Biochemistry and Molecular Biology International

Eosinophilia-myalgia syndrome (EMS), a recently described inflammatory disorder characterized by myalgia, peripheral eosinophilia, and multisystem inflammation is associated with L-tryptophan consumption. Fibrosis of various tissues due to excessive accumulation of type I collagen is a prominent late manifestation of the syndrome. 1,1'-Ethylidenebis[L-tryptophan] (EBT), an impurity distinct from L-tryptophan found in case-associated lots, has been implicated in function in vitro. Incubation of confluent fibroblasts with EBT, but not its hydrolysis product 1-methyl-tetrahydro-beta-carboline-3-carboxylic acid, caused a dose-dependent increase in collagen synthesis and in type I collagen mRNA levels independent of its effect on proliferation. In contrast, expression mRNA for fibronectin was not affected. These findings indicate that EBT stimulates type I collagen production by human fibroblast, and suggest that EBT may be involved in the development of fibrosis in EMS.

Triplet-excited carbonyl species can be generated by photoexcitation of carbonyl compounds or alternatively by thermal decomposition of 1,2-dioxetanes. Such electronically excited species are involved in the oxidative modification of biologically important molecules such as DNA. This study demonstrates that thermal decomposition of 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane (HTMD), which is a chemical source of triplet-excited ketones, is accompanied by infrared photoemission at 1270 nm characteristic for singlet oxygen monomol emission. The intensity of infrared photoemission at 1270 nm was solvent-dependent, in agreement with the reported lifetime of singlet oxygen in the employed solvents. Calibration of monomol emission with the endoperoxide of 1,4-dimethylnaphthalene (DMNO2) showed that HTMD (30 mM) produced 25 microMs of singlet oxygen. Thus, the yield of singlet oxygen production in the thermal decomposition of HTMD in carbon tetrachloride is about 0.1%.

Catechol 1,2-dioxygenase catalyzes the oxygenative ring cleavage of catechol to form cis,cis-muconic acid, and contains a ferric form of iron as its sole cofactor. Pseudomonas arvilla C-1 contains three isozymes of the enzyme, alpha alpha, alpha beta, and beta beta [C. Nakai et al. (1990) J. Biol. Chem. 265, 660-665]. We have determined the amino acid sequence of the alpha alpha isozyme by direct analysis. The sequence shared 77% homology with isozyme beta beta, and had conserved tyrosyl and histidyl residues which are thought to be involved in the binding of ferric ion.

This study examines the effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], serum or forskolin on the proliferation of IMR-90 fetal lung fibroblasts and demonstrates, for the first time, the presence of 1,25(OH)2D3 receptor (VDR) in this cell line. In quiescent, subconfluent cultures neither the treatment with 100 nM 1,25(OH)2D3 nor that with 50 microM forskolin influenced proliferation, while a significant increase was observed after incubation of the cells with 10% fetal bovine serum. Either cell number, determined on growing IMR-90 human fibroblasts after 48 or 72 h incubation with 100 nM 1,25(OH)2D3 or [3H]thymidine incorporation (24, 48 or 72 h incubation) significantly decreased, while protein content per cell increased. Northern blot analysis revealed the expression of the VDR gene, the VDR mRNA bands being prominent in 1,25(OH)2D3, serum or forskolin treated fibroblasts. VDR mRNA levels slightly decreased, when growing fibroblasts were exposed to 100 nM 1,25(OH)2D3 for 48 or 72 h.

1,25-dihydroxy-vitamin D3 induces the synthesis of a 9 kDa calcium binding protein in chick embryo myoblasts. This work revealed comigration of the myoblast protein with rat calbindin-D9K in SDS-polyacrylamide gels. Western-blot analysis with a specific calbindin-D9K antibody showed the presence of an immunoreactive protein of 9 kDa in chick tissues. In addition, higher levels of myoblast mRNA hybridizing to a specific cDNA probe for rat calbindin-D9K were detected in response to 1,25-dihydroxy-vitamin D3. Northern hybridization analysis showed that the increase was related to a single 550 nucleotide mRNA species. These results provide the first evidence on the presence of calbindin-D9K or a closely related protein in avian tissues and its inducible expression in muscle by 1,25-dihydroxy-vitamin D3.

The rapid, non-genomic effects of 1,25(OH)2-vitamin D3 [1,25(OH)2D3] on calcium influx in cultured neonate rat and embryonic chick myoblasts were investigated. The hormone stimulated 45Ca uptake in a time (1-10 min) and dose (10(-11)-10(-7) M)-dependent manner. The effects of 1,25(OH)2D3 on both rat and chick myoblasts were mimicked by the Ca(2+)-channel agonist BAY K8644 and effectively suppressed by the antagonist nifedipine. 1,25(OH)2D3 induction of Ca2+ uptake was dependent on the presence of extracellular calcium, since it was abolished by prior addition of EGTA and was restored upon the addition of 1 mM Ca2+ Cell membrane depolarization with high K+, similarly to the hormone, elicited a rapid increase in Ca2+ uptake (+75% above control). These results suggest the modulation of Ca2+ influx through competent calcium channels in rat and chick embryo myoblasts by 1,25(OH)2D3.

