Dietary menhaden and corn oils and the red blood cell membrane lipid composition and fluidity in hyper- and normocholesterolemic miniature swine

This paper describes the design and synthesis of a tricationic transition state analogue (TSA 1) for the Diels-Alder reaction. TSA 1 contains a bicyclo[2.2.1]heptene ring system that mimics the boat conformation of the Diels-Alder transition state and is designed to bind tightly to antibodies, nucleic acids, and imprinted polymers by means of hydrogen bonds and salt-bridges. This paper also describes the syntheses of the Diels-Alder reaction substrates (diene 2 and dienophile 3) and a sensitive HPLC assay to monitor the formation of Diels-Alder product 4. In contrast to previously reported TSAs and dienophiles for the Diels-Alder reaction that are based upon maleimides, TSA 1 and dienophile 3 are based upon fumaramide. The fumaramide system should destabilize the initially formed boat conformer of Diels-Alder product 4 and stabilize a half-chair conformer. The conversion of the initially formed boat conformer to the half-chair conformer is designed to help prevent Diels-Alder product 4 from binding strongly to catalysts selected to strongly bind TSA 1. This feature should minimize product inhibition, which can be a problem in the catalysis of the Diels-Alder reaction.     

Comparison of sequence preference of tomaymycin- and anthramycin-DNA bonding by exonuclease III and lambda exonuclease digestion and UvrABC nuclease incision analysis

The DNA bonding sites of two pyrrolo[1,4]benzodiazepine derivatives--tomaymycin (Tma) and anthramycin (Atm)--were identified by exonuclease III (exo III) digestion, lambda exonuclease (lambda exo) digestion, and UvrABC nuclease incision analysis. exo III digestion stalls 4-5 bases 3' to a drug-DNA adduct. While this method can recognize most of the Atm-and Tma-DNA modification sites, it is complicated in that exo III digestion is also stalled by certain unmodified sequences and by drug bound to the opposite strand. lambda exo digestion stalls 1-2 bases 5' to a drug-DNA adduct. The lambda exo method also recognizes most of the drug-DNA bonding sites and renders a cleaner background; however, it is also affected by opposite-strand drug bonding. Due to their intrinsic digestion polarities, these two exonucleases tend to be stalled by the drug-DNA adduct at one end of the DNA molecule. Purified UvrA, UvrB, and UvrC proteins acting together make dual incisions 6-8 bases 5' and 4 bases 3' to a Atm- or Tma-DNA adduct. This nuclease complex recognizes all the Tma- and Atm-DNA bonding sites identified by exonuclease digestion methods, and all the UvrABC incisions can be attributed to drug modifications in the incised DNA strand. The degree of UvrABC nuclease incision increases with increasing drug concentrations for DNA modification. Using the UvrABC incision method, we have identified the sequence preference of Tma- and Atm-DNA adduct formation in three DNA fragments, and we have found that these two drugs have different preferred sites for adduction. Both Tma- and Atm-DNA bonding is strongly influenced by the 5' and 3' neighboring bases; the orders of preferred 5' and 3' bases for Tma are A > G, T > C, and A, C > G, T, and for Atm the orders are A > G > T > C and A > G > T, C. The preferred triplets for Tma bonding are -AGA- > -GGC-, -TGC-, and AGC- and for Atm are -AGA-, -AGG- > -GGA-, -GGG-.     

Biosynthesis of pteridines. NMR studies on the reaction mechanisms of GTP cyclohydrolase I, pyruvoyltetrahydropterin synthase, and sepiapterin reductase

GTP cyclohydrolase I catalyzes a ring expansion affording dihydroneopterin triphosphate from GTP. [1',2',3',4',5'-13C5, 2'-2H1]GTP was prepared enzymatically from [U-13C6]glucose for use as enzyme substrate. Multinuclear NMR experiments showed that the reaction catalyzed by GTP cyclohydrolase I involves the release of a proton from C-2' of GTP that is exchanged with the bulk solvent. Subsequently, a proton is reintroduced stereospecifically from the bulk solvent. This is in line with an Amadori rearrangement mechanism. The proton introduced from solvent occupies the pro-7R position in the enzyme product. The data also confirm that the reaction catalyzed by pyruvoyltetrahydropterin synthase results in the incorporation of solvent protons into positions C-6 and C-3' of the enzyme product. On the other hand, the reaction catalyzed by sepiapterin reductase does not involve any detectable incorporation of solvent protons into tetrahydrobiopterin.     

