Iron-dependent production of a heat-modifiable, 23,000-Mr outer membrane protein in Paracoccus denitrificans.

Production of a 23,000-Mr major outer membrane protein of Paracoccus denitrificans ATCC 13543 was dependent upon the addition of iron to a succinate-salts medium. The 23,000-Mr protein was not produced in an iron-deficient medium, but production of five outer membrane proteins in the 85,000- to 72,000-Mr range and of catechol were induced. The 23,000-Mr protein was not produced in a complex medium even when ferric citrate was added to the medium. Production of the protein was influenced by the carbon source and was decreased by peptone.     

B lymphocyte regulation of human hematopoiesis

Epstein Barr virus (EBV)-transformed B lymphoblastoid cell lines (BLCL) were derived from seven different individuals. The ability of BLCL supernatants to stimulate hematopoietic colony formation in vitro was tested in a conventional stem cell assay system. Supernatants promoted the growth of single (GM, E, MK) as well as multi-lineage (GEMM) colonies in bone marrow cultures. Our results indicate that EBV-transformed B lymphocytes produce cytokines that affect in vitro stem cell proliferation and differentiation. These studies demonstrate the regulatory potential of activated B lymphocytes in human hematopoiesis.     

Involvement of GABA in palate morphogenesis and its relation to diazepam teratogenesis in two mouse strains

Previous studies have indicated that serotonin and acetylcholine stimulate palate shelf reorientation. The present studies were undertaken to determine whether gamma-aminobutyric acid (GABA) functions as an inhibitory neurotransmitter in the palate and whether diazepam mimics GABA to inhibit shelf reorientation and cause cleft palate. First, it was shown that 10(-4) M GABA inhibits palate shelf reorientation in day 14.5 AJ embryos cultured for 2 hours. Anterior palate reorientation stimulated by 10(-5) M serotonin was decreased by GABA; 10(-5) M picrotoxin (GABA antagonist) stimulated anterior shelf reorientation and reversed the effect of GABA. Diazepam (10(-4) M) partially inhibited palate shelf reorientation and that stimulated by 10(-5) M serotonin. Diazepam (400 mg/kg) was administered to AJ mice at day 13.5 of gestation and embryos were cultured at day 14.5. The inhibition produced by diazepam was significantly reduced by 10(-5) M picrotoxin. The teratogenic effect of diazepam was compared with AJ and Swiss-Webster Vancouver (SWV) inbred strains. Diazepam produced greater clefting in SWV mice (57% net) than in the AJ (18% net) when compared to their water- and food-starved controls. The greater sensitivity of the SWV strain than the AJ strain to diazepam, as well as to GABA, was also observed in embryo culture. GABA (10(-5) M) markedly inhibited posterior palate reorientation and reversed the stimulation produced by bethanechol in SWV mice. The inhibitory effects of GABA on the posterior palate were partially reversed by picrotoxin. Furthermore, diazepam inhibited palate reorientation either when administered to the pregnant dam or added in embryo culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo effects of monoclonal antibody to ICAM-1 (CD54) in nonhuman primates with renal allografts

