Preparation and chemical properties of the outer membrane of a moderately halophilic gram-negative bacterium.

Outer membranes, almost free from peptidoglycan components, were prepared from a moderately halophilic gram-negative bacterium grown in a medium containing 2 M NaCl. The outer membrane was easily released, leaving mureinoplasts, by mild desalting in a 20% sucrose solution containing 50 mM tris(hydroxymethyl)aminomethane-HCl buffer, pH 7.8. The membrane was recovered by treatment with DNase I and CsCl buoyant density centrifugation. Chemical analyses revealed that the outer membrane was mainly composed of 31% protein, about 20% extractable lipids (mainly phospholipids), and lipopolysaccharides. The proteins had about 18 mol % excess of acidic over basic amino acids. The phospholipids comprised phosphatidyl ethanolamine, phosphatidyl glycerol, cardiolipin, and an unidentified phospholipid containing glucose, which seemed mainly associated with the outer membrane. The content of lipopolysaccharides in the outer membrane was calculated arbitrarily as 30% from the heptose content. A unique feature of these lipopolysaccharides seemed to be higher lipid content than found in lipopolysaccharides of other gram-negative bacteria. The major fatty acids of bound lipids of the outer membrane resembled those of the lipopolysaccharides obtained from cell envelope preparation and contained high concentrations of 3-hydroxy lauric acid.     

Hypothalamic and hormonal control of the photoperiodically induced vernal functions in the White-crowned Sparrow,Zonotrichia leucophrys gambelii

To assess the role of the hypothalamo-hypophysial complex in the control of photoperiodically induced vernal premigratory responses in the White-crowned Sparrow, the effects of hypothalamic lesions and systemic administration of several hormones on these responses were investigated. Lesions that destroyed the posterior median eminence (PME) or the entire median eminence (ME) inhibited photoperiodically induced testicular growth, premigratory fattening and Zugunruhe. Lesions in the basal infundibular nucleus (IN) that resulted in complete inhibition of testicular growth abolished Zugunruhe, but allowed varying degrees of fattening. The systemic administration of prolactin, testosterone propionate (TP) or the combination thereof in the PME-lesioned birds induced fattening similar to that observed in photostimulated controls but did not induce Zugunruhe. It is concluded that testosterone and prolactin are the most important hormones involved in the control of vernal premigratory fattening. The role of these hormones in the induction of vernal Zugunruhe is not positively proven.     

Effects of 4-[beta-(diethylamino)-ethoxy]-benzophenone upon carotenogenesis in Rhodospirillum rubrum.

Carotenoid production was determined in illuminated anaerobically maintained cultures of Rhodospirillum rubrum in media with and without 4-[beta-(diethylamino)-ethoxy]-benzophenone. In treated cultures, lycopene--which normally is not produced by R. rubrum--accumulated as the predominant pigment, and total carotenoids increased five- to sixfold.     

The Immunodetection of the Abnormal Isoform of Prion Protein

Transmissible spongiform encephalopathies such as scrapie in sheep and goats, Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle, are neurodegenerative disorders. A proposed causative agent for these diseases is an infectious protein, the so called prion. An abnormal isoform of prion protein (PrPSc) can be detected according to the prion propagation method used. As PrPSc appears to constitute the main, if not the only, infectious entity its detection for the diagnosis of prion diseases is important. Immunodetection methods for PrPSc analysis are popular tools for diagnosis and research studies. In this paper, a review of the present knowledge concerning immunodetection is presented and the enhancement of the immunoreactivity of antisera to mouse and hamster prion protein peptides using the techniques of Western blotting and immunohistochemistry is summarized.     

