Alteration of the acyl chain composition of free fatty acids,acyl coenzyme A and other liPids by dietary polyunsaturated fats

Dietary alterations were used to demonstrate selective handling of fatty acids during their redistributionin vivo. Differences in the mol Per cent of individual acyl chains in the non-esterified fatty acid, acyl-coenzyme A and PhosPholiPid fractions reflected a result of relative Precursor abundance combined with enzymic selectivities. Selective distributions were observed in the utilization of individual acyl chains between 16:0 and 18:0, 18:1 and 18:2, and among 20:3, 20:4 and 20:5, 22:6 by ligase(s), hydrolase(s) and acyl-transferases. The variations in the mol Per cent of linoleate Present in the acyl-coenzyme A fraction of liver relative to that in the non-esterified fatty acids suggested anin vivo regulation of the level of linoleoyl-coenzyme A that influenced the synthesis of both arachidonoyl-coenzyme A and lipids. The greater abundance of eicosaPentaenoic acid in the free fatty acid fraction relative to that in the acyl-coenzyme A fraction may increase the ability of dietary 20: 5n-3 to be an effective inhibitor of the synthesis of Prostaglandins derived from 20:4n-6.     

Ganglioside-dependent adhesion events of human neuroblastoma cells regulated by the RGDS-dependent fibronectin receptor and proteoglycans

Human neuroblastoma cells (Platt and La-N1) adhere and extend neurites on a ganglioside GM1-binding substratum provided by cholera toxin B (CTB). These adhesive responses, similar to those on plasma fibronectin (pFN), require the mediation of one or more cell-surface proteins [G. Mugnai and L. A. Culp (1987) Exp. Cell Res. 169, 328]. The involvement of two pFN receptor molecules in ganglioside GM1-mediated responses on CTB have now been tested. In order to test the role of cellular FN binding to its glycoprotein receptor integrin, a soluble peptide containing the Arg-Gly-Asp-Ser (RGDS) sequence was added to the medium. It did not inhibit attachment on CTB but completely inhibited formation of neurites; in contrast, the RGDS peptide minimally inhibited attachment or neurite formation on pFN. Once formed, neurites on CTB became resistant to the peptide. In order to test the role of cell-surface heparan sulfate proteoglycan (HS-PG), two approaches were used. First, the HS-binding protein platelet factor-4 (PF4) was used to dilute CTB or pFN on the substratum or, alternatively, added to the medium. Diluting the substratum ligand with PF4 had no effects on attachment on either CTB or pFN. However, neurite formation on CTB was readily inhibited and on pFN partially inhibited; the effects of PF4 were far greater than a similar dilution with nonbinding albumin. When PF4 was added to the medium of cells, attachment on either substratum was unaffected as was neurite outgrowth on pFN, revealing differences in PF4's inhibition as the substratum-bound or medium-borne component. In contrast, PF4 in the medium at low concentrations (1 microgram/ml) was highly inhibitory for neurite formation on CTB. The second approach utilized the addition of bovine cartilage dermatan sulfate proteoglycan (DS-PG), shown to bind to pFN as well as to substratum-bound CTB by ELISA, or cartilage chondroitin sulfate/keratan sulfate proteoglycan (CS/KS-PG) to the substratum or to the medium. At low concentrations, DS-PG but not CS/KS-PG actually stimulated neurite formation on CTB while at higher concentrations DS-PG completely inhibited attachment and neurite formation. While DS-PG partially inhibited attachment on pFN, it had no effect on neurite formation of the attached cells. Neuroblastoma cells adhered to some extent to substrata coated only with DS-PG, indicating "receptors" for PGs that permit stable interaction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia

Proprotein convertase subtilisin kexin type 9 (PCSK9) lowers the abundance of surface low-density lipoprotein (LDL) receptor through an undefined mechanism. The structure of human PCSK9 shows the subtilisin-like catalytic site blocked by the prodomain in a noncovalent complex and inaccessible to exogenous ligands, and that the C-terminal domain has a novel fold. Biosensor studies show that PCSK9 binds the extracellular domain of LDL receptor with K(d) = 170 nM at the neutral pH of plasma, but with a K(d) as low as 1 nM at the acidic pH of endosomes. The D374Y gain-of-function mutant, associated with hypercholesterolemia and early-onset cardiovascular disease, binds the receptor 25 times more tightly than wild-type PCSK9 at neutral pH and remains exclusively in a high-affinity complex at the acidic pH. PCSK9 may diminish LDL receptors by a mechanism that requires direct binding but not necessarily receptor proteolysis.     

