Insulin-specific suppressor T cell factors

Murine antibody responses to heterologous insulins are controlled by MHC-linked immune response genes. Although nonresponder mice fail to make antibody when injected with nonimmunogenic variants of insulin, we have recently shown that nonimmunogenic variants stimulate radioresistant, Lyt- 1+2- helper T cells that support secondary antibody responses. However, the helper activity can not be detected unless dominant, radiosensitive Lyt-1-2+, I-J+ suppressor T cells are removed. In this paper we report that extracts of primed Lyt-2+ suppressor T cells contain insulin-specific suppressor factors (TsF) that are capable of replacing the activity of suppressor T cells in vitro. The activity of these factors is restricted by MHC-linked genes that map to the I-J region, and immunoadsorption studies indicated that they bind antigen and bear I-J-encoded determinants. Insulin-specific TsF consists of at least two chains, one-bearing I-J and the other the antigen-binding site. Furthermore, mixing of isolated chains from different strains of mice indicates that the antigenic specificity is determined by the antigen-binding chain and the MHC restriction by the H-2 haplotype of the source of the non-antigen-binding, I-J+ chain. Moreover, mixtures containing antigen-binding chain from allogeneic cell donors and I-J+ chain from responder cell donors have activity in cultures containing responder lymphocytes. This suggests that preferential activation of suppressor T cells, rather than differential sensitivity to suppression, results in the nonresponder phenotype to insulin.     

Monoclonal antibodies recognizing single amino acid substitutions in hemoglobin

Four monoclonal antibodies (mAb) to non-human primate hemoglobin referred to as Cap-4, Cap-5, Rh-2, and Rh-4, and two mAb to human hemoglobin, referred to as H-1 and H-3 were isolated and were partially characterized. Binding studies with these mAb on a panel of hemoglobins and isolated alpha and beta globin chains revealed a unique reactivity pattern for each mAb. Amino acid sequence analysis of the antigens used to generate the binding data suggests that the specific recognition of certain hemoglobin antigens by each mAb is controlled by the presence of a particular amino acid at a specific position within the epitope. The use of synthetic peptides as antigens confirmed this observation for five of the mAb. No synthetic peptides were tested with the sixth mAb, Rh-2. The amino acids required for binding of mAb Cap-4, Cap-5, Rh-4, and Rh-2 to hemoglobin are alanine at beta 5, threonine at beta 13, glutamine at beta 125, and leucine at alpha 68. The non-human primate hemoglobin antibodies require a specific amino acid that is not present in human hemoglobin. The amino acid required for binding of Cap-4, Cap-5, and Rh-4 could arise by a single base change in the beta globin gene, whereas the amino acid required for Rh-2 binding would only occur if two base changes occurred. Thus these mAb are candidate probes for a somatic cell mutation assay on the basis of the detection of peripheral blood red cells that possess single amino acid substituted hemoglobin as a result of single base substitutions in the globin genes of precursor cells.     

Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae

Summary A gene conferring high-level resistance to tylosin in Streptomyces lividans and Streptomyces griseofuscus was cloned from a tylosin-producing strain of Streptomyces fradiae. The tylosin-resistance (Tyl^r) gene (tlrA) was isolated on five overlapping DNA fragments which contained a common 2.6 Kb KpnI fragment. The KpnI fragment contained all of the information required for the expression of the Tyl^r phenotype in S. lividans and S. griseofuscus. Southern hybridization indicated that the sequence conferring tylosin resistance was present on the same 5 kb SalI fragment in genomic DNA from S. fradiae and several tylosin-sensitive (Tyl^s) mutants. The cloned tlrA gene failed to restore tylosin resistance in two Tyl^s mutants derived by protoplast formation and regeneration, and it restored partial resistance in a Tyl^s mutant obtained by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The tlrA gene conferred resistance to tylosin, carbomycin, niddamycin, vernamycin-B and, to some degree, lincomycin in S. griseofuscus, but it had no effect on sensitivity to streptomycin or spectinomycin, suggesting that the cloned gene is an MLS (macrolide, lincosamide, streptogramin-B)-resistance gene. Twenty-eight kb of S. fradiae DNA surrounding the tlrA gene was isolated from a genomic library in bacteriophage Charon 4. Introduction of these DNA sequence into S. fradiae mutants blocked at different steps in tylosin biosynthesis failed to restore tylosin production, suggesting that the cloned Tyl^r gene is not closely linked to tylosin biosynthetic genes.     