Interleukin 13 inhibits growth of human renal cell carcinoma cells independently of the p140 interleukin 4 receptor chain

Interleukin-13 (IL-13) is a cytokine produced primarily by activated T lymphocytes. It exerts a variety of effects on different cell types, including monocytes, B lymphocytes, mast cells, and keratinocytes. The effects of IL-13 on target cells are often similar to the effects of IL-4, which is another cytokine product of activated T lymphocytes. We recently described the expression of intermediate- to high-affinity receptors for IL-13 (IL-13R) on renal cell carcinoma (RCC) cells. In the present study, we examined the effect of IL-13 on the growth of RCC cells as measured by [3H]thymidine uptake and a clonogenic assay. In addition, we used an IL-4R-specific antibody to examine the specificity of IL-4R and IL-13R binding and function. We observed that IL-13 inhibited RCC cell proliferation by up to 50% and colony formation by up to 32% when compared with cells cultured in medium alone. A combination of IL-4 and IL-13 did not have an additive or synergistic effect on the growth of RCC cells. These cells expressed mRNA for IL-13 and secreted immunoreactive IL-13 protein in culture. The growth-inhibitory effects of IL-13 were specific, because they were not affected by antibodies to IL-4 or to the 140-kilodalton subunit of IL-4R. Furthermore, polyclonal antibodies to IL-4R failed to inhibit the binding of 125I-IL-13 to RCC cells. These results indicate that IL-13 has significant antiproliferative effects on human RCC cells, and the inhibition of IL-13 effects by anti-IL-4R antibody previously reported in lymphoid cells does not occur in RCC cells.     

Evidence for a neurotensin receptor in rat serosal mast cells

OBJECTIVE AND DESIGN: The ability of neurotensin (NT) at nmolar levels to stimulate exocytosis of the mast cell suggested that it could play a role in neuro-immune-endocrine interactions. The inhibition by a specific receptor antagonist of NT's mast cell stimulation suggested the presence of a specific mast cell NT receptor. We have here employed several probes to determine if a specific neurotensin receptor was present on rat serosal mast cells. MATERIAL: Serosal mast cells were isolated from the peritoneal and pleural cavities of male Sprague-Dawley rats. METHODS: Immunocytochemistry with an antibody raised against the C-terminal peptide of the neurotensin receptor was utilized. The same antibody was employed in immunoblotting following SDS gel electrophoresis of mast cell extracts. An RNA probe for ribonuclease protection assays (RPA) was prepared using the rat brain neurotensin receptor cDNA and polymerase chain reaction was carried out using primers based on the rat brain neurotensin receptor sequence. RESULTS: Mast cells showed specific staining with the anti-neurotensin receptor antibody and this same antibody revealed a protein on SDS gels migrating as a 70 kDa species. Ribonuclease protection assays revealed the predicted protected fragment at approximately 450 bp while PCR amplification gave a major product at 843 bp. CONCLUSIONS: These results indicate that a specific neurotensin receptor is present on the rat mast cell.     

Triplet correlation in DNA sequences and stability of heteroduplexes

Rhodococcus equi was isolated from the lungs of six foals with bronchopneumonia. All isolates expressed 15-17-kd antigens by immunoblot analysis and contained a virulence-associated plasmid of 85 or 90 kb. Immunohistochemically, R. equi from all pulmonary lesions showed the expression of 15-17-kd antigens mainly in the phagocytic cells. The specific monoclonal antibody to 15-17-kd antigens of R. equi (MAb 10G5) may be an aid in the diagnosis of R. equi-induced pneumonia.     