These studies test whether allograft rejection can be blocked by interference with leukocyte adhesion, using a murine IgG2a mAb (R6.5) reactive with monkey ICAM-1 (CD54). In 16 Cynomolgus renal allograft recipients, R6.5 was administered prophylactically as the sole immunosuppressive agent for 12 days (0.01 to 2 mg/kg/day). Survival in 14 recipients with technically successful grafts was significantly prolonged (24.2 +/- 2.4 vs 9.2 +/- 0.6 days for controls; p less than 0.001). Intercellular adhesion molecule-1 (CD54) (ICAM-1) was expressed on vascular endothelium in the kidney and other organs in the monkey in a pattern similar to that in humans. During cellular rejection in controls, ICAM-1 expression increased on endothelial cells, infiltrating mononuclear leukocytes and tubular cells. Biopsies during R6.5 administration showed decreased T cell infiltration (CD2, CD8, CD4) compared with controls and decreased arterial endothelial inflammation. No changes occurred in circulating T cells, aside from variable coating with mIgG. In six of eight other recipients R6.5 administration (0.5 to 2 mg/kg/day for 10 days) reversed preexisting rejection that resulted from taper of Cyclosporine to subtherapeutic levels. Responding grafts showed decreased edema and hemorrhage but no consistent change in the infiltrate. At 1 h after the first dose, mouse IgG deposited primarily on the graft vascular endothelium without any change in the inflammatory infiltrate. Mouse IgG also deposited on the endothelium of normal organs without eliciting an inflammatory response and was cleared from the endothelium within 4 days. Inasmuch as the principal site of binding was the vascular endothelium, we hypothesize that the antibody blocks adhesion to graft ICAM-1 molecules on the vessels. Anti-ICAM-1 also binds to recipient cells and may interfere with Ag presentation and/or T cell interactions. Whatever the mechanism(s), these studies indicate that an anti-ICAM-1 antibody inhibits T cell mediated injury in vivo, and that ICAM-1 is a critical molecule in the pathogenesis of allograft rejection.     

Fine needle aspiration biopsy of hepatocellular carcinoma. Some unusual features.

The cytologic features of fine needle aspiration smears from 28 hepatocellular carcinomas (HCC) were reviewed. All aspirations except one were guided. There were 14 well-, 11 moderately and 3 poorly differentiated HCC. The better-differentiated HCC were characterized by similarity of the tumor cells to hepatocytes (83%), cohesive cell clusters with a trabecular arrangement (72%) and presence of sinusoidal endothelial cells (66%). Other features included bile production (38%), atypical hepatocytic naked nuclei (52%), acinar formation (31%), intracytoplasmic vacuoles (14%) and abnormal vascular patterns (14%). Poorly differentiated HCC showed dyshesive pleomorphic cells. Unusual cytologic features from a well-differentiated HCC with fatty change and an HCC with a prominent acinar component are described. The identification of fatty change in dissociated well-differentiated hepatocytes or cytologic features suggestive of an adenocarcinoma do not preclude the diagnosis of HCC. The usefulness of cell blocks is emphasized.     

Divergent effects of peripheral and global neuropeptide Y deletion

Objectives:Neuropeptide Y (NPY) is involved in the coordination of bone mass and adiposity. However, multiple NPY sources exist and their individual contribution to the skeleton and adiposity not known. The objectives of our study were to evaluate the effects of peripheral mesenchymal derived NPY to the skeleton and adiposity and to compare them to the global NPY^KO model.Methods:To study the role of mesenchymal-derived NPY, we crossed conditional NPY (NPY^fl/fl) mice with Prx1cre to generate PrxNPY^KO mice. The bone phenotype was assessed using micro-CT. The skeletal phenotype of PrxNPY^KO mice was subsequently compared to global NPY^KO model. We evaluated body weight, adiposity and functionally assessed the feeding response of NPY neurons to determine whether central NPY signaling was altered by Prx1cre.Results:We identified the increase in cortical parameters in PrxNPY^KO mice with no changes to cancellous bone. This was the opposite phenotype to global NPY^KO mice generated from the same conditional allele. Male NPY^KO mice have increased adiposity, while PrxNPY^KO mice showed no difference, demonstrating that local mesenchymal-derived NPY does not influence adiposity.Conclusion:NPY mediates both positive and negative effects on bone mass via separate regulatory pathways. Deletion of mesenchymal-derived NPY had a positive effect on bone mass.     