Growth, dry matter partitioning, and diurnal activities of RuBP carboxylase in citrus seedlings maintained at two levels of CO2

The long-term response of citrus rootstock seedlings to CO₂ enrichment was examined in Carrizo estrange ( Poncirua trifoliata (L.) Raf. x Citrus sinensis (L.) Osbeck] and Swingle citrumelo ( P. trifoliate x C. parodist Macf.]. Plaotlets 14 weeks old were transferred to outdoor controlled-environment chambers and maintained for 5 months from Feb. 14 to July 21. During this period, new growth (cm) of citrange and citrumelo shoots at 660 μl1⁻¹ was 94 and 69% greater, respectively, than at 330 μ1 1⁻¹. Total dry weight of both rootstock shoots had increased by over 100%. Growth of few species is affected this markedly by elevated CO₂ levels.
More carbon was partitioned to above-ground organs in CO₂-enriched citrus seedlings. Stem dry matter per unit length was also 32 and 44% greater in citrange and citrumelo, respectively. Total leaf area was increased by 124% in citrange and 85% in citrumelo due to greater leaf number and size. Variations in overall relative growth rate appeared to be related to the rapid, sequential, flush-type growth in citrus, in which an entire shoot segment with its associated leaves remains an active sink until fully expanded. RuBP carboxylase (EC 4.1.1.39) activity in leaves of recently-expanded flushes was higher in citrumelo plants grown at 660 vs 330 μ1 1⁻¹ CO₂ and changed diurnally for citrange (but not citrumelo) leaves at both CO₂ levels. The results are consistent with the hypothesis that positive long-term effects of CO² enrichment may be greater in species or during growth periods where sink capacity for carbon utilization is high.     

Comparative action of glyphosate as a trigger of energy drain in eubacteria.

Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa, each possessing a 5-enolpyruvylshikimate 3-phosphate synthase that is sensitive to inhibition by glyphosate [N-(phosphonomethyl)glycine], provide a good cross-section of organisms exemplifying the biochemical diversity of the aromatic pathway targeted by this potent antimicrobial compound. The pattern of growth inhibition, the alteration in levels of aromatic-pathway enzymes, and the accumulation of early-pathway metabolites after the addition of glyphosate were distinctive for each organism. Substantial intracellular shikimate-3-phosphate accumulated in response to glyphosate treatment in all three organisms. Both E. coli and P. aeruginosa, but not B. subtilis, accumulated near-millimolar levels of shikimate-3-phosphate in the culture medium. Intracellular backup of common-pathway precursors of shikimate-3-phosphate was substantial in B. subtilis, moderate in P. aeruginosa, and not detectable in E. coli. The full complement of aromatic amino acids prevented growth inhibition and metabolite accumulation in E. coli and P. aeruginosa where amino acid end products directly control early-pathway enzyme activity. In contrast, the initial prevention of growth inhibition in the presence of aromatic amino acids in B. subtilis was succeeded by progressively greater growth inhibition that correlated with rapid metabolite accumulation. In B. subtilis glyphosate can decrease prephenate concentrations sufficiently to uncouple the sequentially acting loops of feedback inhibition that ordinarily link end product excess to feedback inhibition of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase by prephenate. The consequential unrestrained entry is an energy-rich substrates into the aromatic pathway, even in the presence of aromatic amino acid end products, is an energy drain that potentially accounts for the inability of end products to fully reverse glyphosate inhibition in B. subtilis. Even in E. coli after glyphosate inhibition and metabolite accumulation were allowed to become fully established, a transient period where end products were capable of only partial reversal of growth inhibition occurred. The distinctive metabolism produced by dissimilation of different carbon sources also profound effects upon glyphosate sensitivity.     