Differential behavior of (25R)-5,6-epoxyspirostan-22 alpha-O-3 beta-ol and (25R)-5,6-epoxyspirostan-22 alpha-O-3 beta, 4 beta-diol toward Dowex

The acid-catalyzed hydrolytic cleavage of the 5,6-epoxyspirostane derivatives by the cation exchange resin Dowex 50W X8 has been exploited with the goal of developing synthetic protocols toward 3,4,5,6-polyhydroxyspirostane analogs that can serve as intermediates to potential biologically active compounds. Whereas the diastereomers (25R)-5 alpha, 6 alpha-epoxyspirostan-22 alpha-O-3 beta-ol and (25R)-5 beta, 6 beta-epoxyspirostan-22 alpha-O-3 beta-ol yield two products, (25R)-6 beta-methoxyspirostan-22 alpha-O-3 beta, 5 alpha-diol and (25R)-spirostan-22 alpha-O-3 beta, 5 alpha, 6 beta-triol on Dowex treatment in water-methanol, the alpha- and beta-diastereomers of the 5,6-epoxy derivative of 3 beta, 4 beta-diol provide a single product, (25R)-3 beta, 6 beta-dihydroxy-5 alpha-spirostan-4-one, in good yields. The structures of these products have been confirmed using 1H NMR, 13C NMR, and 1H-1H J-correlated spectroscopies. Multifunctional product formation suggests tremendous utility of Dowex in steroid synthesis. The product formation has been rationalized on the basis of differential conformational constraints of the A/B rings of the different epoxides in directing the reaction course. The reaction shows an interesting example of stereoelectronic effect of a single hydroxy group in discriminating solvent participation.     

An established hybridoma clone producing a monoclonal antibody against Vibrio anguillarum

Vibrio anguillarum is a pathogenic microorganism of vibriosis, an infectious disease found in various fish species. A mouse hybridoma clone, named C5, that produced a monoclonal antibody to V. anguillarum was established. The specific reaction of C5 antibody with V. anguillarum was confirmed by the pre-adsorption effect of the V. anguillarum cells in ELISA and a cell immunoprecipitation experiment. Western blotting analysis indicated that the C5 antibody recognized a high molecular weight substance extracted from cells with detergents.     

Expression and characterization of the rat D3 dopamine receptor: pharmacologic properties and development of antibodies

Serotonin (5-HT) potently contracts the fundus of the rat stomach; however, the associated transduction pathway has not been described fully. Experiments were performed in an attempt to gain insight into the coupling mechanism associated with this fundal 5-HT receptor. 5-HT-stimulated [35S]GTP gamma S binding to a protein which was recognized by anti-G alpha Z antiserum in a Mg(++)-dependent fashion. 5-HT increased [35S]GTP gamma S binding in the fundus, but not in the corpus of the rat stomach. 5-HT also enhanced the binding of [alpha-32P]GTP to the fundal protein and increased the hydrolysis of GTP to GDP in fundal membranes. The fundal protein which binds GTP is 25 to 29 kDa in size whereas the brain G alpha Z protein which is recognized by the anti-G alpha Z antibody is a 41 kDa protein. Mixing experiments revealed that the fundal guanine nucleotide binding protein does not appear to be a proteolytic product of the 41 kDa G alpha Z protein. Activating protein kinase C with phorbol-12-myristate, 13-acetate induced a concentration-dependent, noncompetitive inhibition of [35S]GTP gamma S binding to the fundal protein, and of 5-HT-induced contraction of fundal strips. Phorbol-12-myristate, 13-acetate did not alter carbachol- or KCl-mediated fundus contraction. Furthermore, the activation of [35S]GTP gamma S binding by serotonergic agonists and its inhibition by pharmacological antagonists corresponded to the known actions of these agents on contraction of fundal muscle. The results provide evidence that the 5-HT receptor in the rat stomach fundus is coupled directly or indirectly to a G alpha z-like protein which may mediate 5-HT-induced contraction in this tissue.     

