Variations in the carbohydrate regions of Bordetella pertussis lipopolysaccharides: electrophoretic, serological, and structural features.

Structural and immunological differences between the two components that are usually present in unequal quantities in Bordetella pertussis endotoxin preparations and are visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis have been studied by using strains 1414, A100, and 134, all in phase I. According to analyses by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thin-layer chromatography, the minor (8%) component of the endotoxin of strain 1414 (endotoxin 1414) appeared to be the predominating component of endotoxins A100 and 134. The masses of the carbohydrate chains isolated from endotoxin A100 and from the major component of endotoxin 1414 were 1,649 and 2,311 atomic mass units, respectively, as determined by 252Cf plasma desorption mass spectrometry. Comparison of the 1H nuclear magnetic resonance spectra of these chains established that four N-acetyl groups, an N-methyl group, and a 6-deoxy function, which characterize the nonreducing, distal trisaccharide of the glycose chain of strain 1414, were absent from that of strain A100. The antigenicity of endotoxin 1414, as measured by enzyme-linked immunosorbent assay, was higher than that of endotoxin A100, but fell below it when the glycose chain of endotoxin 1414 was deprived of seven sugars by treatment with nitrous acid. This observation suggests that at least three (distal, proximal, and intermediate) regions of the glycose chain of endotoxin 1414 carry antigenic determinants. One of these, located in the distal trisaccharide, is absent from both endotoxins A100 and 134.     

Structure, multiple site binding, and segmental accommodation in thymidylate synthase on binding dUMP and an anti-folate

The structure of Escherichia coli thymidylate synthase (TS) complexed with the substrate dUMP and an analogue of the cofactor methylenetetrahydrofolate was solved by multiple isomorphous replacement and refined at 1.97-A resolution to a residual of 18% for all data (16% for data greater than 2 sigma) for a highly constrained structure. All residues in the structure are clearly resolved and give a very high confidence in total correctness of the structure. The ternary complex directly suggests how methylation of dUMP takes place. C-6 of dUMP is covalently bound to gamma S of Cys-198(146) during catalysis, and the reactants are surrounded by specific hydrogen bonds and hydrophobic interactions from conserved residues. Comparison with the independently solved structure of unliganded TS reveals a large conformation change in the enzyme, which closes down to sequester the reactants and several highly ordered water molecules within a cavernous active center, away from bulk solvent. A second binding site for the quinazoline ring of the cofactor analogue was discovered by withholding addition of reducing agent during crystal storage. The chemical change in the protein is slight, and from difference density maps modification of sulfhydryls is not directly responsible for blockade of the primary site. The site, only partially overlapping with the primary site, is also surrounded by conserved residues and thus may play a functional role. The ligand-induced conformational change is not a domain shift but involves the segmental accommodation of several helices, beta-strands, and loops that move as units against the beta-sheet interface between monomers.     

Regulation of eicosanoid synthesis in microvessel endothelium: glucocorticoids do not affect arachidonyl CoA synthetase activity

The properties of acyl coenzyme A (CoA) synthetase activity were characterized in cultured rabbit coronary microvessel endothelial cells. We report here that microvessel endothelial cells contain two long-chain acyl CoA synthetases. One shows activity with a variety of fatty acids, similar to long-chain non-selective fatty acyl CoA synthetases described previously. The other activity was selective for arachidonic acid and other structurally related substrates. Both activities required ATP, Mg2+ and CoA for optimal activity. The arachidonyl CoA and the non-selective acyl CoA synthetases showed different thermolabilities. Arachidonyl CoA formation was inhibited by greater than 50% after 1 min at 45 degrees C, whereas a 15 min heating treatment was necessary to produce the same relative inhibition of oleoyl CoA synthesis. Glucocorticoid pretreatment (10(-7) M dexamethasone) of the RCME cells did not affect the apparent Km or Vmax, nor the fatty acid selectivity for either acyl CoA synthetase. Therefore, although fatty acyl CoA synthetases may be involved in limiting eicosanoid formation, these activities do not appear to be glucocorticoid-responsive.     

Regulation of ribosomal protein mRNA content and translation in growth-stimulated mouse fibroblasts

When resting (G₀) mouse 3T6 fibroblasts are serum stimulated to reenter the cell cycle, the rates of synthesis of rRNA and ribosomal proteins increase, resulting in an increase in ribosome content beginning about 6 h after stimulation. In this study, we monitored the content, metabolism, and translation of ribosomal protein mRNA (rp mRNA) in resting, exponentially growing, and serum-stimulated 3T6 cells. Cloned cDNAs for seven rp mRNAs were used in DNA-excess filter hybridization studies to assay rp mRNA. We found that about 85% of rp mRNA is polyadenylated under all growth conditions. The rate of labeling of rp mRNA relative to total polyadenylated mRNA changed very little after stimulation. The half-life of rp mRNA was about 11 h in resting cells and about 8 h in exponentially growing cells, values which are similar to the half-lives of total mRNA in resting and growing cells (about 9 h). The content of rp mRNA relative to total mRNA was about the same in resting and growing 3T6 cells. Furthermore, the total amount of rp mRNA did not begin to increase until about 6 h after stimulation. Since an increase in rp mRNA content did not appear to be responsible for the increase in ribosomal protein synthesis, we determined the efficiency of translation of rp mRNA under different conditions. We found that about 85% of pulse-labeled rp mRNA was associated with polysomes in exponentially growing cells. In resting cells, however, only about half was associated with polysomes, and about 30% was found in the monosomal fraction. The distribution shifted to that found in growing cells within 3 h after serum stimulation. Similar results were obtained when cells were labeled for 10.5 h. About 70% of total polyadenylated mRNA was in the polysome fraction in all growth states regardless of labeling time, indicating that the shift in mRNA distribution was species specific. These results indicate that the content and metabolism of rp mRNA do not change significantly after growth stimulation. The rate of ribosomal protein synthesis appears to be controlled during the resting-growing transition by an alteration of the efficiency of translation of rp mRNA, possibly at the level of protein synthesis initiation.     