CXCR4 is a major chemokine receptor on glioma cells and mediates their survival

Chemokines were described originally in the context of providing migrational cues for leukocytes. They are now known to have broader activities, including those that favor tumor growth. We addressed whether and which chemokines may be important promoters of the growth of the incurable brain neoplasm, malignant gliomas. Analyses of 16 human glioma lines for the expression of chemokine receptors belonging to the CXCR and CCR series revealed low to negligible levels of all receptors, with the exception of CXCR4 that was expressed by 13 of 16 lines. All six resected human glioma specimens showed similarly high CXCR4 expression. The CXCR4 on glioma lines is a signaling receptor in that its agonist, stromal cell-derived factor-1 (SDF-1; CXCL12), produced rapid phosphorylation of mitogen-activated protein kinases. Furthermore, SDF-1 induced the phosphorylation of Akt (protein kinase B), a kinase associated with survival, and prevented the apoptosis of glioma cells when serum was withdrawn from the culture medium. SDF-1 also mediated glioma chemotaxis, in accordance with this better known role of chemokines. We conclude that glioma cells express a predominant chemokine receptor, CXCR4, and that this functions to regulate survival in part through activating pathways such as Akt.     

Properties and Expression of Na+/K+-ATPase α-Subunit Isoforms in the Brain of the Swamp Eel,Monopterus albus,Which Has Unusually High Brain Ammonia Tolerance

The swamp eel, Monopterus albus, can survive in high concentrations of ammonia (>75 mmol l⁻¹) and accumulate ammonia to high concentrations in its brain (∼4.5 µmol g⁻¹). Na⁺/K⁺-ATPase (Nka) is an essential transporter in brain cells, and since NH₄ ⁺ can substitute for K⁺ to activate Nka, we hypothesized that the brain of M. albus expressed multiple forms of Nka α-subunits, some of which might have high K⁺ specificity. Thus, this study aimed to clone and sequence the nka α-subunits from the brain of M. albus, and to determine the effects of ammonia exposure on their mRNA expression and overall protein abundance. The effectiveness of NH₄ ⁺ to activate brain Nka from M. albus and Mus musculus was also examined by comparing their Na⁺/K⁺-ATPase and Na⁺/NH₄ ⁺-ATPase activities over a range of K⁺/NH₄ ⁺ concentrations. The full length cDNA coding sequences of three nkaα (nkaα1, nkaα3a and nkaα3b) were identified in the brain of M. albus, but nkaα2 expression was undetectable. Exposure to 50 mmol l⁻¹ NH₄Cl for 1 day or 6 days resulted in significant decreases in the mRNA expression of nkaα1, nkaα3a and nkaα3b. The overall Nka protein abundance also decreased significantly after 6 days of ammonia exposure. For M. albus, brain Na⁺/NH₄ ⁺-ATPase activities were significantly lower than the Na⁺/K⁺-ATPase activities assayed at various NH₄ ⁺/K⁺ concentrations. Furthermore, the effectiveness of NH₄ ⁺ to activate Nka from the brain of M. albus was significantly lower than that from the brain of M. musculus, which is ammonia-sensitive. Hence, the (1) lack of nkaα2 expression, (2) high K⁺ specificity of K⁺ binding sites of Nkaα1, Nkaα3a and Nkaα3b, and (3) down-regulation of mRNA expression of all three nkaα isoforms and the overall Nka protein abundance in response to ammonia exposure might be some of the contributing factors to the high brain ammonia tolerance in M. albus.     

Effect of fermentation inhibitors in the presence and absence of activated charcoal on the growth of Saccharomyces cerevisiae

The acidic hydrolysis of biomass generates numerous inhibitors of fermentation, which adversely affect cell growth and metabolism. The goal of the present study was to determine the effects of fermentation inhibitors on growth and glucose consumption by Saccharomyces cerevisiae. We also conducted in situ adsorption during cell cultivation in synthetic broth containing fermentation inhibitors. In order to evaluate the effect of in situ adsorption on cell growth, five inhibitors, namely 5-hydroxymethylfurfural, levulinic acid, furfural, formic acid, and acetic acid, were introduced into synthetic broth. The existence of fermentation inhibitors during cell culture adversely affects cell growth and sugar consumption. Furfural, formic acid, and acetic acid were the most potent inhibitors in our culture system. The in situ adsorption of inhibitors by the addition of activated charcoal to the synthetic broth increased cell growth and sugar consumption. Our results indicate that detoxification of fermentation media by in situ adsorption may be useful for enhancing biofuel production.