Development of a monoclonal-based enzyme-linked immunoassay for saxitoxin-induced protein

A monoclonal antibody was generated against saxitoxin-induced protein (SIP) from the small shore crab Hemigrapsus oregenesis. SIP was induced by saxitoxin injection and could be detected in the crude crab extracts with both polyclonal and monoclonal antibody preparations. On Western blots, the polyclonal serum reacted against several bands which were induced by saxitoxin in the crude extracts. These bands represented proteins related to SIP. The monoclonal (4G5), however, was specific for the 79,000 mol. wt subunit of SIP. A triple antibody sandwich ELISA was developed in which polyclonal anti-SIP IgG was used as a trapping layer and monoclonal 4G5 was used as the detection layer. This assay was shown to be more specific and more accurate than a direct bind assay which employed the polyclonal antiserum alone. Although the polyclonal serum was more sensitive than the monoclonal on Western blots, the triple antibody sandwich and direct bind ELISAs were of comparable sensitivity.     

Serologic correlates of immunity in a tetravalent reassortant rotavirus vaccine trial. US Rotavirus Vaccine Efficacy Group

The correlation of antibody responses (serum rotavirus IgA and neutralizing antibody to serotype G1-G4 human rotaviruses and rhesus rotavirus [RRV]) in a reassortant rotavirus vaccine trial with protection against rotavirus infection or disease was investigated. Most subjects administered 4 x 10(5) pfu of either the serotype G1 monovalent or serotype G1-G4 tetravalent vaccine seroconverted for at least one of the six antibodies (85% and 91%, respectively). However, fewer than one-third seroconverted to any prototype G1-G4 human rotavirus. Analyses of covariance indicated that higher prevaccination neutralizing antibody titers negatively affected postvaccination titers. Significant relationships were found between several postvaccination rotavirus antibody titers and protection, and serotype-specific correlates of protection were identified between anti-Wa titers and G1 illnesses (P = .03) and between anti-RRV titers and G3 illnesses (P < .001). Overall, however, serotype-specific immunity was no more significant than heterotypic immunity, and no specific titer of any antibody analyzed was a reliable indicator of protection.     

7-Methoxy-2-nitronaphtho[2,1-b]furan (R7000)-induced mutation spectrum in the lacI gene of Escherichia coli: influence of SOS mutagenesis

The mutagenic specificity of 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), a very potent genotoxic 2-nitrofuran, was investigated in the lacI gene of E.coli. To analyze the influence of SOS-mutagenesis on R7000-induced mutations, 86 and 84 LacI- mutants were respectively isolated from umuC+ and umuC strains. Treatment of bacteria with increasing concentrations of R7000, affected 2-4 times more the survival rate in the umuC context, as compared to umuC+. 80% of all mutations occurred primarily at G:C base pairs and were substitution events and single-base frameshifts (-1) in the same proportions. The six possible substitution events were observed in both strains. In the umuC+ context, they were dominated by G:C-->T:A transversions. 38% of substitutions at G:C base pairs occurred in the consensus sequence 5'TGGCG3' or 5'TGGC3' where the G was mutated. When umuC was deficient G:C-->C:G transversions were mainly observed. The proportions of substitution mutations were very similar to those that have been reported for apurinic (AP) sites, suggesting strongly that one mechanism for R7000-induced mutations is the formation of intermediate abasic sites that serve as a substrate for error-prone repair. Single frameshift events consisted essentially of deletions of one (G:C) base pair in runs of contiguous G or C residues. Frameshift frequency increased with the length of the reiterated sequence, suggesting a strand-slippage process. Other mutational classes were recovered to a lower extent, including double-base frameshifts and large deletions. In addition, 10% of the mutants presented two proximate mutations. Comparison of the mutations induced by R7000 in the umuC+/umuC backgrounds suggests an influence of the umuC product on strand specificity of R7000-induced mutations, particularly in the case of frameshift events.     

