.

We report the sequence of the active site tryptic peptide of penicillin-binding protein 3 from Escherichia coli. Purified penicillin-binding protein 3 was labeled with [14C]penicillin G and digested with trypsin, and the resulting radioactive peptides were isolated by a combination of gel filtration and high-pressure liquid chromatography. The major radioactive peak from high-pressure liquid chromatography was sequenced, and the peptide Thr-Ile-Thr-Asp-Val-Phe-Glu-Pro-Gly-Ser-Thr-Val-Lys, which comprises residues 298 to 310 in the amino acid sequence, was identified. This sequence is compared with the active site sequences from other penicillin-binding proteins and beta-lactamases.     

Fimbriae from the oral anaerobe Bacteroides gingivalis: physical, chemical, and immunological properties.

Circular dichroism spectra indicated the predominance of beta-sheet structure in Bacteroides gingivalis fimbriae regardless of the presence of sodium dodecyl sulfate. By using a computer program, the alpha-helix, beta-sheet, and beta-turn contents and the remainder were estimated to be 0, 55, 18, and 27%, respectively, judging from the circular dichroism spectra of the fimbriae. Heating for 5 min at 100 degrees C in sodium dodecyl sulfate was necessary to denature the fimbriae into their constituent protein (fimbrilin) monomers with a reduced content of beta-sheet structure. The amino-terminal amino acid sequence of the fimbrilin was different from partial or complete amino acid sequences of fimbrilins so far determined from Bacteroides nodosus, which falls into the same nonfermentative species of the genus Bacteroides as B. gingivalis, and from various other bacteria. Fimbrilin monomers had an isoelectric point of 6.0. Examination of antibodies against fimbriae and sodium dodecyl sulfate-denatured fimbrilin by enzyme-linked immunosorbent assay reinforced a previous notion (F. Yoshimura, K. Takahashi, Y. Nodasaka, and T. Suzuki, J. Bacteriol. 160:949-957, 1984) that different sets of antigenic determinants seemed to be exposed on their surfaces.     

Nucleotide sequence of the extracellular glucoamylase gene STA1 in the yeast Saccharomyces diastaticus.

The complete nucleotide sequence of the extracellular glucoamylase gene STA1 from the yeast Saccharomyces diastaticus has been determined. A single open reading frame codes for a 778-amino-acid protein which contains 13 potential N-glycosylation sites. In the 5'- and 3'-flanking regions of the gene, there are striking sequence homologies to the corresponding regions of ADH1 for alcohol dehydrogenase and MAT alpha 2 for mating type control in the yeast Saccharomyces cerevisiae. The putative precursor begins with a hydrophobic segment that presumably acts as a signal sequence for secretion. The presumptive signal sequence showed a significant homology to that of Bacillus subtilis alpha-amylase precursor. The next segment, of ca. 320 amino acids, contains a threonine-rich tract in which direct repeat sequences of 35 amino acids exist, and is bordered by a pair of basic amino acid residues (Lys-Lys) which may be a proteolytic processing signal. The carboxy-terminal half of the precursor is a presumptive glucoamylase which contains several peptide segments showing a high degree of homology with alpha-amylases from widely diverse organisms including a procaryote (B. subtilis) and eucaryotes (Aspergillus oryzae and mouse). Analysis of both the nucleotide sequence of the STA1 gene and the amino acid composition of the purified glucoamylase suggested that the putative precursor is processed to yield subunits H and Y of mature enzyme by both trypsin-like and chymotrypsin-like cleavages.     

Two positional isomers of sialylheptasaccharides isolated from the urine of a patient with sialidosis

We describe the structures of two positional isomers of sialylheptasaccharide isolated from the urine of a patient with sialidosis with partial deficiency of beta-galactosidase. Based on structural studies including compositional sugar analysis, exoglycosidase digestion, chemical ionization mass spectrometry, proton nuclear magnetic resonance spectrometry, and methylation analysis, their structures were deduced to be as follows: AcNeu alpha 2----6Gal beta 1----4GlcNac beta 1----2Man alpha 1----3(Man alpha 1----6)Man beta 1----4GlcNac; AcNeu alpha 2----6Gal beta 1----4GlcNac beta 1----2Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNac. Sialyloligosaccharide 1 has previously been found in the urine and liver of patients with mucolipidosis I and II and sialidosis, but sialyloligosaccharide 2 has not been found yet in human urine. These two sialyloligosaccharides could not be completely separated by any chromatographic procedures tested. The analytical techniques, including methylation study and NMR spectroscopy, could not clearly detect the differences between them. However, alpha-mannosidase treatment gave important information for the structural analyses of these sialyloligosaccharides.     

Introduction of mouse C epsilon genes into Cos-7 cells and fertilized mouse eggs

A fragment of the cloned gene for the mouse C epsilon chain, coding for the first, second, third, and fourth domains, has been coupled to the SV40 promotor region (pSV2-mC epsilon). About 50 copies of pSV2-mC epsilon or its PvuII-EcoRI fragments were introduced into Cos-7 cells. Expression of PvuII-EcoRI fragments of pSV2-mC epsilon was observed in about 50% of the Cos-7 cells by indirect fluorescence staining. However, no expression of circular pSV2-mC epsilon was observed. About 200 copies of linearized pSV2-mC epsilon with EcoRI were introduced into fertilized mouse eggs. Two of 78 mice born from these eggs had integrated mouse C epsilon genes. Mouse C epsilon gene was shown to be integrated in a tandem array and as intact structures without undergoing gross deletions or rearrangements, judged from the Southern blotting patterns from several restriction enzymes. The first transgenic mouse was mated to a normal male to examine whether mouse C epsilon gene were stably transmitted to progeny. Among 5 mice to which the C epsilon gene had been transmitted, one deleted 5 copies of this gene and another deleted one junction fragment, thus demonstrating relatively unstable transmission. No C epsilon mRNA was detected in the liver, kidney, brain, lung, skeletal muscle, heart, testis, or spleen of a transgenic mouse.