The combined antimitogenic effects of IFN-beta and 1,25-dihydroxyvitamin D3 (vit. D3) were investigated by treating the androgen-independent JCA-1 cells, established from the primary prostatic tumor site prior to anti-hormonal therapy, with IFN-beta (1000 IU/ml), vit. D3 (100 nM), and both agents. Cell growth, changes in overall RNA and protein contents, and cell cycle regulatory proteins pRB/p53 were determined. After a 24 h exposure, a significant reduction in cell proliferation was observed in all three conditions. IFN-beta, vit D3, and their combination elicited, respectively, a 1.7-, 1.6- and 2.5-fold increase in total RNA and a corresponding 1.4-, 1.2- and 1.7-fold increase in soluble proteins. The IFN-inducible 2-5A synthetase activity was elevated by 15-, 1.4- and 21-fold, respectively. No differences in cell cycle phase distribution were found between control and treated samples. However, a significant change in pRB and p53 expression was observed upon exposure to these agents. A progressive increase in total pRB was observed in untreated JCA-1 cells, with the 48 h culture showing a 1.9-fold increase over the 6 h culture. The ratio of phosphorylated to the nonphosphorylated forms of pRB, however, decreased from 3.00 at 6 h to 1.2 at 48 h. The overall pRB increase as well as the modified:unmodified protein ratio change were both markedly decreased when the cells were treated with IFN-beta, vit. D3, or their combination. With p53, a similar progressive increase was also observed in control cells, which was largely abolished by IFN-beta but only partially blocked by vit. D3. The combination of IFN-beta and vit. D3 gave results similar to samples receiving vit. D3 alone suggesting that the effects of IFN-beta, insofar as p53 modulation is concerned, is distal to the effects of vit. D3.

The intact and the truncated endo-beta-1,4-glucanase expressed in Bacillus megaterium by a B. subtilis gene were purified and its adsorption characteristics to cellulosic materials were investigated. The intact enzyme was purified by affinity towards insoluble cellulose and Mono-Q chromatography. The truncated enzyme was purified by gel filtration and chromatofocusing. The molecular activities of these enzymes towards the soluble substrates were identical. Optimum pH and temperature of these two types of enzyme were same, 5.5 and 60 degrees C, respectively. However, the insoluble substrate, Avicel, was hydrolyzed slowly by the intact endoglucanase while the truncated endoglucanase did not hydrolyze the Avicel. Isoelectric points of the intact and the truncated enzyme were 6.2 and 5.6, respectively. In a Sephadex column the large form of intact endoglucanase was eluted later than the small form of truncated enzyme. Only the intact enzyme was strongly adsorbed to Avicel. The practical maximum adsorption was about 20 mg/g of Avicel at pH 6.0 and 4 degrees C.

1,4-Dihydropyridine derivatives possess various physiological activities but their mechanism of membranotropic action is not completely investigated yet. We have examined the membranotropic effects of 4-beta-pyridyl-1,4-Dihydropyridine derivatives containing different length alkyl chain substituent at N-quaternised 4-beta-pyridyl moiety. The results show the relation between incorporation of 1,4-dihydropyridine derivatives in the liposomal membranes and influence on bilayer fluidity. The compound with hexadecyl (C16H33) substituent in the 4-beta-pyridyl moiety possess the most pronounced incorporation ability and fluidizing effect. This compound causes the remarkable release of fluorescent probe calcein from liposomes and induces the hemolysis of human erythrocytes as well. The obtained results suggest that the length of alkyl chain at quaternized 4-beta-pyridyl moiety is significant for the expression of membranotropic effects of tested compounds.

A cDNA library from the filamentous fungus Aspergillus aculeatus was constructed in the yeast expression vector pYES2.0 and used to isolate 57 full length cDNA's encoding beta-1,4-mannanase by expression in S. cerevisiae. The positive clones were identified on agar plates containing 0.2% azurine dyed cross-linked mannan by the formation of blue halos around the colonies. All clones represented transcripts of the same mannanase gene (man1). The gene was sub-cloned into an Aspergillus expression vector and transformed into Aspergillus oryzae for overexpression and purification of the enzyme. The recombinant enzyme had a molecular weight of 45 kDa, an isoelectric point of pH 4.5, a pH optimum of pH 5.0 and a temperature optimum of 60-70 degrees.

The concentration of inositol 1,4,5-trisphosphate (InsP3) in erythrocytes from volunteers has been found to modify following strenuous physical exercise. The basal value was almost regained within some 75 min after the completion of the effort. The concurrent variations of pH and blood lactate have also been evaluated. Our results represent, to our knowledge, the first evidence of in vivo induced intraerythrocyte InsP3 modification. They reinforce the idea of the participation of its precursor phosphatidylinositol 4,5-bisphosphate (PtdIns4,5-P2) in red bood cell shape regulation by contributing to the interactions between membrane and cytoskeleton components.

When the synthetic hydroxyl radical generator, N, N'-bis (2-Hydroperoxy-2-methoxyethyl)-1,4,5,8-naphthalene-tetracarboxylic diimide, NP III, was photoirradiated in the presence of ferric-cytochrome c (Fe3+), the fragmentation peak corresponding to the specific oxidative modification at the histidine site of cytochrome c was observed in the Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectroscopy (MALDI-TOF) measurement. Without photoirradiation, no such protein oxidation was observed in the experimental conditions employed. This result suggests the possible usage of NP-III as an oxidative protein-modifying reagent.