Oxygen concentration determines regiospecificity in soybean lipoxygenase-1 reaction via a branched kinetic scheme

The effect of oxygen concentration on the regiospecificity of the soybean lipoxygenase-1 dioxygenation reaction was studied. At low oxygen concentrations (<5 microM), a dramatic change in the regiospecificity of the enzyme was observed with the hydroperoxy-octadecadienoic acid (HPOD) 13:9 ratio closer to 50:50 instead of the generally reported 95:5. This alteration of regiospecificity is not an isolated phenomenon, since it occurs during a reaction carried out under "classical" conditions, i.e. in a buffer saturated with air before the reaction. beta-carotene bleaching and electronic paramagnetic resonance findings provided evidence that substrate-derived free radical species are released from the enzyme. The kinetic scheme proposed by Schilstra et al. (Schilstra, M. J., Veldink, G. A. & Vliegenthart, J. F. G. (1994) Biochemistry 33, 3974-3979) was thus expanded to account for the observed variations in specificity. The equations describing the branched scheme show two different kinetic pathways: a fully enzymatic one leading to a regio-isomeric composition of 13-HPOD:9-HPOD = 95:5, and a semienzymatic one leading to a regio-isomeric composition of 13-HPOD:9-HPOD = 50:50. The ratio between the two different pathways depends on oxygen concentration, which thus determines the overall specificity of the reaction.     

Turnover of inositol polyphosphate pyrophosphates in pancreatoma cells

There is little information concerning the intracellular function of inositol 1,3,4,5,6-pentakis- and hexakisphosphate, despite their being the most abundant inositol polyphosphates. Current opinions that they play passive roles as antioxidants (Graf, E., Mahoney, J. R., Bryant, R. G., and Eaton, J. W. (1987) J. Biol. Chem. 259, 3620-3624) or "housekeeping" molecules (Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205) arises from belief in their metabolic lethargy. However, we have discovered that cell homogenates, incubated with 5 mM fluoride and 5 mM ATP, converted both inositol hexakisphosphate (Km = 2 +/- 0.5 microM, Vmax = 9 +/- 2 pmol/mg of protein/min) and inositol 1,3,4,5,6-pentakisphosphate (Km = 13 +/- 4 microM, Vmax = 11 +/- 5 pmol/mg of protein/min) to more polar products. These reactions were also observed in intact cells treated with 0.5-20 mM fluoride, and the precursor/product relationships were confirmed by comparing the effects of fluoride on cells differentially labeled with [3H]inositol in either short-term or pulse-chase protocols. The novel products were determined to be inositol pyrophosphates because of their relatively specific hydrolysis by tobacco pyrophosphatase and alkaline phosphatase. The pyrophosphates were metabolized rapidly by cell homogenates back to their pentakisphosphate and hexakisphosphate precursors. This endogenous pyrophosphatase activity was inhibited by up to 99% by 5 mM fluoride in vitro. In intact cells incubated with 10 mM fluoride, about 20% of the inositol 1,3,4,5,6-pentakisphosphate pool, and 50% of the inositol hexakisphosphate pool were each converted to pyrophosphate derivatives within 1 h.     

Cytosolic Sec13p complex is required for vesicle formation from the endoplasmic reticulum in vitro

The SEC13 gene of Saccharomyces cerevisiae is required in vesicle biogenesis at a step before or concurrent with the release of transport vesicles from the ER membrane. SEC13 encodes a 33-kD protein with sequence homology to a series of conserved internal repeat motifs found in beta subunits of heterotrimeric G proteins. The product of this gene, Sec13p, is a cytosolic protein peripherally associated with membranes. We developed a cell-free Sec13p-dependent vesicle formation reaction. Sec13p-depleted membranes and cytosol fractions were generated by urea treatment of membranes and affinity depletion of a Sec13p-dihydrofolate reductase fusion protein, respectively. These fractions were unable to support vesicle formation from the ER unless cytosol containing Sec13p was added. Cytosolic Sec13p fractionated by gel filtration as a large complex of about 700 kD. Fractions containing the Sec13p complex restored activity to the Sec13p- dependent vesicle formation reaction. Expression of SEC13 on a multicopy plasmid resulted in overproduction of a monomeric form of Sec13p, suggesting that another member of the complex becomes limiting when Sec13p is overproduced. Overproduced, monomeric Sec13p was inactive in the Sec13p-dependent vesicle formation assay.     

