Chemical and biological properties of a protein-rich fraction of bacterial lipopolysaccharides. II. The in vitro rat peritoneal mast cell response.

Lipid A-associated protein (LAP) isolated from preparations of bacterial lipopolysaccharides (LPS, endotoxins) has been demonstrated to initiate the release of amines from rat peritoneal mast cells. The release at low concentrations of LAP requires both cellular energy and calcium, and thus appears to be a true secretory response. At higher concentrations the release is independent of these variables. The time required for maximal response is approximately 10 to 15 min at 37 degrees C. The response appears to be a general property of Escherichia coli LAP preparations since LAP isolated from three serotypes of these organisms all have similar activity. On the basis of heat lability at 100 degrees C, the ability of LAP to initiate mast cell secretion appears to be independent of its ability mitogenically to stimulate murine B lymphocytes.     

Evidence of a limited schizogonous cycle for Cytauxzoon felis in bobcats following exposure to infected ticks

Schizogonous tissue stages of Cytauxzoon felis (Apicomplexa: Theileridae) were not observed by microscopic evaluation of impression smears of liver, spleen, lung and lymph nodes in 10 bobcats (Lynx rufus) from Oklahoma with naturally occurring piroplasm infections. Schizogonous stages were observed in similar tissues from experimentally-infected bobcats at 11 days postexposure to infected Dermacentor variabilis, but not at 30 days following tick feeding. The schizogonous cycle of this parasite appears to be short, although the bobcat appears to be a long-term carrier.     

Subcellular localization of immunoreactive dynorphin and vasopressin in rat pituitary and hypothalamus

Dynorphin is found mainly in the particulate fraction of rat pituitary gland and hypothalamus homogenates. Dynorphin-like immunoreactivity (DYN-LI) from neurointermediate lobe (NIL) homogenates migrates at the same rate as vasopressin-like immunoreactivity (AVP-LI), in sucrose density gradients, whereas DYN-LI from the hypothalamus appears to migrate principally in a less dense region of the gradient. This suggests that dynorphin and vasopressin from pituitary are present in organelles of similar size and density, while the bulk of the dynorphin in the hypothalamus appears to be stored in a different subcellular organelle. Anterior lobe (AL) dynorphin appears to migrate in two separate bands on density gradients: the less dense band (slower) migrates at a similar rate to that of dynorphin and vasopressin from NIL. When alpha-neo-endorphin was measured in sucrose gradients of NIL and hypothalamus, it was found to co-migrate with DYN-LI.     

Conversion in the peptides coating cadmium:sulfide crystallites in Candida glabrata.

Cultures of Candida glabrata treated with CdCl2 form intracellular Cd(II) complexes that evolve with the time of culturing. Initially, glutathione (gamma ECG) appears to be the major buffering component. One type of Cd(II)-glutathione complex exists as a cadmium:sulfide (CdS) crystallite coated with glutathione. A time dependent change in the coating of the CdS particles occurs with a decrease in the (gamma ECG) content and a corresponding increase in the abundance of (gamma EC)nG peptides with (gamma EC)2G becoming the predominant peptide. The des-Gly variant (gamma EC)2 appears in significant concentration only in late cultures. The evolution in isopeptide coating appears to be dependent on the sulfide content of the CdS particles. Cellular conditions that enhance the generation of sulfide ions facilitate the conversion from gamma ECG to (gamma EC)2G.     

Alloalbuminemia in North India.

Electrophoretic screening of sera from 550 individuals from Punjab, North India, revealed four cases of alloalbuminemia. Two albumin variants migrated slower and two migrated faster than the common albumin A. These variants were further analyzed by electrophoresis of their cyanogen bromide fragments to localize their molecular differences. One of the slow variants appears similar to, if not identical with, albumin B, with an altered cyanogen bromide fragment CNBr VII. The other slow variant appears to be a new variant (proposed name albumin Punjab) differing from albumin A in an altered fragment CNBr VI (which also occurs in albumins Kashmir and Adana) and in an altered fragment CNBr I. Among the fast variants, one has the same altered fragment CNBr V as albumin Naskapi, while the other appears to be a new variant (proposed name albumin Patiala) having an altered fragment CNBr VI. The presence of albumin Naskapi in Punjabis, North American Indians, and Eti Turks (previously reported) is consistent with the existence of a common ancestral population in which the mutation to Naskapi occurred before the migrations eastward and westward.     

