Specific inhibitors of tyrosine-specific protein kinase, synthetic 4-hydroxycinnamamide derivatives

Several newly synthesized 4-hydroxycinnamamide derivatives such as 3-(3',5'-di-isopropyl-4'-hydroxybenzylidene)-2-oxindol (ST 280), 3-(3',5'-di-methylthiomethyl-4'-hydroxybenzylidene)-2-oxindole (ST 458), alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638) and 3-(3'-ethoxy-4'-hydroxy-5'-phenylthiomethylbenzylidene)-2-pyrol idinone (ST 642) were found to inhibit tyrosine-specific protein kinase activity of the epidermal growth factor (EGF) receptor with IC50 values of 0.44 microM, 0.44 microM, 0.37 microM and 0.85 microM, respectively. None of them showed inhibitory effect on the enzyme activities of serine- and/or threonine-specific protein kinases such as cAMP-dependent protein kinase, Ca2+/phospholipid-dependent protein kinase C, casein kinase I and casein kinase II. In addition, none of them had effect on Na+/K+-ATPase or 5'-nucleotidase. The results suggest that the compound ST 280, ST 458, ST 638 and ST 642 are potent and specific inhibitors of tyrosine-specific protein kinase.     

An antibody and anti-idiotypic antibody against the extra signal peptide of pre-aspartate aminotransferase

A peptide (extra signal peptide) comprising amino acids 1-29 of pig liver pre-mitochondrial aspartate aminotransferase (p-mAAT) was synthesized chemically. The peptide was found to block the import of rat liver p-mAAT into rat liver mitochondria. An antibody raised against the peptide immunoprecipitated rat liver p-mAAT synthesized in a rabbit reticulocyte cell-free translation system. These results suggested that the extra signal peptide sequence of p-mAAT is essential for import of p-mAAT into the mitochondria and that there is structural homology between the extra signal peptides of pig and rat liver p-mAAT. An anti-idiotypic antibody against the peptide was also prepared and purified by affinity chromatography on an Affi-Gel 10 anti-peptide IgG column and was then characterized.     

Interleukin-1 derived from human monocytic leukemia cell line JOSK-I acts as an autocrine growth factor

Interleukin-1 (IL-1) enhances the growth of human monocytic leukemia cell line JOSK-I cells, which were recently established in our laboratory and which were demonstrated to produce a high level of IL-1 constitutively, in liquid as well as semisolid culture systems. Concomitantly, IL-1 stimulated the prostaglandin E2 synthesis and nitroblue tetrazolium dye-reducing capacity of JOSK-I cells. This indicates that IL-1 may act as autocrine growth factor for monocytes, and also suggests the possibility that this autocrine stimulation may play an important role in the pathophysiology of monocytic leukemia in vivo.     

Decrease of palmitoyl-CoA elongation in platelets and leukocytes in the patient of hereditary methemoglobinemia associated with mental retardation

Effect of the deficiency of NADH-cytochrome b5 reductase on fatty acid elongation was studied in the platelets and leukocytes taken from a patient of hereditary methemoglobinemia associated with mental retardation. The activity of fatty acid elongation was determined by measuring the incorporation of [2-14C]malonyl-CoA into palmitoyl-CoA. The de novo biosynthesis of fatty acids was blocked by the addition of phosphotransacetylase, and the elongation system could be assayed in the homogenates separated from de novo biosynthesis. As compared to normal subjects approximately 40% decrease of fatty acid elongation was observed both in the platelets and leukocytes from the patient.     

Saturated and trans-unsaturated fatty acids elicit high levels of superoxide generation in intact and cell-free preparations of neutrophils

Saturated and trans-unsaturated fatty acids, such as laurate and elaidate, elicited O2- generation in intact porcine and human neutrophils and also in a cell-free preparation of porcine neutrophils. The activities thus induced were comparable to those induced by cis-unsaturated fatty acids. However, the activation by saturated or trans-unsaturated fatty acids was depressed almost completely in the presence of Ca2+ at around 1 mM, which is usually contained in the media for phagocytes. In contrast, the activation by cis-unsaturated fatty acids such as arachidonate was scarcely affected by Ca2+. These findings appear to demand reevaluation of the effects of long chain fatty acids on the respiratory burst system in phagocytes.     

