Characterization and cDNA cloning of phospholipase C-gamma, a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase.

Heparin-binding growth factors (HBGFs) bind to high-affinity cell surface receptors which possess intrinsic tyrosine kinase activity. A Mr 150,000 protein phosphorylated on tyrosine in response to class 1 HBGF (HBGF-1) was purified and partially sequenced. On the basis of this sequence, cDNA clones were isolated from a human endothelial cell library and identified as encoding phospholipase C-gamma. Phosphorylation of phospholipase C-gamma in intact cells treated with HBGF-1 was directly demonstrated by using antiphospholipase C-gamma antibodies. Thus, HBGF-1 joins epidermal growth factor and platelet-derived growth factor, whose receptor activation leads to tyrosine phosphorylation and probable activation of phospholipase C-gamma.     

Ligand-induced transphosphorylation between different FGF receptors.

Recent evidence shows that different fibroblast growth factors (FGF) bind with similar high affinities to two FGF receptors (FGFR) called flg and bek. In order to explore the mechanism of FGFR tyrosine autophosphorylation, we have generated cell lines which co-express a kinase-negative mutant of FGFR and an active form of FGFR. The following transfected NIH 3T3 cells were generated: (i) cells which express a shorter truncated form of bek (two Ig domains) together with a kinase-negative mutant of full length bek (bek K517A), (ii) cells which express wild-type bek together with kinase-negative flg (flg K514A) and (iii) cells co-expressing wild-type flg together with bek K517A. Immunoprecipitations with either bek-or flg-specific antisera followed by immunoblotting indicated that the double transfectants express the desired receptor species. The addition of acidic FGF (aFGF) to the various cell lines followed by immunoprecipitation with anti-FGFR antibodies and immunoblotting with anti-phosphotyrosine specific antibodies indicated that aFGF induces tyrosine phosphorylation of the kinase-negative FGFR mutants. These results show that tyrosine autophosphorylation of the kinase-negative FGFR is mediated by a transphosphorylation mechanism and that both homologous (bek----bek) and heterologous (bek----flg and flg----bek) transphosphorylation occurs in living cells. Recent evidence shows that tyrosine autophosphorylation of receptors with tyrosine kinase activities is essential for mediating interactions with signaling molecules. Therefore, heterologous transphosphorylation could amplify the response of cells to various forms of FGFs and their cognate receptors.     

Nuclear-encoded subunits of human cytochrome c oxidase: SstI restriction fragment length polymorphism

A DNA polymorphism of the nuclear-encoded subunit Va of the human cytochrome c oxidase (COX), a mitochondrial respiratory enzyme, is reported. No polymorphism was detected in genes for the subunits IV and Vb of the same enzyme.     

The Human Obesity Gene Map: The 1996 Update

An update of the human obesity gene map up to October 1996 is presented. Evidence from Mendelian disorders exhibiting obesity as a clinical feature, single-gene mutation rodent models, quantitative trait loci uncovered in crossbreeding experiments with mouse, rat, and pig models, association and case-control studies with candidate genes, and linkage studies with genes and other markers is reviewed. All chromosomal locations of the animal loci are converted into human genome locations based on syntenic relationships between the genomes. A complete listing of all these loci reveals that only 4 of the 24 human chromosomes are not yet represented, i.e., 9, 18, 21, and Y. Several chromosome arms are characterized by the presence of several putative loci. The following arms include at least three such loci: 1p, 1q, 3p, 4q, 6p, 7q, 8p, 8q, 11p, 11q, 15q, 20q, and Xq. Studies with negative association and linkage results are also reviewed.     

The distance between S1, S21, and the 3' end of 16S RNA in 30S ribosomal subunits. The effect of poly(uridylic acid) and 50S subunits on these distances

The apparent distances between probes covalently attached to the cysteine thiols of S1 or S21 and the 3' end of 16S RNA in Escherichia coli 30S ribosomal subunits were determined by non-radiative energy transfer to be: S21-16S RNA, 5.1 nm; S21-S1, 6.9 nm; S1-16S RNA, 6.8 nm. Binding of poly(uridylic acid) to 30S subunits causes the apparent distances between S1 and 16S RNA or S21 and 16S RNA to increase by more than 1.2 nm and 0.5 nm, respectively, but has little or no effect on the S1-S21 distance. Binding of 50S subunits causes an apparent increase in the S21-16S RNA and S21-S1 distances by 1.0 nm and 0.8 nm, respectively, but has little or no effect on the S1-16S-RNA distance.     

Chemosensory responses in isolated olfactory receptor neurons from Necturus maculosus

Olfactory receptor neurons were isolated without enzymes from the mudpuppy, Necturus maculosus, and tested for chemosensitivity. The cells responded to odorants with changes in firing frequency and alterations in excitability that were detected with tight-seal patch electrodes using on-cell and whole-cell recording conditions. Chemosensitive cells exhibited two primary response characteristics: excitation and inhibition. Both types of primary response were observed in different cells stimulated by mixtures of amino acids as well as by the single compound L-alanine, suggesting that there may be more than one transduction pathway for some odorants. Using the normal whole-cell recording method, the chemosensitivity of competent cells washed out rapidly; a resistive whole-cell method was used to record odorant responses under current-clamp conditions. In response to chemical stimulation, excitability appeared to be modulated in several different ways in different cells: odorants induced hyperpolarizing or depolarizing receptor potentials, elicited or inhibited transient, rhythmic generator potentials, and altered excitability without changing the membrane potential or input resistance. These effects suggest that olfactory transduction is mediated through at least three different pathways with effects on four or more components of the membrane conductance. Polychotomous pathways such as these may be important for odor discrimination and for sharpening the "odor image" generated in the olfactory epithelium.     

Effect of heparin on the binding affinity of acidic FGF for the cloned human FGF receptors, flg and bek.

Heparin potentiates the mitogenic activity of acidic fibroblast growth factor (aFGF) by 20-100 fold but mechanisms detailing this potentiation have not yet been fully elucidated. We report that heparin increases the binding affinity of aFGF for the two cloned and overexpressed human FGF receptors, flg and bek, by 2-3 fold. This increase in binding affinity, together with previous data demonstrating a 3-5 fold increase in the stability of aFGF, are likely to account for a significant portion of heparin's potentiation of aFGF activity observed in biological assay systems.     

The gene for human acidic fibroblast growth factor encodes two upstream exons alternatively spliced to the first coding exon

We have isolated two cDNA clones encoding human acidic fibroblast growth factor (aFGF) which represent the utilization of alternative upstream exons in aFGF mRNA. Isolation and sequence analysis of genomic clones spanning the first coding exon and each of the upstream sequences confirms that the divergent 5' sequences are separate exons, spliced alternatively to the first coding exon 34 nucleotides upstream of the initiator AUG codon. Restriction mapping of the genomic clones provides a minimum size estimate of 45 kilobase pairs for the aFGF locus.     

A novel bufadienolide synthesis

A simple two-step synthesis of bufadienolides is reported. It consists in the addition of the dimethyl acetal of chloroketene to a steroidal 20-methylene 21-aldehyde and in the treatment of the resulting 2,2-dimethoxy 3-chloro 2,3-dihydropyran with sodium methoxide in dimethylsulfoxide. This method is exemplified by the synthesis of 3β-hydroxy-5α,14α-bufadienolide from 3β-acetoxy-20-methylene-5α-pregnan-21-al, prepared from 3β-acetoxy-5α-androstan-17-one. The new procedure represents the most efficient bufadienolide synthesis yet known.