Gut- and bronchus-associated lymphoid tissue

Bronchus-associated and gut-associated lymphoid tissues (BALT and GALT) have both functional and morphologic similarities and are involved in seeding lung, gut, and other mucosal sites with predominantly IgA-containing B cells. Both types of lymphoid tissue are engaged in the regulation and the controlled amplification of immune responses, which vary from positive mucosal responses in both mucosae and peripheral tissues to local mucosal responses and systemic tolerance. Their further involvement in provision of cells destined to reside in the epithelial compartment of the body appears likely but requires further investigation. Their role in the provision of precursors of mucosal mast cells must also be explored further, but some participation in this event appears likely. The mucosa-associated lymphoid tissue (MALT) system appears to be integrated with the systemic immune system but may be considered as separate from it in several functional ways.     

Copper-containing nitrite reductase: a DFT study of nitrite and nitric oxide adducts

Copper-containing nitrite reductases (Cu-NIRs) reduce nitrite to NO. Reported here are DFT (density functional theory) results on models of the Cu-NIR active site bound to nitrite and nitric oxide. The Cu-NIR active site appears to have been designed to exclude N-nitrite binding even though N-O bond cleavage would be equally facile in the N- and O-isomers. The active site also appears to force a side-on coordination of the end-product, nitric oxide. The latter feature has to rely on the sterics of the active site to destabilize, thermodynamically speaking, the Cu-NO adduct; under these conditions, the absence of N-nitrite coordination is proposed to be merely a side-effect. For the Cu(II)-NO adduct, sterical crowding appears to also favour the Cu-NO electromer over Cu(I)-NO+, helping to avoid the potentially damaging chemistry associated with an NO+ moiety. These conclusions are in reasonable agreement with previous conclusions drawn from experiment [Science 304 (2004) 867].     

A high molecular weight RNA fraction in chromatin.

Utilizing a new chromatin isolation and fractionation technique we have obtained a high molecular weight RNA fraction from L-929 cell chromatin. The synthesis of this RNA is not greatly inhibited by concentrations of 0.04 mug/ml actinomycin D in the medium. Its synthesis appears to be strongly inhibited by 2 mug/ml of alpha-amanitin. The RNA appears to be quickly degraded (or removed from the chromatin) and does not contain a poly(A) sequence at its 3'-OH terminal end. Our working hypothesis is that this RNA is "nascent" heterogenous nuclear RNA partially transcribed from regions of the chromatin.     

The disappearance of a cyclin-like protein and the appearance of statin is correlated with the onset of differentiation during myogenesis in vitro

We have used monoclonal antibodies to statin (S-44) and a cyclin-like protein (S-132) to examine the distribution of these two antigens in proliferating and in nonproliferating populations of cells. We have found that this cyclin-like protein is present in proliferating fibroblasts, whereas statin is absent from these same cell populations; in contrast, in senescent populations of fibroblasts the cyclin-like antigen disappears and statin labeling of nuclei appears. During myogenesis in rat muscle cell cultures, S-132 labeling is present in proliferating myoblasts and disappears after cells fuse and differentiate as multinucleated myotubes. In contrast, statin is absent from proliferating myoblasts, but appears when these cells become postmitotic and begin to differentiate. Similar results were seen during chick myogenesis. We have also found similar results during serum-starvation-induced differentiation in neuroblastoma cells. These results indicate that the cyclin-like protein disappears and statin appears upon commitment to differentiation in vitro, and the presence or the absence of these proteins appears to provide cellular markers for the transition from the proliferative to the nonproliferative state during differentiation.     