The values of molecular carboxylase activity kcat and carboxylation specificity factor tau for mutant ribulose 1,5-bisphosphate carboxylase (rubisco) from Anacystis nidulans decreased as compared to those of the wild type recombinant rubisco. The substitution of five amino acid residues in rubisco large subunit Lys,Ala,Ser,Thr,Leu(339-343)Phe,Leu,Met,Ile,Lys had kcat decreased by 90% and tau by 36.3%. The same parameters for mutants with the single replacements decreased: for Thr342Ile kcat by 40.5% and tau by 16.7%, and for mutant Leu343Lys kcat by 48.1% and tau by 18.5%. Mutant rubisco with three amino acid residues changed Val,Asp,Leu(346-348)Tyr,His,Thr was inactive. The substitution Leu326Ile decreased kcat by 54.4% and tau by 34.2%; and change Ser328Ala decreased kcat only by 5.6% but tau by 41.5%. Replacement Asn123His decreased kcat by 16.5%. Significance of the non conservative amino acid residues for carboxylase activity and ribulose-1,5-bisphosphate partition is discussed.

Fructose 1,6-bisphosphatase [EC. 3.1.3.11] is activated by the treatment with 0.1 mM cystamine up to about 400% compared to its original activity (dithiothreitol-reduced form). Thiol compounds (0.1 mM of cysteamine and dithiothreitol) can restore its activity effectively. Reduced glutathione, at 0.2 mM, also restores fructose 1,6-bisphosphatase activity only in the presence of cystamine. When excess cystamine is removed, the addition of 1.0 U/ml thioltransferase is able to restore FBPase activity very efficiently coexistence with 0.2 mM reduced glutathione though reduced glutathione alone does not work.

The effect of amphotericin B on rat heart sarcoplasmic reticular membrane Na(+)-K+ and Ca2+ ATPase activities in vitro was investigated. Amphotericin B in selected concentrations of 100-1000 ng significantly inhibited the sarcoplasmic reticular membrane ATPase activities studied. Fructose-1,6-diphosphate (1000 microM concentration) completely reversed the inhibition of Ca2+ ATPase activity in particular, but failed to reverse that of Na+(-)K+ATPase activities at 1000 microM concentration. Fructose-1,6-diphosphate may afford some protection against 1000 ng amphotericin B-induced myocardial toxicity. These damages may vary depending upon the dose of amphotericin B used in experimental studies.

The activity of chicken liver fructose 1,6-bisphosphatase increases dramatically after incubation with allicin, a major biologically active compound produced by garlic. Activation is more pronounced when the enzyme is assayed with Mn2+ than Mg2+. Maximum activation is accompanied by the disappearance of 4 highly reactive sulfhydryl groups per molecule of enzyme. This modification also leads to loss of activation by K+, and reduced sensitivity to inhibition by AMP, fructose 2,6-bisphosphate, and high concentration of fructose 1,6-bisphosphate. All the altered properties induced by allicin can be reversed by dithiothreitol or tris(2-carboxyethyl)phosphine, the latter being much more effective.

Complete activation of chloroplast fructose-1, 6-bisphosphatase by dithiothreitol involves the reduction of its four disulfide bonds as revealed by thiol titration and activity measurement. Both before and after reduction, the enzyme is inhibited by the thiol-specific reagent 5,5'-dithiobis(2-nitro-benzoic acid) with complete inactivation upon modifications of the four accessible thiols. However, oxidative modification of the enzyme facillitates the reduction of the four mentioned disulfide bonds as the process of activation by DTT is accelerated.

The expression of the fructose 1,6-bisphosphatase gene in HL-60 cells was induced by retinoic acid. The levels of mRNA, enzyme activity and enzyme protein in the cell line began to rapidly increase after culturing with retinoic acid for 72 h. Retinoic acid dose-dependently increased the enzyme activity with maximal stimulation at 1 microM. The responses of the fructose 1,6-bisphosphatase gene expression by retinoic acid were markedly slower than those of the enzyme expression by 1alpha,25-dihydroxyvitamin D3. When HL-60 cells were cultured in the presence of both retinoic acid and 1alpha,25-dihydroxyvitamin D3, the effects of the two agents on enzyme activity, protein and mRNA were additive.

cFBP is studied for its affinity to Mg++ and Fru-1,6-P2. The affinity for Mg++ is not very high with a Km of 0.24 +/- 0.01 mM. High concentrations of Mg++ are inhibitory. The saturation curve for Fru-1,6-P2 is hyperbolic with a Km of 0.54 +/- 0.014 microM. The presence of citrate (10 mM) induces a sigmoidal curve, modifying both Vmax and S0.5. Citrate affects the allosteric properties of cFBPase: at low substrate concentration cooperativity becomes negative while at higher concentration it is positive. Addition of higher concentrations of Mg++ shows a synergistic effect with citrate, decreasing of the affinity for Fru-1,6-P2: S0.5 equals 7.6 +/- 0.25 mM, 9.0 +/- 0.86 mM and 21.5 +/- 1.46 mM in presence of 5, 7.5 and 10 mM Mg++, respectively.

