Procedures for the production and separation of H and M antigens in histoplasmin: Chemical and serological properties of the isolated products

Two strains of Histoplasma capsulatum were required to prepare maximum yields of H and of M antigen from histoplasmin. The antigens were separated and partially purified by a series of procedures yielding an overall recovery of 70 to 90% of the individual antigens. Stable products suitable for use as reference products were obtained when the final purification step employed DEAE-cellulose with phosphate buffer elution at increasing molarity and decreasing pH. A final step of purification of each antigen with slab acrylamide gel electrophoresis gave products which were highly reactive and specific in a variety of serological tests with sera from persons with proven cases of histoplasmosis and with natural infections of heterologous deep mycoses. These antigens were maximally active at concentrations of 2 to 16 g protein in the complement fixation, capillary precipitin, microimmunodiffusion, or immunoelectrophoresis tests; 0.5 g gave a maximum delayed cutaneous hypersensitivity reaction in homologously infected animals and caused no appreciable reaction in control animals. Although these antigens appeared to be specific when tested with sera from persons with natural infections, the M and H antigens demonstrated the presence of an additional antigen reacting with sera of rabbits immunized with cell membrane and cell particulate fractions of Blastomyces dermatitidis. After purification by electrophoresis, both the H and M antigens of some preparations showed some decomposition and loss of reactivity after storage at 5 C for more than six months. The overall results suggest that the purified H and M antigens of Heiner (12) have multiple serological reactivity and may function in precipitin reactions, complementfixing reactions, hemagglutination of formalin-fixed goose red blood cells, and as antigens for delayed cutaneous tests.     

Medical and surgical approach to laryngeal air sacculitis in a baboon caused by Pasteurella multocida.

Air sacculitis was diagnosed in a chronically chaired baboon, Papio anubis. Pasteurella multocida was repeatedly isolated from the air sac for a period of 1 year. The condition was characterized by the continuous accumulation of either mucoserous or purulent fluid. Biopsies of the air sac taken during the course of the disease initially revealed goblet cell hyperplasia. Later a subepithalial mononuclear or polymorphonuclear infiltrate and necrosis was the predominate finding. Medical therapy was only intermittently successful and consisted of frequent drainage and antibiotics administered locally and intravenously. The condition was resolved after surgical resection of the air sac lining membrane.     

Studies on dehydro-l-ascorbic acid 2-arylhydrazone 3-oximes: Conversion into substituted triazoles and isoxazolines

l-threo-2,3-Hexodiulosono-1,4-lactone 2-(arylhydrazones) (2) were prepared by condensation of dehydro-l-ascorbic acid with various arylhydrazines. Reaction of 2 with hydroxylamine gave the 2-(arylhydrazone) 3-oximes (3). On boiling with acetic anhydride, 3 gave 2-aryl-4-(2,3-di-O-acetyl-l-threo-glycerol-l-yl)-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones (4). On treatment of 4 with liquid ammonia, 2-aryl-4-(l-threo-glycerol-l-yl)-1,2,3-triazole-5-carboxamides (5) were obtained. Acetylation of 5 with acetic anhydride-pyridine gave the triacetates, and vigorous acetylation with boiling acetic anhydride gave the tetraacetyl derivatives. Periodate oxidation of 5 gave the 2-aryl-4-formyl-1,2,3-triazole-5-carboxamides (8), and, on reduction, 8 gave the 2-aryl-4-(hydroxymethyl)-1,2,3-triazole-5-carboxamides, characterized as the monoacetates and diacetates. Controlled reaction of 2 with sodium hydroxide, followed by neutralization, gave 3-(l-threo-glycerol-l-yl)-4,5-isoxazolinedione 4-(arylhydrazones), characterized by their triacetates. Reaction of 2 with HBr-HOAc gave 5-O-acetyl-6-bromo-6-deoxy-l-threo-2,3-hexodiulosono-1,4-lactone 2-(arylhydrazones); these were converted into 4-(2-O-acetyl-3-bromo-3-deoxy-l-threo-glycerol-l-yl)-2-aryl-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones on treatment with acetic anhydride-pyridine.     

Effect of food-deprivation on rat pancreatic islet cells

Somatostatin, insulin and glucagon concentrations in rat pancreas were measured following various intervals of food-deprivation. Tissue concentrations, as measured by radioimmunoassay, were correlated with A-, B-, and D-cell number and size using a scanning integrating image analyzer (Quantimet 720). Alterations in total islet hormone content were not correlated to changes in size or distribution of cells. This implies that changes in tissue content reflect changes in turnover of peptides rather than changes in cell size or number.     

Platelet-derived growth factor isoforms AA, AB, and BB differentially activate poly r(I):r(C)-induced genes in human fibroblast FS4 cells.

Polyribocytidylic-polyriboinosinic acid [poly r(I):r(C)]-inducible genes were isolated by a differential screening procedure from a human fibroblast cell (FS-4) cDNA bank. Among yet unidentified genes (gene 274), one codes for a protein with multiple finger motifs and has previously been detected in endothelial cells after tumor necrosis factor-alpha (TNF-alpha) treatment (A20; Opipari et al., 1990), the second one codes for a variant of the I kappa B family (Haskill et al., 1991), and a third one for the Ca2+ ATPase (isoform 1). Platelet-derived growth factor (PDGF) isoforms (AA, AB, and BB) stimulated the expression of these immediate-early genes. But the extent of the respective induction correlated neither with the number of the two receptors alpha or beta nor with the level of PDGF-stimulated receptor autophosphorylation on tyrosine. Although alpha-receptors were less abundant than beta-receptors (12,500 binding sites were estimated for PDGF-AA, KD 0.03 nM; 20,000 for PDGF-AB, KD 0.03 nM; 35,000 for PDGF-BB KD 0.16 nM) and tyrosine phosphorylation induced by PDGF-AA was significantly less than that evoked by PDGF-BB, some of the investigated genes were more strongly induced by PDGF-AA. We discuss how the differences in the biological potency of the PDGF isoforms may reside in different functions of the two receptors by activation of alternative signaling pathways.     

