Effects of ionic concentration and pH upon the state of aggregation of Neurospora mitochondrial malate dehydrogenase

Five active isoenzymes of $\text{Neurospora}$ mitochondrial malate dehydrogenase of 105,000, 91,000, 78,000, 65,000 and 39,000 daltons were observed when the enzyme was extracted from mycelia and centrifuged in a sucrose gradient with 5 mM tris-Cl at pH 9. Only one active species of 65,000 daltons was observed in either 100 mM tris-Cl, pH 9, or 5 or 100 mM sodium citrate at either pH 4 or 6. The addition of 10 mM of either MgCl₂ or CaCl₂ to the 5 mM tris-Cl pH 9 buffer reversed the aggregation and led to the occurrence of only the 65,000 daltons species. The addition of either 10, 50 or 100 mM KCl to the 5 mM tris-Cl pH 9 buffer yielded 4, 3 and 1 isoenzymes, respectively. The latter's molecular weight was 65,000. Thus, in alkaline solution, monovalent cations at 100 mM and divalent cations at 10 mM prevent the formation of multiple molecular weight species.     

Partial Transcription of Murine Type C Viral Genomes in BALB/c Cell Lines

The mouse cell line, BALB/c 3T3, and its derivatives transformed either spontaneously or by treatment with a variety of external agents, were analyzed for cytoplasmic RNA complementary to DNA products prepared from the Kirsten strain of murine sarcoma-leukemia virus, and from an endogenous type C virus of BALB/c 3T3. Although none of these cell lines spontaneously releases complete type C virions, they all contain RNA which is partially homologous to a portion of the 35S RNA isolated from these viruses. The parental cell line, BALB/c 3T3, contains a low level of viral-related RNA, and there is an increased amount of this RNA in some of the transformed cells. The RNA detected represents only a fraction of the viral RNA found in virus-producing cells. The formation of RNA:DNA hybrids was detected by equilibrium centrifugation in Cs(2)SO(4) density gradients and by analysis with a single-strand-specific nuclease from Aspergillus oryzae. Viral DNA products prepared either from an endogenous reaction with whole virus in the presence of actinomycin D or from purified 70S viral RNA as template using avian myeloblastosis virus DNA polymerase yield comparable data. In addition, all of the BALB/c lines examined produce detectable levels of murine type C virus group-specific antigen.     

Changes Induced in Astrocyte Cathepsin D by Cytokines and Leupeptin

Cathepsin D is widely, but unevenly, distributed among cells and is capable of degrading a number of neural peptides and proteins. The present study was undertaken to examine the level of cathepsin D in astrocytes that might be relevant to its induction in inflammatory demyelination. Primary astrocytes were cultured from neonatal rat cerebrums according to the method of McCarthy and de Vellis. Based on staining for cell markers, cultures were greater than 95% astrocytes and less than 3% microglia. Under serum-free conditions, leupeptin induced a 1.4- to 2.0-fold increase, maximal by 48 hours, in cathepsin D protein quantified by a radioimmunoassay. Cathepsin D enzymatic activity, inhibitable by pepstatin, also increased. Northern blot analysis demonstrated that leupeptin also increased cathepsin D mRNA expression. Kinetic analysis indicated that maximal cathepsin D mRNA levels are detected 24 h after stimulation with leupeptin. Exposure of astrocytes under the same conditions to rat recombinant interferon-gamma, human recombinant tumor necrosis factor-alpha, human recombinant interleukin-1 beta, lipopolysaccharide, calcium ionophore, or a combination of these reagents did not increase the level of cathepsin D above controls. These results indicate that astrocytic cathepsin D mRNA and protein can be induced by selected materials. Furthermore, the effects attributed to leupeptin as a proteinase inhibitor may be modified by its ability to increase cathepsin D activity.     

Overexpression of epidermal growth factor receptor in NIH-3T3- transfected cells slows its lateral diffusion and rate of endocytosis

Interactions between membrane proteins are believed to be important for the induction of transmembrane signaling. Endocytosis is one of the responses which is regulated by both intracellular and extracellular signals. To study such interactions, we have measured the lateral mobility and rate of endocytosis of epidermal growth factor receptor in three transfected NIH-3T3 cell lines (HER84, HER22, and HER82) expressing 2 X 10(4), 2 X 10(5) and 1.5 X 10(6) EGF-receptors per cell, respectively. Using rhodamine-labeled EGF (Rh-EGF) and rhodamine-labeled monoclonal anti-EGF-receptor antibody (Rh-mAb-108), we measured twofold decreases in the lateral diffusion coefficients for each approximately 10-fold increase in EGF-receptor concentration. Since steric effects cannot account for such dependence, we propose that protein mobility within the membrane, which is determined by the rate of motion between immobile barriers, decreases due to aggregate formation. The rate of endocytosis also decreases twofold between the HER84 (2 X 10(4) receptors/cell) and HER22 (2 X 10(5) receptors/cell) cell lines, suggesting that it is diffusion limited. The comparable rates of endocytosis of the HER82 and HER22 cell lines suggest that at high receptor density endocytosis may be limited by the total number of sites for receptors in coated-pits and by their rate of recycling.     