An enzyme fraction with catalytic activities for the biosynthesis of the pipecolic acid containing cyclopeptolide SDZ 90-215 was partially purified and characterized from the genus Septoria. The crude cell homogenate was subjected to polyethyleneimine precipitation, ammonium sulfate precipitation and FPLC gel filtration. The denatured enzyme shows an apparent molecular mass of 1.2 MDa in 3% SDS-PAGE. Peptolide SDZ 90-215 synthetase catalyzes the ATP-PPi exchange reaction dependent on all substituent amino and hydroxy acids. SDZ 90-215 synthetase synthesizes the peptolide in vitro when incubated together with D-lactic acid, all constitutive amino acids in their N-unmethylated form, ATP, magnesium chloride and S-adenosyl-L-methionine. The yield of SDZ 90-215 is higher when O-methyl-L-tyrosine instead of L-tyrosine is used, indicating that O-methylation of tyrosine is not carried out by the synthetase.

Arachin subunit (mol. wt. 29,100) from peanut of variety G-201 was separated and isolated, its purity and homogeneity were ascertained. The subunit was cleaved with trypsin, chymotrypsin and Staphylococcus aureus V8 protease. The fragments obtained were separated and isolated by PAGE, gel filtration & ion exchange chromatography, these were subjected to amino acid analysis, and Edman degradation. The PTH amino acids obtained were identified by spectroscopy and TLC. The complete amino acid sequence of the subunit (226 residues) was established, and the structural class of arachin was predicted from the amino acid composition.

EGR (early growth response) genes represent a family of the proteins whose structure includes a zinc finger domain(s) interacting with the specific site in regulatory sequences of different genes. Using as an antigen the short sequence RSNHLTTHIR from the middle of the zinc finger domain of EGR-1 we have generated a clone of the hybridoma cells producing the monoclonal antibody (mAb) that binds to 102 kD and 58 kD proteins, but apparently does not bind EGR-1 protein. The 102 kD protein is probably made of the 58 kD subunit and another one of 44 kD. Expression of these proteins is strongly induced in serum stimulated mouse fibroblasts.

We investigated subcellular distribution of ERK2 in osteoblast-like UMR-106 cell and explored to determine if its activities are regulated by insulin. 23%, 34% and 43% of total ERK2 were distributed in membrane, cytosol and nucleus, respectively. Insulin caused 40% increase of ERK2 content in membrane in 10 min whereas it induced approximately 50% decrease of ERK2 in cytosol in 10 min. In terms of kinase activity, insulin stimulated phosphorylation of the membrane-associated ERK2 by 2-fold and 1.8-fold in 1 min and 10 min and cytosolic ERK2 by 2.7-fold and 2.3-fold in 1 min and 10 min, respectively. In contrast, the phosphorylation of nuclear ERK2 was stimulated by insulin in time-dependent manner with maximal (3-fold) activity observed at 30 min. Insulin also increased the content of MEK2 in membrane by 2.2- to 2.6-fold in 10 min. MEK2 translocated into membrane in response to insulin may play a role in the activation of the membrane-associated ERK2 via phosphorylation.

Osteopontin (OPN) is a secreted glycoprotein implicated in cell adhesion. It contains the arginine-glycine-asparatic acid (RGD) cell adhesive domain and the thrombin cleavage sequence. Although thrombin cleavage of OPN has been shown to be of physiological importance, the function of C-terminal OPN fragment cleaved by thrombin remains unknown. To determine its role, we performed cell adhesion assays using glutathione S-transferase-OPN fusion protein fragments and full-length OPN fusion protein. The N-terminal fragment containing RGD motif promoted enhanced adhesion of mouse and human fibroblasts by 2.9 and 2.8 folds in comparison with full-length OPN, respectively. The enhanced adhesion of both cells mediated by N-terminal fragment was significantly suppressed by addition of C-terminal fragment lacking RGD motif that has less cell adhesive property than full-length OPN. These results suggest that the C-terminal domain may play a pivotal role in regulating OPN functions by suppressing the RGD-dependent cell adhesion.

DNA polymerase alpha-primase complexes in extracts of MVM-infected murine cells were dissociated in the presence of 0.3M KCl to generate a 12S DNA primase and a 10S DNA polymerase alpha that were readily separated by sedimentation in glycerol gradients. A 12S DNA polymerase alpha-primase complex refractory to dissociation in 0.3M KCl was identified in extracts of MVM-infected HeLa cells. In extracts of mock-infected murine and HeLa cells DNA primase and DNA polymerase alpha were not dissociated from each other in 0.3M KCl but remained in a stable complex that sedimented at 10S. We propose that a novel 12S DNA polymerase alpha-primase complex prone to disruption by salt is induced by MVM infection and that the DNA primase component of the complex is modified.

During chick limb development the expression of Hoxa-11 specifically associated with the prospective zeugopod (radius + ulna) region. A series of micromass cultures were performed using mesenchyme from whole stage 18/19 or 20/21 limbs or mesenchyme from the tips of stage 22/23, 23/24 or 25 limbs. In such cultures, limb mesenchyme undergoes chondrogenesis paralleling its fate in vivo. Hoxa-11 and collagen type II mRNA were examined by dot blot and in situ analysis at different time points. The highest level of Hoxa-11 mRNA was found in micromass cultures of the tips of stage 22/23 and 23/24 limbs at 48 hours. Between stages 22 and 24, the determining events occur which subsequently lead to the formation of the zeugopod by the mesenchyme present in the tips at these stages. This correlation further supports a possible role of Hoxa-11 in the specification of the zeugopod.