A laboratory study on biochemical degradation and microbial utilization of organic matter comprising a marine diatom,land grass,and salt marsh plant in estuarine ecosystems

We studied the biochemical degradation of organic matter comprising marine diatom, land grass, and salt marsh plant in estuarine ecosystems in two laboratory microcosms consisting of estuarine sediments and coastal seawater. The materials were incubated separately and together under controlled oxic and anoxic conditions to test effects of co-metabolism and redox on overall degradation of organic matter. We followed variations of bulk parameters [total organic carbon (TOC), total nitrogen (TN), C/N ratio, δ¹³C_TOC, and δ¹⁵N_TN], fatty acid concentrations, and compound-specific δ¹³C values over 3 months. Coexistence of marine diatom (relatively labile) with land grass/salt marsh plant (relatively refractory) in the microcosms yielded a negative co-metabolism effect (retardation rather than acceleration) on the overall degradation of organic matter. The ratios of oxic to anoxic degradation rate constants (k _ox/k _an) of TOC and most fatty acids were in a range of 1.1–1.7, implying that redox conditions per se had a limited influence on degradation of fresh organic materials in estuarine ecosystems. Variations of two bacteria-specific fatty acids (iso- and anteiso-15:0) and their δ¹³C values indicated that bacterial metabolism could use organic carbon (OC) from any available material when only one single-source material was dominant in the ecosystems. However, bacteria probably utilized OC preferentially from labile marine diatom when multiple-source materials were almost equally present in the ecosystems.     

PDA: Pooled DNA analyzer

Background  

Association mapping using abundant single nucleotide polymorphisms is a powerful tool for identifying disease susceptibility genes for complex traits and exploring possible genetic diversity. Genotyping large numbers of SNPs individually is performed routinely but is cost prohibitive for large-scale genetic studies. DNA pooling is a reliable and cost-saving alternative genotyping method. However, no software has been developed for complete pooled-DNA analyses, including data standardization, allele frequency estimation, and single/multipoint DNA pooling association tests. This motivated the development of the software, 'PDA' (Pooled DNA Analyzer), to analyze pooled DNA data.     

c-mos and cdc2 cooperate in the translational activation of fibroblast growth factor receptor-1 during Xenopus oocyte maturation

During oocyte maturation in Xenopus, previously quiescent maternal mRNAs are translationally activated at specific times. We hypothesized that the translational recruitment of individual messages is triggered by particular cellular events and investigated the potential for known effectors of the meiotic cell cycle to activate the translation of the FGF receptor-1 (XFGFR) maternal mRNA. We found that both c-mos and cdc2 activate the translation of XFGFR. However, although oocytes matured by injection of recombinant cdc2/cyclin B translate normal levels of XFGFR protein, c-mos depletion reduces the level of XFGFR protein induced by cdc2/cyclin B injection. In oocytes blocked for cdc2 activity, injection of mos RNA induced low levels of XFGFR protein, independent of MAPK activity. Through the use of injected reporter RNAs, we show that the XFGFR 3' untranslated region inhibitory element is completely derepressed by cdc2 alone. In addition, we identified a new inhibitory element through which both mos and cdc2 activate translation. We found that cdc2 derepresses translation in the absence of polyadenylation, whereas mos requires poly(A) extension to activate XFGFR translation. Our results demonstrate that mos and cdc2, in addition to functioning as key regulators of the meiotic cell cycle, cooperate in the translational activation of a specific maternal mRNA during oocyte maturation.     

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

Adhesive responses of fibroblast and neuroblastoma cells to substrata coated with polyvalent or monoclonal antibody to fibronectin

Both polyvalent and hybridoma-produced antibodies to fibronectin (Fn) were used to ‘map’ the immunoaccessible subsets of cell surface fibronectin on virus-transformed murine fibroblast SVT2 and rat neuroblastoma B104 cells. As one approach to this end, attachment and spreading responses of cells were measured on tissue culture substrata coated with antibody or with plasma fibronectin to compare their adhesive responses. Both SVT2 and B104 cells adhere poorly to polyvalent anti-Fn-coated substrata over short time intervals, but within several hours changes occur which permit cells to attach and spread as well on anti-Fn as on Fn (post-adsorption of the anti-Fn with Fn also generates a maximal response). This adhesive response could be completely prevented by predigesting the cells with Flavobacterium heparanase, but not with chondroitinase ABC, indicating that the cell surface Fn responsible for antibody-mediated adhesion is associated with heparan sulfate proteoglycans on the cell surface. The compositions of the substratum-attached material (left bound after EGTA-mediated detachment of cells) from cells attaching to anti-Fn or Fn were analysed by SDS-PAGE and found to be identical within the same cell type for the two different substrata. Three hybridoma-produced antibodies, which recognize different determinants on Fn, generated different adhesive responses for SVT2 or B104 cells when adsorbed to the substratum. SVT2 cells adhered well to antibody no. 32-coated substrata but poorly to antibodies 92 or 136; on the other hand, B104 cells responded similarly to all three antibodies over short times of attachment but much better to no. 32 after a several hour incubation. These experiments indicate that (1) much of the cell surface fibronectin is complexed with heparan sulfate proteoglycan and is initially inaccessible to bind to polyvalent antibody on the substratum to promote adhesion; (2) the surface of neuroblastoma cells contains a fibronectin-like molecule which is important in their substratum adhesion; and (3) monoclonal antibodies are valuable tools in ‘mapping’ the orientation of cell surface molecules like fibronectin by measuring adhesive responses to antibody-coated substrata.