Induction of the differentiation of human HL-60 promyelocytic leukemia cell line by succinoyl trehalose lipids

Four analogs of succinoyl trehalose lipid-3 (STL-3)with saturated even-number or odd-number carbonchains, and unsaturated or halogenated fatty acidswere examined for their ability to inhibit the growthand induce the differentiation of HL-60 humanpromyelocytic leukemia cells. The optimalconcentration of STL-3 at which such activities wererecognized was closed to the critical micelleconcentration of STL-3. Analog of STL-3 witheven-number or odd-number carbon chain and unsaturatedfatty acids strongly inhibited growth and induced thedifferentiation of HL-60 cells, as evaluated in termsof nitroblue tetrazilium-reducing activity and theappearance of the CD36 antigen. An analog of STL-3with halogenated fatty acids significantly inhibitedproliferation but only induced the differentiation ofHL-60 cells. Our results indicate that the effects ofSTL-3 and its analogs on HL-60 cells depend on thestructure of the hydrophobic moiety of STL-3.These authors contributed equally to this work     

Type I IFN-mediated enhancement of anti-leukemic cytotoxicity of gammadelta T cells expanded from peripheral blood cells by stimulation with zoledronate

BACKGROUND: In order to establish efficient gammadelta T-cell based tumor immunotherapy, we explored a method to enhance the cytotoxicity of gammadelta T cells against leukemia cells by stimulating gammadelta T cells with type I IFN. METHODS: Gammadelta T cells were expanded from normal PBMC by culturing with zoledronate and a low concentration of IL-2 for 2 weeks. For the activation of gammadelta T cells, gammadelta T cells were cultured with type I IFN (HLBI, IFN-alpha2b and IFN-beta) for 1-3 days. The cytotoxicity of HLBI-activated gammadelta T cells against leukemia cell lines and fresh leukemia cells was evaluated by 51Cr-release assay. RESULTS: Gammadelta T cells, which were expanded and purified with magnetic beads using an anti-gammadelta TCR MAb, were demonstrated to be cytotoxic against leukemia cell lines of both lymphoid and myeloid origin and fresh myeloid leukemia cells. By culturing expanded gammadelta T cells with type I IFN, the expression of the activation marker CD69 was increased and the cytometric bead array showed an elevated production of IFN-gamma by gammadelta T cells. In addition, the cytotoxicity of gammadelta T cells against leukemia cells was definitely enhanced by culturing gammadelta T cells with HLBI. DISCUSSION: The present study has demonstrated that type I IFN could enhance the anti-leukemic cytotoxicity of expanded gammadelta T cells, which implies that in vitro bisphosphonate (such as zoledronate)-expanded and type I IFN-activated gammadelta T cells could be applied to immunotherapy for hematologic malignancies such as leukemia and lymphoma.     

Immune regulatory functions of DOCK family proteins in health and disease

DOCK proteins constitute a family of evolutionarily conserved guanine nucleotide exchange factors (GEFs) for Rho family of GTPases. Although DOCK family proteins do not contain the Dbl homology domain typically found in GEFs, they mediate the GTP–GDP exchange reaction through DHR-2 domain. Accumulating evidence indicates that the DOCK proteins act as major GEFs in varied biological settings. For example, DOCK2, which is predominantly expressed in hematopoietic cells, regulates migration and activation of leukocytes through Rac activation. On the other hand, it was recently reported that mutations of DOCK8, another member of the DOCK family proteins, cause a combined immunodeficiency syndrome in humans. This article reviews the structure, functions and signaling of DOCK2 and DOCK8, especially focusing on their roles in immune responses.     

Quantitative determination of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane

Quantitative determination of the sulfated glycoproteins present in tissue and secretion fluid was performed. After digestion of the specimen with pronase in order to convert glycoproteins to glycopeptides, the sulfated glycopeptides were separated from a mixture of acidic glycans (glycosaminoglycans, sialoglycopeptides and sulfated glycopeptides) by two-dimensional electrophoresis on cellulose acetate membrane [(1986) J. Biochem. Biophys. Methods 12, 239-246]. After staining with alcian blue, the spot of sulfated glycopeptide on the cellulose acetate membrane was cut out, and then only the dye bound to the sulfated glycopeptide was extracted with a 5% cetylpyridinium chloride solution at 100 degrees C for 15 min. The extract was then measured by absorbance at 615 nm using an authentic sulfated glycopeptide as a standard. This method facilitated the determination of sulfated glycopeptides, which were separated from other acidic glycans, within the range 0-25 micrograms.