Two forms of cDNA clones corresponding to Hoxd-11 were obtained by screening a chicken limb bud cDNA library. Comparison of the two cDNA sequences with the mouse cDNA and genomic sequence, shows that the shorter cDNA is missing 3 nucleotides, which encode an alanine residue, at the junction between exons 1 and 2. Analysis of the chicken genomic sequence shows two tandem CAG repeats at the intron 1-exon 2 junction, both of which could serve as splice acceptor sequences. Thus we hypothesize that the two spliced forms result from alternative use of two tandem splice acceptor sequences. Using reverse transcription polymerase chain reaction we examined expression of the two forms in 3 to 14-day chick embryos. All samples showed both species of RNA with approximately 2 times more of the larger from compared with the smaller form.

We have used synthetic peptide to study a conserved RNA binding motif in eukaryotic poly(A) binding protein (PABP) as well as its functions. We synthesized an 11 amino-acid peptide based on the consensus sequence (GKSKGFGFV), which is found in all the sequenced eukaryotic PABPs. The synthetic peptide was found to be preferentially bound to the poly(A) alone or the poly(A) attached to the 3'-end of mRNA and not the deadenylated mRNA. Additionally, the 11-mer had strong affinity to bind to ATP. In vitro translation of rabbit globin mRNA poly(A) in the presence of the 11-mer resulted in stimulation of globin synthesis by two-fold as compared to translation in the presence of either deadenylated globin mRNA or globin mRNA poly(A) but in the absence of the 11-mer.

To better delineate the antioxidant potential of Bio-Catalyzer alpha.rho No.11 (Bio-Normalizer), a natural food supplement recently proposed as an antioxidant agent, we investigated the efficacy of Bio-Normalizer supplementation to protect rat organ homogenates against oxidative damage induced in vitro by peroxyl radicals generated in the hydrophobic or in the hydrophilic phase. Bio-Normalizer supplementation efficiently protected rat kidney homogenates against the accumulation of thiobarbituric reactive substances (TBARS), the formation of protein carbonyl derivatives and the depletion of alpha-tocopherol induced by peroxyl radicals generated from the hydrophobic azo-initiator 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN). It also protected the heart but not the liver or the brain homogenates. Bio-Normalizer supplementation did not have effect in any organ homogenates when peroxyl radicals were generated from the hydrophilic azo-initiator 2,2'-azobis (2-amidinopropane) dihydrochloride) (AAPH). In vitro direct addition of aqueous solutions of Bio-Normalizer to the organ homogenates was ineffective against AMVN or AAPH-induced oxidative damage. Our findings expand previous reports on the antioxidant activity of Bio-Normalizer. They confirm that supplemented Bio-Normalizer protects against peroxyl radical-induced oxidative damage and suggest that its antioxidant action depends on in vivo bioactivation, it is organ specific and it is limited to damage induced by peroxyl radicals generated in the hydrophobic phase.

Bio-Catalyzer alpha . rho No. 11 (Bio-Normalizer), a natural health food product prepared by yeast fermentation of medicinal plants, has been recently reported to possess antioxidant properties. To better define its antioxidant action, we investigated the effects of orally supplemented Bio-Normalizer on oxidative damage in the rat heart. Hearts were isolated from control or Bio-Normalizer supplemented animals and 1) exposed to ischemia-reperfusion using the Langendorff technique, or 2) homogenized and exposed to peroxyl radicals generated from (2,2'-azobis (2,4'-dimethylvaleronitrile) (AMVN). During reperfusion following 40 minutes of ischemia, leakage of lactate dehydrogenase from hearts isolated from Bio-Normalizer supplemented rats was significantly lower than from hearts of control animals. Furthermore, lower levels of AMVN-induced accumulation of thiobarbituric acid reactive substances and of protein carbonyl derivatives were measured in homogenates prepared from hearts isolated from Bio-Normalizer supplemented rats than in samples from control animals. Our findings confirm an antioxidant action of Bio-Normalizer and show that it protects the heart against ischemia-reperfusion induced damage.

Interleukin-11/adipogenesis inhibitory factor (IL-11/AGIF) inhibits adipogenesis and suppresses lipoprotein lipase (EC3.1.1.34, LPL) activity in adipocytes (1,2). We investigated the mechanism of suppression of LPL activity in 3T3-L1 adipocytes by IL-11/AGIF. Incubation of adipocytes with 50 ng/ml of IL-11/AGIF led to a 75% decrease in LPL activity within 8 hours, whereas LPL mRNA level decreased by less than 30%. The LPL synthesis, as judged by the incorporation of 35S-label into immunoprecipitable LPL, decreased at almost the same rate over the same time period as enzyme activity. The degradation rate was not significantly affected by IL-11/AGIF. These data suggest that regulation of the synthesis of the enzyme protein is at least one of the main steps in the suppression of LPL by IL-11/AGIF in 3T3-L1 adipocytes.

Brain capillaries contain a great variety of membrane proteins involved in the transport of hydrophilic nutrients or in the reception of hormonal signals. The use of Triton X-114 fractionation to purify membrane proteins according to their degree of hydrophobicity was investigated. Analysis by polyacrylamide gel electrophoresis showed a distinct polypeptide composition for each fraction. Most of the proteins (68%) were solubilized by Triton X-114 and, of these proteins, the majority (74%) was found in the detergent-poor phase. Alkaline phosphatase which possesses a glycosyl-phosphatidylinositol anchor partitioned in the pellet of insoluble proteins where it was enriched 2.3-fold. In contrast, gamma-glutamyltranspeptidase, the GLUT1 glucose transporter and P-glycoprotein, three integral membrane proteins, and p21ras and a 42 kDa G protein alpha subunit, both covalently modified by lipids, were efficiently solubilized and fractionated in the detergent-rich fraction where they were enriched 3.5-, 4.8-, 4.4-, 4.5- and 4.7-fold, respectively. Triton X-114 fractionation could therefore be used as a first step in the purification of many blood-brain barrier membrane proteins.