The Azotobacter vinelandii mannuronan C-5-epimerase AlgE1 consists of two separate catalytic domains

The Azotobacter vinelandii enzyme AlgE1 is a member of a family of secreted mannuronan C-5-epimerases. These enzymes convert beta-D-mannuronic acid residues (M) to alpha-L-guluronic acid residues (G) at the polymer level in the industrially important polysaccharide alginate, leading to altered physical and immunological properties of the polymer. The reaction product of AlgE1 was found to be a mixture of blocks of continuous G residues (G-blocks) and blocks containing alternating M and G residues (MG-blocks). The enzyme is dependent on Ca2+ for activity, and only Sr2+ of those tested was able to replace Ca2+. Zn2+ blocked the activity even at low concentrations. algE1 has been divided into two parts based on the modular type of structure previously reported to be a characteristic of the secreted epimerases, and each part has been expressed in Escherichia coli. These experiments showed that AlgE1 contains two catalytic domains, AlgE1-1, which introduces both G-blocks and MG-blocks, and AlgE1-2, which only introduces MG-blocks. AlgE1-1 has a much lower specific activity than both AlgE1-2 and AlgE1. However, the two halves of AlgE1 seem to cooperate in such a way that they contribute approximately equally to the overall epimerization reaction.     

Roles of TraI protein with activities of cleaving and rejoining the single-stranded DNA in both initiation and termination of conjugal DNA transfer

BACKGROUND: The plasmid R100 encodes the TraI protein, which is required for conjugal DNA transfer. TraI has the activity of site- and strand-specific nicking of the supercoiled plasmid DNA. The molecular mechanism of this specific nicking, which is supposed to be the initiation reaction of DNA transfer, is not understood. RESULTS: We have demonstrated that TraI has the ability to cleave the single-stranded DNA at the same site as the nicking site (nic) in a region, which we here refer to as sbi. The product contained the TraI protein which was covalently linked to the newly generated 5' end of the nicking reaction. Both the cleaving and nicking reactions took place under almost the same conditions and required the presence of the sbi region. DNase I-footprinting analysis revealed that the TraI bound to the single-stranded DNA of the sbi region. TraI did not cleave the double-stranded DNA fragment, but it did cleave the double-stranded DNA with a single-stranded DNA portion in the sbi region. KMnO4 mapping analysis revealed that TraI can melt the sbi region in the supercoiled DNA to generate a single-stranded portion. We have also demonstrated that TraI was able to rejoin the cleaved products. The rejoining reaction required the 5' end of one cleaved product with the TraI covalently attached and the 3' end of the other product containing the sbi region. CONCLUSIONS: Our results demonstrate that the nicking reaction-the initiation reaction of DNA transfer-is actually the cleaving reaction of the single-stranded DNA. TraI, which has both cleaving and rejoining activities, is thought to be involved in the termination of DNA transfer, to give a copy of the conjugative plasmid by joining the 5' end, which is generated by the initiation reaction, with the 3' end, which will be generated upon cleavage of the sbi region appearing after one round of the rolling circle replication of the plasmid.     

Envelope formation is blocked by mutation of a sequence related to the HKD phospholipid metabolism motif in the vaccinia virus F13L protein

The outer envelope of the extracellular form of vaccinia virus is derived from Golgi membranes that have been modified by the insertion of specific viral proteins, of which the major component is the 37-kDa, palmitylated, nonglycosylated product of the F13L gene. The F13L protein contains a variant of the HKD (His-Lys-Asp) motif, which is conserved in numerous enzymes of phospholipid metabolism. Vaccinia virus mutants with a conservative substitution of either the K (K314R) or the D (D319E) residue of the F13L protein formed only tiny plaques similar to those produced by an F13L deletion mutant, were unable to produce extracellular enveloped virions, and failed to mediate low-pH-induced fusion of infected cells. Membrane-wrapped forms of intracellular virus were rarely detected in electron microscopic images of cells infected with either of the mutants. Western blotting and pulse-chase experiments demonstrated that the D319E protein was less stable than either the K314R or wild-type F13L protein. Most striking, however, was the failure of either of the two mutated proteins to concentrate in the Golgi compartment. Palmitylation, oleation, and partitioning of the F13L protein in Triton X-114 detergent were unaffected by the K314R substitution. These results indicated that the F13L protein must retain the K314 and D319 for it to localize in the Golgi compartment and function in membrane envelopment of vaccinia virus.