In vitro mutated beta subunits from the F1-ATPase of the thermophilic bacterium, PS3, containing glutamine in place of glutamic acid in positions 190 or 201 assembles with the alpha and gamma subunits to produce inactive complexes

Using site-directed mutagenesis, Glu-190 or Glu-201 of the beta subunit of the F1-ATPase from the thermophilic bacterium PS3 were replaced with glutamine. It was possible to reconstitute complexes of the mutated beta subunits with alpha and gamma subunits, but the complexes did not have ATPase activity. It is concluded that carboxylic acid side chains of Glu-190 and Glu-201 of the beta subunit are essential for catalytic activity of F1-ATPase.     

Three distinct forms of rat brain protein kinase C: differential response to unsaturated fatty acids

Although the three distinct forms of protein kinase C isolated from rat brain soluble fraction are structurally very similar, they respond differently to free unsaturated fatty acids such as arachidonic acid to exhibit their catalytic activity. Type I enzyme encoded by gamma-sequence, as predicted by cDNA clone analysis, responds to these fatty acids only slightly, whereas Type III enzyme determined by alpha-sequence is activated by free unsaturated fatty acids in the presence of Ca2+ in a comparable manner to phosphatidylserine plus diacylglycerol. Type II, a mixture of two enzymes encoded by beta I- and beta II-sequence, resulting from alternative splicing, shows properties in between those of Type I and Type III. Some of these forms of protein kinase C may function at a relatively later phase of cellular responses when large amounts of unsaturated fatty acids and Ca2+ are mobilized.     

Substitution of an arginyl residue for the active site lysyl residue (Lys258) of aspartate aminotransferase

The active site lysyl residue (Lys258) of E. coli aspartate amino transferase was substituted for an arginyl residue by oligonucleotide-directed, site-specific mutagenesis. The mutant enzyme was obviously unable to form an aldimine bond with pyridoxal 5'-phosphate but firmly bound the coenzyme. The finding that the mutation did not lead to entire loss in the enzymic activity suggests that Lys258 may not be essential but auxiliary for enzymic catalysis. It is also conceived that the positive charge provided by Arg258 may contribute to the enzymic catalysis.     

In vivo 31P NMR in crustacean muscles: fatigue and recovery in the tail musculature from the prawn Palaemon elegans

31P NMR has been used to observe the in vivo phosphometabolite concentrations in the tail musculature from the prawn Palaemon elegans, at rest and after escape swimming and subsequent recovery. Muscular fatigue corresponds to a 60% breakdown of phosphoarginine, and a 45% increase of sugar phosphates. The pHi fell from 7.10 to 6.86. During recovery, the sugar phosphates and arginine phosphate are replenished after 20 minutes. The ATP concentration did not change throughout the experiment. The pHi was restored within 20 minutes.     

The induction of a specific form of cytochrome P-450 (P-450j) by fasting

In previous work we have demonstrated that liver microsomal N-nitrosodimethylamine demethylase (NDMAd) activity is increased in rats by fasting, and we have postulated that this is due to the induction of a specific form of cytochrome P-450. This communication provides evidence for such a hypothesis. Fasting for 24 and 48 h caused 59 and 116% increases, respectively, in NDMAd activity in male rats, and fasting for 48 h caused a 63% increase in female rats. These increases were accompanied by corresponding increases of cytochrome P-450j (P-450ac) determined by immunoblotting. Fasting for 24 and 48 h also increased the mRNA for P-450j by 153 to 250%, as determined by hybridization with a cDNA probe of this cytochrome. The results suggest that fasting affects the gene expression of P-450j.