Presence of paf-acether in human thymus

Paf-acether (paf) is a phospholipid mediator of inflammation endowed with major immunoregulatory properties. The present study demonstrates that human thymus contains large amounts of paf, as well as paf precursors. In addition, isolated thymic cells produced paf under ionophore stimulation. Paf from thymus exhibited the same biological and physiochemical properties as synthetic paf. The purity and molecular structure of paf from thymus were further characterized by reverse-phase HPLC and gas chromatography with electron-capture detection. These findings may have important implications since thymus microenvironment is essential in the proper development of bone marrow progenitors committed to the T cell lineage into thymocytes capable of emigrating to the periphery as functional T lymphocytes.     

IL-10 inhibits lipopolysaccharide-induced CD40 gene expression through induction of suppressor of cytokine signaling-3

Costimulation between T cells and APCs is required for adaptive immune responses. CD40, an important costimulatory molecule, is expressed on a variety of cell types, including macrophages and microglia. The aberrant expression of CD40 is implicated in diseases including multiple sclerosis, rheumatoid arthritis, and Alzheimer's disease, and inhibition of CD40 signaling has beneficial effects in a number of animal models of autoimmune diseases. In this study, we discovered that IL-10, a cytokine with anti-inflammatory properties, inhibits LPS-induced CD40 gene expression. We previously demonstrated that LPS induction of CD40 in macrophages/microglia involves both NF-kappaB activation and LPS-induced production of IFN-beta, which subsequently activates STAT-1alpha. IL-10 inhibits LPS-induced IFN-beta gene expression and subsequent STAT-1alpha activation, but does not affect NF-kappaB activation. Our results also demonstrate that IL-10 inhibits LPS-induced recruitment of STAT-1alpha, RNA polymerase II, and the coactivators CREB binding protein and p300 to the CD40 promoter, as well as inhibiting permissive histone H3 acetylation (AcH3). IL-10 and LPS synergize to induce suppressor of cytokine signaling (SOCS)-3 gene expression in macrophages and microglia. Ectopic expression of SOCS-3 attenuates LPS-induced STAT activation, and inhibits LPS-induced CD40 gene expression, comparable to that seen by IL-10. These results indicate that SOCS-3 plays an important role in the negative regulation of LPS-induced CD40 gene expression by IL-10.     

CYP86B1 Is Required for Very Long Chain ω-Hydroxyacid and α,ω-Dicarboxylic Acid Synthesis in Root and Seed Suberin Polyester

Suberin composition of various plants including Arabidopsis (Arabidopsis thaliana) has shown the presence of very long chain fatty acid derivatives C20 in addition to the C16 and C18 series. Phylogenetic studies and plant genome mining have led to the identification of putative aliphatic hydroxylases belonging to the CYP86B subfamily of cytochrome P450 monooxygenases. In Arabidopsis, this subfamily is represented by CYP86B1 and CYP86B2, which share about 45% identity with CYP86A1, a fatty acid ω-hydroxylase implicated in root suberin monomer synthesis. Here, we show that CYP86B1 is located to the endoplasmic reticulum and is highly expressed in roots. Indeed, CYP86B1 promoter-driven β-glucuronidase expression indicated strong reporter activities at known sites of suberin production such as the endodermis. These observations, together with the fact that proteins of the CYP86B type are widespread among plant species, suggested a role of CYP86B1 in suberin biogenesis. To investigate the involvement of CYP86B1 in suberin biogenesis, we characterized an allelic series of cyp86B1 mutants of which two strong alleles were knockouts and two weak ones were RNA interference-silenced lines. These root aliphatic plant hydroxylase lines had a root and a seed coat aliphatic polyester composition in which C22- and C24-hydroxyacids and α,ω-dicarboxylic acids were strongly reduced. However, these changes did not affect seed coat permeability and ion content in leaves. The presumed precursors, C22 and C24 fatty acids, accumulated in the suberin polyester. These results demonstrate that CYP86B1 is a very long chain fatty acid hydroxylase specifically involved in polyester monomer biosynthesis during the course of plant development.     

Influence of the cystic fibrosis transmembrane conductance regulator on expression of lipid metabolism-related genes in dendritic cells

Background

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Infections of the respiratory tract are a hallmark in CF. The host immune responses in CF are not adequate to eradicate pathogens, such as P. aeruginosa. Dendritic cells (DC) are crucial in initiation and regulation of immune responses. Changes in DC function could contribute to abnormal immune responses on multiple levels. The role of DC in CF lung disease remains unknown.

Methods

This study investigated the expression of CFTR gene in bone marrow-derived DC. We compared the differentiation and maturation profile of DC from CF and wild type (WT) mice. We analyzed the gene expression levels in DC from naive CF and WT mice or following P. aeruginosa infection.

Results

CFTR is expressed in DC with lower level compared to lung tissue. DC from CF mice showed a delayed in the early phase of differentiation. Gene expression analysis in DC generated from naive CF and WT mice revealed decreased expression of Caveolin-1 (Cav1), a membrane lipid raft protein, in the CF DC compared to WT DC. Consistently, protein and activity levels of the sterol regulatory element binding protein (SREBP), a negative regulator of Cav1 expression, were increased in CF DC. Following exposure to P. aeruginosa, expression of 3β-hydroxysterol-Δ7 reductase (Dhcr7) and stearoyl-CoA desaturase 2 (Scd2), two enzymes involved in the lipid metabolism that are also regulated by SREBP, was less decreased in the CF DC compared to WT DC.

Conclusion

These results suggest that CFTR dysfunction in DC affects factors involved in membrane structure and lipid-metabolism, which may contribute to the abnormal inflammatory and immune response characteristic of CF.