Depletion of epinephrine in rat hypothalamus by a dopamine agonist,pergolide

The i.p. injection of pergolide mesylate, a dopamine agonist, at doses of 0.3–0.6 mg/kg led to a decrease in epinephrine concentration in rat hypothalamus. After a 0.6 mg/kg dose of pergolide mesylate, epinephrine concentration in hypothalamus decreased within 2 hr, reached a minimum concentration at about 8 hrs, and then returned toward control values. Norepinephrine N-methyltransferase activity was not decreased after pergolide injection in vivo nor was it inhibited by pergolide added in vitro at concentrations as high as 10^–3 M. Higher i.p. doses of less potent dopamine agonists, apomorphine (10 mg/kg) and lergotrile (3 mg/kg), also decreased epinephrine concentration in hypothalamus. The pergolideinduced decrease in hypothalamic epinephrine concentration was prevented by pretreatment with haloperidol or spiperone., antagonists of dopamine receptors. Activation of dopamine receptors appears to result in a decrease in epinephrine concentration in rat brain, possibly due to, enhanced release of epinephrine.     

Rats chronically injected with urine from Huntington's chorea patients do not striatal damage

Rats were injected subcutaneously for 147 consecutive days with large volumes of urine from control subjects and from patients with Huntington's chorea (HC) in an effort to test for presence of a possible neurotoxic substance in HC. No evidence of illness was observed in animals treated with HC urine, and their behavior did not differ from animals treated with control urine. After rats were sacrificed, striatum was examined for the biochemical and neuropathological changes seen in human striatum in HC. No deficiency of γ-aminobutyric acid content, nor reduction in activities of glutamic acid decarboxylase and choline acetyltransferase, was found in striatum of rats chronically treated with HC urine. Also, no significant differences were found between striatum of control and experimental rats by light or electron microscopy. These results neither support for exclude the possibility of a neurotoxic mechanism for the neuronal loss characteristic of HC.     

The effect of histone hyperacetylation on the nuclease sensitivity and the solubility of chromatin

We have examined the effects of histone hyperacetylation upon nuclease digestion of nuclei and subsequent fractionation of chromosomal material in the presence of MgCl2. DNase I shows a maximum sensitivity towards hyperacetylated nuclei at somewhat elevated ionic strengths (150-200 mM NaCl), whereas micrococcal nuclease exhibits no specificity for acetylated nuclei over a broad range of ionic strengths. Fractionation in the presence of MgCl2 of hyperacetylated nuclei digested with micrococcal nuclease results in a substantial increase in the amount of soluble chromatin relative to that obtained with control nuclei. This increased yield of Mg2+-soluble chromatin results from the recruitment into this fraction of oligonucleosomes containing extremely hyperacetylated histones. These results suggest that contiguous nucleosomes containing highly acetylated histones may be altered in their ability to interact with themselves and with other nucleosomes.     

Cytochalasin B blocks sperm incorporation but allows activation of the sea urchin egg

Cytochalasin B (CB) (2 × 10⁻⁶ M) prevents the incorporation of sperm into the eggs of Lytechinus pictus and Strongylocentrotus purpuratus as judged by light and transmission electron microscopy (TEM). At lower concentrations of CB (2 × 10⁻⁷ M), sperm are successfully incorporated into the egg, but their migration in the area of the egg cortex is impaired. The site of action of CB on the sperm may be on the initial rotation of the sperm nucleus in the cortex; the subsequent migration is not affected by CB. Although sperm incorporation is prevented at the higher CB concentrations, the eggs become activated—as judged by cortical reaction, increased protein synthesis and increased respiration. These findings raise the concept that egg activation by sperm could result from some pre-fusion event and hence that sperm-egg fusion would not be a prerequisite for the triggering of development. An alternative hypothesis is that fusion occurs between the acrosome process membrane and egg membrane, but since CB has destroyed the integrity of the cortex actin, the fusion bridge is so weak that it cannot be maintained without some contractile or cytoskeletal support by the cortex. The sperm may activate the CB-treated egg in the same manner as pricking with a microelectrode sometimes does.     

Regulation of the immune response to tumor antigen. VIII. The effects of host specific anti-I-J antibodies on the immune response to tumors of different origin

The efficiency of in vivo therapy using alloantisera produced to interact specifically with I-J subregion encoded determinants has been investigated in two etiologically distinct syngeneic tumor systems, both of which have been shown to evoke suppressor T-cell host responses. Administration of 2 μl/day of anti-I-J^k alloantisera caused a significant reduction in the growth of the P815 methylcholanthrene (MCA)-induced mastocytoma or the 1316 ultraviolet (uv) radiation-induced fibrosarcoma in the syngeneic host. Inhibition of tumor growth with anti-I-J antibody treatment occurred in normal as well as in subcarcinogenically uv-treated hosts given the uv-induced 1316 fibrosarcoma, even though the normal host is capable of spontaneously rejecting the tumor graft in the absence of external manipulation. Evidence is also provided that the effects of anti-I-J antibody treatment are not due to direct interactions with the tumor cells, or to contaminating antiviral antibody activity within the antiserum. We have previously demonstrated the reduction of tumor growth in two antigenically distinct MCA-induced tumor systems (S1509a, SAI) using similar treatments. The data presented herein thus reinforce the possibility that such means of therapy may be beneficial to the treatment of a wide variety to tumor types where suppression represents a detrimental component of the host response, and may also provide some insight into the mechanisms underlying the effects of uv radiation on the immune response to tumor antigen.