Tetrahydrobiopterin synthesis. An absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle.

Nitric oxide (NO) synthesis is induced in vascular smooth muscle cells by lipopolysaccharide (LPS) where it appears to mediate a variety of vascular dysfunctions. In some cell types tetrahydrobiopterin (BH4) synthesis has also been found to be induced by cytokines. Because BH4 is a cofactor for NO synthase, we investigated whether BH4 synthesis is required for LPS-induced NO production in rat aortic smooth muscle cells (RASMC). The total biopterin content (BH4 and more oxidized states) of untreated RASMC was below our limit of detection. However, treatment with LPS caused a significant rise in biopterin levels and an induction of NO synthesis; both effects of LPS were markedly potentiated by interferon-gamma. 2,4-Diamino-6-hydroxypyrimidine (DAHP), a selective inhibitor of GTP cyclohydrolase I, the rate-limiting enzyme for de novo BH4 synthesis, completely abolished the elevated biopterin levels induced by LPS. DAHP also caused a concentration-dependent inhibition of LPS-induced NO synthesis. Inhibition of NO synthesis by DAHP was reversed by sepiapterin, an agent which circumvents the inhibition of biopterin synthesis by DAHP by serving as a substrate for BH4 synthesis via the pterin salvage pathway. The reversal by sepiapterin was overcome by methotrexate, an inhibitor of the pterin salvage pathway. Sepiapterin, and to a lesser extent BH4, dose-dependently enhanced LPS-induced NO synthesis, indicating that BH4 concentration limits the rate of NO production by LPS-activated RASMC. Sepiapterin also caused LPS-induced NO synthesis to appear with an abbreviated lag period phase, suggesting that BH4 availability also limits the onset of NO synthesis. In contrast to the stimulation of LPS-induced NO synthesis, observed when sepiapterin was given alone, sepiapterin became a potent inhibitor of NO synthesis in the presence of methotrexate. This is attributable to a direct inhibitory action of sepiapterin on GTP cyclohydrolase I, an activity which is only revealed after blocking the metabolism of sepiapterin to BH4. Further studies with sepiapterin, methotrexate, and N-acetylserotonin (an inhibitor of the BH4 synthetic enzyme, sepiapterin reductase) indicated that the BH4 is synthesized in RASMC predominantly from GTP; however, a lesser amount may derive from pterin salvage. We demonstrate that BH4 synthesis is an absolute requirement for induction of NO synthesis by LPS in vascular smooth muscle. Our findings also suggest that pterin synthesis inhibitors may be useful for the therapy of endotoxin- and cytokine-induced shock.     

A synthetic sialic acid analogue is recognized by influenza C virus as a receptor determinant but is resistant to the receptor-destroying enzyme.

Synthetic sialic acid analogues varying in the substitutents at position C-9 were analyzed for their ability to replace the natural receptor determinant for influenza C virus, N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). By incubation of erythrocytes with sialyltransferase and the CMP-activated analogues, the cell surface was modified to contain sialic acid with one of the following C-9 substituents: an azido, an amino, an acetamido, or a hexanoylamido group. Among these, only 9-acetamido-N-acetylneuraminic acid (9-acetamido-Neu5Ac) was able to function as a receptor determinant for influenza C virus as indicated by the ability of the virus to agglutinate the modified red blood cells. In contrast to the natural receptors, 9-acetamido-Neu5Ac-containing receptors were found to be resistant against the action of sialate 9-O-acetylesterase, the viral receptor-destroying enzyme. No difference in the hemolytic activity of influenza C virus was detected when analyzed with erythrocytes containing either Neu5,9Ac2 or 9-acetamido-Neu5Ac on their surface. This finding indicates that cleavage of the receptor is not required for the viral fusion activity. The sialic acid analogues should be useful for analyzing not only the importance of the receptor-destroying enzyme of influenza C virus, but also other biological processes involving sialic acid.     

DNA insertions distinguish the duplicated renin genes of DBA/2 andM. hortulanus mice

In a survey of inbred and wild mouse DNAs for genetic variation at the duplicate renin loci,Ren-1 andRen-2, a variantNot I hybridization pattern was observed in the wild mouseM. hortulanus. To determine the basis for this variation, the structure of theM. hortulanus renin loci has been examined in detail and compared to that of the inbred strain DBA/2. Overall, the gross features of structure in this chromosomal region are conserved in bothMus species. In particular, the sequence at the recombination site between the linkedRen-1 andRen-2 loci was found to be identical in both DBA/2 andM. hortulanus, indicating that the renin gene duplication occurred prior to the divergence of ancestors of these mice. Renin flanking sequences inM. hortulanus, however, were found to lack four DNA insertions totaling approximately 10.5 kb which reside near the DBA/2 loci. The postduplication evolution of the mouse renin genes in thus characterized by a number of insertion and/or deletion events within nearby flanking sequences. Analysis of renin expression showed little or no difference between these mice in steady state renin RNA levels in most tissues examined, suggesting that these insertions do not influence expression at those sites. A notable exception is the adrenal gland, in which DBA/2 andM. hortulanus mice exhibit different patterns of developmentally regulated renin expression.