To clarify the characteristics of possible synergestic mitochondrial DNA (mtDNA) mutations associated with Leber's hereditary optic neuropathy (LHON), we analyzed the entire nucleotide sequences of mitochondrial genome of two Japanese patients from independent pedigrees harboring the 11778 mtDNA mutation, and compared their sequences with those of 47 disease and 6 normal controls. We have detected several unique mutations in the mtDNA in addition to the 11778 mutation. Two nucleotide substitutions, an A-to-G transition at position 856 in the 12S rRNA gene and an A-to-G transition at 14692 in the T psi C loop of the tRNA(Glu) gene, occurred at highly conserved sites among various species. These mutations in combination with the 11778 mutation might synergetically contribute to the pathogenesis of LHON.

The metabolism (uptake and degradation) of peroxynitrite-modified beta-VLDL and native beta-VLDL labeled with [125I]iodine was studied in monocytes isolated from peripheral blood of hypercholesterolemic (HC) and normolipidemic rabbits (NC). The peroxynitrite-modified beta-VLDL uptake and degradation was up to 2-fold higher in monocytes from HC rabbits than monocytes from NC rabbits. In addition, monocytes from HC rabbits took up and degraded significantly more peroxynitrite-modified beta-VLDL than native beta-VLDL. In contrast, monocytes from NC rabbits took up both lipoproteins in a similar rate and degraded more native beta-VLDL than peroxynitrite-modified beta-VLDL. Our results suggest that hypercholesterolemia can affect monocyte metabolism enhancing foam cell formation by increasing the influx of peroxynitrite-modified beta-VLDL.

In order to elucidate the biochemical roles of imidazol-containing dipeptides, we have studied quenching of singlet molecular oxygen (1O2) by carnosine (beta-alanyl-L-histidine), its structural components (L-histidine, imidazole, and beta-alanine), and related natural free-radical scavengers-L-anserine (beta-alanyl-1-methyl-histidine), ergothioneine (2-thiol-L-histidine-betaine), and taurine (2-aminoethanesulfonic acid) in aqueous (D2O, pD 7) solutions by using monitoring of 1O2-phosphorescence (1270-nm). The rate constants of 1O2 quenching (Kq) by carnosine, anserine, and ergothioneine were shown to be similar [(3 +/- 1) x 10(7) M-1s-1]. Their values resembled those of free-L-histidine [Kq = (4 +/- 1) x 10(7) M-1s-1] and imidazole [Kq = (2 +/- 1) x 10(7) M-1s-1]. Non-aromatic amino acids-taurine and beta-alanine-showed very low quenching activities (Kq < 3 x 10(3) M-1c-1). The Kq values did not correlate with the literature data on abilities of the tested compounds to stimulate muscle working capacities and inhibit myeloperoxidase-catalyzed oxygenation. Thus, the dipeptides can be used as potent water-soluble protectors against 1O2 attack whereas their natural biochemical functions are most probably determined by the processes of different nature.

The purpose of this study was to examine the effects of 13-cis-retinoic acid (13-cis-RA) on the growth regulation of DU-145 human prostatic cancer cells. The results of these experiments demonstrate that cell growth and metastatic potential of DU-145 cells were significantly inhibited by 13-cis-RA (10 microM). In order to elucidate the possible molecular mechanisms of 13-cis-RA action on prostate cancer cells, we examined the expression of nuclear receptor genes (hRXR alpha) and found that 13-cis-RA treated cells showed higher mRNA expression for hRXR alpha nuclear receptors compared to untreated cells. To elucidate further the possible biochemical mechanisms associated with these alterations, we analyzed the phosphorous metabolites by MR spectroscopy and found that the major metabolites were PME, (PC, PE) and DPDE (UDP-GalNAc, UDP-GLcNAc). The DU-145 cells and xenografts, which were both treated with 13-cis-RA, showed a two-fold decrease in DPDE's, compared to their controls. The higher resolution spectra of perfused cells revealed that phosphocholine levels were twice as high in 13-cis-RA-treated DU-145 cells as compared to untreated cells. These investigations demonstrate for the first time that 13-cis-RA inhibits the growth of human prostatic cancer cells, and this inhibition is associated with an increase in hRXR alpha nuclear receptor gene expression and alterations in phosphorous metabolites detected by 31P MR spectroscopy.

The proliferation of the human prostatic cancer JCA-1 cells showed a complex response to different concentrations of TPA. Whereas cells exposed for 24 h had growth reduction which was proportional to the concentration of TPA added to the culture media, they showed resistance to low (0.016 and 0.16 nM) but not high (> or = 1.6 nM) doses of TPA after 72 h. Growth-inhibited, treated cells also displayed a distinct cell morphology compared with the controls. Since c-myc expression has previously been shown to be correlated with cellular proliferation, we determined changes in its steady-state level in control and treated cells by Northern analysis. Following a 24h, 48h, and 72h treatment, with 16 nM TPA, c-myc mRNA was suppressed by 91%, 83%, and 78%, respectively, in good agreement with the extent of growth reduction observed. At the low dose of TPA (0.16 nM), however, the c-myc mRNA expression remained inhibited by 85% even though cell growth was only reduced by 10-14%. No difference in the total amount of c-myc protein could be detected between control and treated cells by Western analysis.