Rate studies of polysaccharide biosynthesis from guanosine diphosphate α-d-glucose and guanosine diphosphate α-d-mannose

With enzyme preparations from Phaseolus aureus seedlings, the initial rate of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]glucose is not increased by additions of GDP-alpha-d-mannose. However, final incorporation is increased by addition of GDP-alpha-d-mannose, since the total reaction-time is extended. In contrast, the initial rate of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]mannose is increased by all concentrations of GDP-alpha-d-glucose that are less than that of the GDP-alpha-d-[(14)C]mannose. Maximum stimulation of the initial rate occurs at a GDP-alpha-d-[(14)C]mannose/GDP-alpha-d-glucose concentration ratio of about 4:1. However, eventual incorporation from GDP-alpha-d-[(14)C]mannose is decreased by the addition of GDP-alpha-d-glucose, since the reaction rate falls off sharply after about 2min. Reciprocal plots of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]mannose result in biphasic graphs. The two straight-line portions of the plot are joined by a curved line in the concentration range between 2-3 and 50mum. Extrapolated K(m) values for the two linear components are 0.4-1.0 and 700-1500mum. The effect of GDP-alpha-d-glucose on the kinetics of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]mannose is complex, and depends on relative concentrations of the two sugar nucleotides. (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]glucose also results in biphasic reciprocal plots. One component appears to have K(m) about 2-3mum, the other about 200-400mum. In this reaction, GDP-alpha-d-mannose appears to be a competitive inhibitor with K(i) 20-30mum. With particulate preparations of P. aureus, GDP-alpha-d-[(14)C]glucose appears to be a precursor for the synthesis of one polysaccharide, a glucomannan, the mannose moieties of which are derived from an intermediate existing in the particulate preparation. From the rate results, GDP-alpha-d-[(14)C]mannose appears to be a precursor for at least two polysaccharides, one of which is a glucomannan.     

Cloning and localization of the repressor gene (c) of the Mu-like transposable phage D108

We have localized the D108 thermosensitive (cts) repressor gene to a region of DNA approx. 600 base pairs (bp) in length by sub-cloning an RsaI restriction endonuclease fragment (bp 200 to bp 802 from the left-end of the D108 genome). We determined that the gene product from this fragment appears to be the same size (19 kDa) as that expressed from clones containing larger fragments of D108 DNA. Results from in vitro gel electrophoresis band-retardation and in vivo immunity assays show that the sub-cloned repressor appears to be fully functional.     

Announcement

The mechanism(s) by which lentiviruses and related non-oncogenic retroviruses (e.g. HTLV-III, the etiologic agent of AIDS) escape immune surveillance, and thereby create long term progressive disease conditions, has been unknown until recently. Studies with two lentiviruses have begun to illuminate the mechanisms. In one, antigenic drift in the virus appears to be the primary mechanism of escape from immune surveillance; in the second, selective masking of the viral envelope glycoprotein epitope, which normally elicits neutralizing anti-body, appears to provide the means of escape.     

In vitro lymphocyte cytotoxicity. II. Unstable lymphotoxins (beta-LT) secreted and inactivated by mitogen-stimulated human lymphocytes.

Supernatants from phytohemagglutinin (PHA)-activated human lymphocytes contain two major classes of cytotoxins (α-LT and β-LT). While α-LT appears to be stable, and the major component in 5-day culture supernatants, the majority of cytolytic activity at earlier intervals in these cultures is due to a “family” of highly unstable cytotoxins which are both secreted and destroyed at a rapid rate. The inactivation of the unstable LT molecules appears to be due to: (a) inherent instability o β-LT molecules, and (b) a lymphocyte-mediated inactivation mechanism(s) which involves serum.     

Mapping out the roles of MAP kinases in plant defense.

Arabidopsis contains 20 MAP kinase genes, but their roles in plant physiology have remained largely unknown because of a lack of mutants. Recent papers from two groups have shed new light on the function of two different MAP kinases. The Arabidopsis MPK4 gene appears to negatively regulate salicylic acid-mediated defense responses and positively regulate jasmonic acid-induced responses. The tobacco SIPK gene (orthologous to Arabidopsis MPK6) appears to positively regulate programmed cell death.     

Protein kinases of rabbit and human erythrocyte membranes. Solubilization and characterization.