Analysis of the spatial organization of microtubule-associated proteins

We have developed microdensitometer-computer correlation techniques to analyze the arrangement of microtubule arms and bridges (i.e., microtubule-associated proteins [MAPs]). A microdensitometer was used to scan immediately adjacent to the wall of longitudinally sectioned microtubules in positive transparency electron micrographs. Signal enhancement procedures were applied to the digitized densitometer output to produce a binary sequence representing the apparent axial spacing of MAP projections. These enhanced records were analyzed in two ways. (a) Autocorrelograms were formed for each record and correlogram peaks from a group of scans were pooled to construct a peak frequency histogram. (b) Cross-correlation was used to optimize the match between each enhanced record and templates predicted by different models of MAP organization. Seven symmetrical superlattices were considered as well as single axial repeats. The analyses were repeated with randomly generated records to establish confidence levels. Using the above methods, we analyzed the intrarow bridges of the Saccinobaculus axostyle and the MAP2 projections associated with brain microtubules synthesized in vitro. We confirmed a strict 16-nm axial repeat for axostyle bridges. For 26 MAP2 records, the only significant match was to a 12-dimer superlattice model (P less than 0.002). However, we also found some axial distances between MAP2 projections which were compatible with the additional spacings predicted by a 6-dimer superlattice. Therefore, we propose that MAP2 projections are arranged in a "saturated 12-dimer, unsaturated 6-dimer" superlattice, which may be characteristic of a wide variety of MAPs.     

31P nuclear magnetic resonance study of enzymatically phosphorylated glycophorin A

Glycophorin A was phosphorylated using protein kinases and the new protein was investigated using³¹P NMR spectroscopy. Most of these ～30 moles of phosphate were found to be attached to Ser and Thr. Some of these phosphate residues appear to be affected by the carbohydrate residues present. The phosphorylated protein appears to be in a severe state of aggregation, with the degree of aggregationpH-dependent.     

Distribution and molecular forms of peptides containing somatostatin immunodeterminants in extracts from the entire gastrointestinal tract of man and pig

Specimens from human porcine mucosal and muscular tissue from the entire gastrointestinal tract were extracted in acid ethanol, subjected to chromatography and analysed for somatostatin-like immunoreactivity by region-specific radioimmunoassays. The concentration of somatostatin-like immunoreactivity from man and pig ranged from 1.13 +/- 0.37 to 101.15 +/- 33.93 pmol/g wet weight, and from 7.64 to 159.48 +/- 23.79 pmol/g wet weight, respectively. In both species the highest concentrations were found in the jejunum. The immunoreactivity in intestinal mucosal extracts was distributed among four major peaks, two of which were identified by HPLC as somatostatin 1-28 and somatostatin 1-14, respectively. A peak of approx. 10 kDa was resolved by ion exchange plus HPLC into three components, two containing at least part of the somatostatin 1-14 sequence as well as (part of) the somatostatin 1-28(1-14) sequence (but differing in charge), the third containing only the 1-28(1-14) sequence. These peptides probably represent uncleaved and partially cleaved prosomatostatin. The fourth component to be identified by gel filtration was slightly larger than somatostatin 1-14. Extracts from the antrum, the pancreas and from muscular tissues contained almost exclusively somatostatin 1-14, and very little somatostatin 1-28, indicating that the somatostatin precursor is processed differently at these sites. Furthermore, extracts of porcine gastric antrum, analysed for somatostatin 1-28(1-14) immunoreactivity, showed two immunoreactive forms in the mucosa and three major forms in the muscular layers.     

The binding of Ruthenium red to tubulin

The inhibition of microtubule assembly by Ruthenium red (Deinum, J., Wallin, M., Kanje, M. and Lagercrantz, C. (1981) Biochim. Biophys. Acta 675, 209-213) could be counteracted by either taxol or dimethyl sulfoxide. Ruthenium red remained bound to the assembled microtubules. Microtubules assembled in the presence of Ruthenium red and taxol showed the typical taxol-dependent stability. The dimethyl sulfoxide-induced microtubules showed normal assembly characteristics, e.g., were GTP dependent, could be disassembled by cold, colchicine and Ca2+ and had no alterations in ultrastructure. The absolute disassembly induced by Ca2+ in the presence of dimethyl sulfoxide and Ruthenium red was dependent on the microtubule protein concentration, but independent in the absence of Ruthenium red. Ruthenium red was strongly bound to purified tubulin also in the presence of 8% (v/v) dimethyl sulfoxide. The dimethyl sulfoxide-induced assembly of purified tubulin in the presence of Ruthenium red was slightly stimulated, although the critical protein concentration was the same. It was found by resonance Raman spectroscopy with a flow technique that Ruthenium red did not bind to a specific calcium binding site on tubulin, although binding to a GTP binding site cannot be excluded. The wavenumbers of the lines in the region 375-500 cm-1 differ from those found for Ruthenium red bound to typical calcium-binding proteins such as calmodulin. Although Ruthenium red binds to serum albumin as well, the spectrum with albumin resembled that of the free dye.