Transcription factors are activated in response to a wide variety of extracellular stimuli. The present study investigated which step Gingko biloba extract (EGb 761), a flavonoid phytochemical rich preparation can affect signal transduction during AP-1 activation. Kinetic experiments with human Jurkat T-cell lines demonstrate that it takes 1 to 1.5 h to activate AP-1 DNA binding activity. These data indicate that c-fos mRNA is expressed and then AP-1 DNA binding activity is activated. Pretreatment of Jurkat T cells with 10 micrograms/ml EGb 761 suppresses AP-1 DNA activation and c-fos mRNA expression. These results suggest that the step in the signal transduction pathway for AP-1 activation that is inhibited by EGb 761 is upstream to c-fos mRNA expression.

The present study was designed to investigate the effects of 13-cis-retinoic acid (13-cis-RA) (100 micrograms/mouse/day) and androgen ablation (castration) alone and in combination on growth of a human prostatic carcinoma line (LNCaP) transplanted to athymic nude mice as an experimental model. The results of these studies suggest that; (1) androgen ablation (castration) significantly decreased the size of LNCaP xenograft as compared to untreated animals; (2) when 13-cis-RA was administered to nude mice carrying established tumors (0.51 +/- 0.04 cm3), the tumor size was significantly reduced as compared to untreated controls (0.65 +/- 0.06 cm3 versus 1.63 +/- 0.12 cm3). About 50% of the animals in this group showed xenografts necrosis followed by complete regression of tumors by five months; (3) the combination of androgen ablation and 13-cis-RA treatment to nude mice carrying tumors showed synergistic effect in decreasing the tumor size. These results indicate that combination therapies based on androgen ablation and retinoid administration may be a useful approach for the treatment of prostate cancer.

A 15-ketoprostaglandin delta 13-reductase was purified to homogeneity from rat liver. The enzyme used NADPH much more effectively than NADH as an electron donor. The molecular weight was estimated to be 39,500 by electrophoresis and 42,000 by gel filtration. The Km apparent for 15-ketoprostaglandin F2 alpha was 213 nM. The enzyme was markedly inhibited by dicumarol, quercitrin, p-chloromercuribenzoic acid and indomethacin. The enzyme had an isoelectric point at pH 4.5 and a broad pH optimum. The enzyme also exhibited the double bond reductase activity toward several xenobiotics with the double bond adjacent to the carbonyl group.

Mutations of p53 tumor suppressor gene are the most common genetic alterations in a variety of human carcinomas. The sites of p53 mutations, however, vary in different cancers. The present study was designed to characterize p53 mutations in 40 primary human renal cancer specimens using hot-start-PCR-single-strand conformation polymorphism (SSCP) analysis, sequencing of PCR product and immunohistochemistry. DNA extracted from microdissected paraffin-embedded sections was amplified by hot-start-PCR using oligonucleotide primers specific for exons 4-9 of p53. The mutations were analyzed by PCR-SSCP technique and the generated fragments were denatured and analyzed by 6% polyacrylamide gel electrophoresis. The samples showing a band shift were denatured and sequenced using the Sequenase Version 2.0 DNA Sequencing Kit (US Biochemical, Cleveland, Ohio). Genomic DNA from control samples containing wild-type p53 alleles was sequenced in parallel for confirming mutations in samples that were positive for p53 in the PCR-SSCP analysis. The results of these experiments demonstrate that: (1) there were mutations in p53 exon 5 and 8 in 35% (14 out of 40 samples) of human renal cancer tissues as revealed by PCR-SSCP analysis; (2) DNA sequencing of samples showing frame-shift have hot spot of p53 mutation on exon 8 at codon 244 (GGC-->TGC) and exon 5 at codon 132 [AAG (Lys)-->AGG (Arg)]. This mutation in p53 exon 5 at codon 132 is novel and has not yet been reported; (3) immunohistochemical staining of p53 in renal cancer tissue using mouse anti-human p53 monoclonal antibody, clone PAb 1801, correlated with the p53 mutation assessed by PCR-SSCP. No correlation was found between p53 mutations and tumor stage and grade of renal cancer.

It has been shown that human growth hormone (hGH) is attacked and digested at Arg(134)-Thr(135) by thrombin and plasmin, facilitating its further degradation in the plasma and tissues. To investigate the roles of the amino acids residues on the 134/135 site in the stability and half-life of the hormone in vivo, we have synthesized a mutant hGH, hGH(R134H, T135E), where Arg and Thr are replaced by His and Glu respectively. The mutant hGH showed an altered half-life time (7 min) as compared to that (11 min) for native form hGH, while the biological activity was not affected by the mutation.

Specific binding of wheat nuclear proteins to 135 bp subrepeat element of the ribosomal DNA intergenic spacer was studied by electrophoretic mobility shift assays. A number of specific DNA-protein complexes are readily formed upon incubation of the labelled 135 bp subrepeat element with wheat nuclear protein extracts. Some of these complexes have various DNA-binding specificities as evidenced by their optimal detection in the presence of different non-specific competitor polynucleotides. One of the wheat nuclear proteins was found to bind specifically both to 135 bp subrepeat element and an intergenic spacer region located downstream of the transcription initiation site (between +192 and +366 bp). No apparent effects of the 135 bp element methylation with methylase HpaII on protein binding were found.