Two protein kinases (EC 2.7.1.37) from rabbit and one from human erythrocyte membranes have been solubilized with 0.5 M NaCl. These enzymes have been partially purified by (NH4)2SO4 fractionation and gel filtration. The rabbit membrane enzymes have apparent Mr values of 100 000 and 30 000, as determined in the presence of 0.4 M NaCl. In the absence of salt, these enzymes aggregate into high molecular weight species. The kinase from human erythrocyte membranes has an apparent Mr of 30 000 and appears to have properties similar to those of the 30 000-dalton rabbit kinase. All three enzymes catalyze the phosphorylation of casein and phosvitin in salt-stimulated reactions. None of these enzymes appears to be related to cyclic AMP-dependent protein kinases.     

Hydroxysteroid dehydrogenases of Pseudomonas testosteroni. Separation of a 17 beta-hydroxysteroid dehydrogenase from the 3(17) beta-hydroxysteroid dehydrogenase and comparison of the two enzymes.

When a crude extract of Pseudomonas testosteroni induced with testosterone was subjected to polyacrylamide gel electrophoresis, six bands that stained for 17 beta-hydroxysteroid dehydrogenase activity was observed. A protein fraction containing the enzyme corresponding to the fastest migrating band and devoid of the other hydroxysteroid dehydrogenase activities has been obtained. This preparation appears to be distinct from the previously isolated 3(17) beta-hydroxysteroid dehydrogenase (EC 1.1.1.51) in its chromatography properties on DEAE-cellulose, substrate and cofactor specificity, immunological properties and heat stability. The preparation appears devoid of 3alpha-, 3beta-, 11beta-, 17alpha-, 20alpha-, and 20beta-hydroxysteroid dehydrogenase activities. The enzyme transfers th 4-pro-S-hydrogen of NADH from estradiol-17beta (1,3,5(10)estratriene-3,17beta-diol) to estrone (3-hydroxy-1,3,5(10)-estratriene-17-one).     

A novel bovine spinal cord endoprotease with high specificity for dynorphin B

An endopeptidase that converts the opioid peptide dynorphin B (Tyr-Gly-Gly-Phe-Leu-Arg-aRg-Gln-Phe-Lys-Val-Val-Thr) to its bioactive fragment Leu-enkephalin-Arg6 was isolated from bovine spinal cord. The enzyme was purified about 230-fold from a concentrated spinal cord extract. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it stained as a protein of Mr 55,000. The purified enzyme is optimally active at around pH7 and has essential thiol groups. It appears to be highly specific for dynorphin B (Km = 11 microM) but not for alpha-neoendorphin or dynorphin A, two other opioids included in the prodynorphin precursor. From its specificity, molecular size, and inhibitory spectrum, this enzyme is different from other known dynorphin-converting or -degrading enzymes and appears to be a unique and novel endoprotease.     

Na,K-ATPase expression in the developing brine shrimp Artemia. Immunochemical localization of the alpha- and beta-subunits.

Developing brine shrimp are a good experimental model for study of gene expression during development. Development is initiated on suspension of brine shrimp cysts in seawater. Only 48 hr are required for progression from cyst to the larval stage. We have localize the alpha- and beta-subunits in different cells by immunostaining as development progresses. Both alpha- and beta-subunits are first detected in epidermal cells in the trunk region at the emergence 2 stage (16-hr incubation). At the nauplius 1 stage (24 hr) the enzyme appears in the brain and epidermal regions, as well as in mesenchymal cells, with weaker staining in the salt gland. After further development (nauplius 2 stage, 36 hr) stronger staining appears in the salt gland and in the epidermal region. At the nauplius 3 stage (48 hr) the enzyme appears in the midgut mucosa. Co-localization of the alpha- and beta-subunits appears in all positive cells during development. In the epidermal and salt gland cells the enzyme is mainly localized on the basolateral membrane. The basolateral localization of the Na,K-ATPase in epidermal and salt gland cells suggests that Na+ is actively transported into the epidermal and salt gland cells and passively diffuses out from the apical region.