The subcellular distribution and binding of 241Am and 137Cs in the visceral mass of the oyster Crassostrea gigas were investigated following exposure to sea water contaminated with these radionuclides. 241Am was predominantly sequestered by the lysosomal system. Approximately, 10% of 241Am was associated with soluble macromolecules. 241Am was bound to lipofuscin, ferritin and to unidentified ligands of 60 to 15 kdaltons mol. wt. No evidence was found for binding of 241Am to metallothionein synthesized de novo. In contrast, only small amounts of 137Cs were present in lysosomes and 137Cs was not associated with soluble cellular proteins. These results indicate that they enter complete separate metabolic pathways.

L-methionine (Met) oxidation products in tripeptides, in Met-enkephalin and in the bovine basic pancreatic trypsin inhibitor have been identified by 1H and 13C NMR spectroscopy. The oxidation of Met residues by stoichiometric amounts of chloramine-B, H2O2 and I2 yields a mixture of L-methionine sulfoxide (Met (O)) and dehydro-L-methionine (DH-Met), Met (O) and DH-Met, respectively, at pH 7.0 and 25.0 degrees C. The formation of DH-Met occurs only if the amino-group of Met is not derivatized. The analysis of 1H and 13C NMR spectra allows us to quantitate Met oxidation products, to ascertain the relative proportion of the R and S forms of Met (O) and DH-Met, and to reveal the presence of a Met residue at the N-terminal position in peptides and proteins.

Molecular mechanism of the interaction between human serum albumin and cloramphenicol was studied by 1H and 13C NMR-spectroscopy. It was found that the main role belongs to [formula: see text] groups of chloramphenicol. A schematic model of the complex-formation between serum albumin and chloramphenicol was proposed.

We have cloned the cDNA of the rat liver 13k protein, that has glutathione binding activity, from a rat liver cDNA library. The nucleotide sequence of this cDNA predicts a protein of 115 amino acids. This protein has 99.1%, 89.6% and 73.0% homology with mouse, human and chicken macrophage migration inhibitory factor (MIF), respectively. This indicates that the rat liver 13k protein is homologue of the MIF. The N-terminal 25 amino acids show 35% sequence similarity with that of the rat glutathione transferase Yb subunit. Although the remaining C-terminal sequence has no sequence identity with glutathione transferase Yb subunit, about 50% homology was found, if conservative substitutions and gaps were taken into account. Northern blot analysis indicates that this gene is expressed in a wide variety of organs including brain, spleen, liver, muscle and kidney. Southern blot analysis suggests that the MIF gene has many pseudo-genes or/and closely related genes.

Auxin binding by tobacco plasma membrane proteins was investigated. After photolabeling with [3H]IAA, 350 polypeptides were resolved on 2D gels and analyzed. Thirteen polypeptides were selected according to physico-chemical criteria. The labeling of three of them was further shown to increase, after treatment of cells with auxin, specifically in that plasma membrane subfraction where the sensitivity to the hormone of the H(+)-ATPase is enhanced by the treatment of cells. These polypeptides were those that exhibited the more specific labeling features according to physico-chemical criteria. They had similar apparent molecular weight (ca 14 kDa) that distinguished them from other auxin-binding proteins described up to now, and exhibited similar amino acid compositions. These 14 kDa polypeptides are proposed to constitute a group of new auxin-binding proteins, potentially involved, within specialized plasma membrane domains, in the stimulation of the proton pump by the hormone.

The subunit structure of soluble goat hepatic lectin was studied by determining molecular weight under nondenaturing conditions by gel filtration and denaturing conditions by SDS PAGE. Affinity purified lectin was subjected to HPLC on asahipack column equilibrated with 10 mM Tris-HCl pH 7.5, containing 1 mM CaCl2, 1mM 2-mercaptoethanol and 0.1M NaCl. The lectin was eluted under single peak at retention time of 12 min. corresponding to molecular weight of 38Kd. On SDS-PAGE in the presence and absence of 2-mercaptoethanol protein moved as single band with Rm 0.45, which corresponds to molecular weight of 20 Kd. The results suggest that soluble goat hepatic lectin is a dimmer of identical subunits which are linked together by noncovalent interactions. The interaction of monoclonal antibodies raised against soluble goat hepatic lectin (MGHL/20) with hemagglutinin from different species as sheep, human, rat, bovine and chicken was studied in PBS by solid phase binding assay. MGHL/20 showed 29.89% binding with these lectin. However no binding was found with Ca++ dependent membrane bound lectin. These results indicate that soluble goat hepatic lectin possesses antigenic structural relationship with soluble 14 K lectin family.

The active component that stimulates fibroblast growth was purified from cultured Clostridium perfringens FERM P-14028, and a quantitative assay method for the biological activity was established. The active component was named cell growth-stimulating factor (CGSF), and the molecular weight of this factor was estimated to be 420 kDa on gel permeation chromatography. CGSF(50-100 ng/ml) stimulated the growth rate of BHK-21 (C-13) cells in the logarithmic growth phase, and shortened the doubling time by 16-18%, but had no effect on confluent cells. These actions were indicated only with medium containing fetal bovine serum. These results suggest that CGSF is a novel protein that regulates cell growth via a mechanism of action different from that of other growth factors.