A gene for universal congenital alopecia maps to chromosome 8p21-22.

Complete or partial congenital absence of hair (congenital alopecia) may occur either in isolation or with associated defects. The majority of families with isolated congenital alopecia has been reported to follow an autosomal-recessive mode of inheritance (MIM 203655). As yet, no gene has been linked to isolated congenital alopecia, nor has linkage been established to a specific region of the genome. In an attempt to map the gene for the autosomal recessive form of the disorder, we have performed genetic linkage analysis on a large inbred Pakistani family in which affected persons show complete absence of hair development (universal congenital alopecia). We have analyzed individuals of this family, using >175 microsatellite polymorphic markers of the human genome. A maximum LOD score of 7.90 at a recombination fraction of 0 has been obtained with locus D8S258. Haplotype analysis of recombination events localized the disease to a 15-cM region between marker loci D8S261 and D8S1771. We have thus mapped the gene for this hereditary form of isolated congenital alopecia to a locus on chromosome 8p21-22 (ALUNC [alopecia universalis congenitalis]). This will aid future identification of the responsible gene, which will be extremely useful for the understanding of the biochemistry of hair development.     

Interaction of rat glutathione S-transferases 7-7 and 8-8 with gamma-glutamyl- or glycyl-modified glutathione analogues.

Analogues of GSH in which either the gamma-glutamyl or the glycyl moiety is modified were synthesized and tested as both substrates for and inhibitors of glutathione S-transferases (GSTs) 7-7 and 8-8. Acceptor substrates for GST 7-7 were 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETA) and for GST 8-8 CDNB, ETA and 4-hydroxynon-trans-2-enal (HNE). The relative ability of each combination of enzyme and GSH analogue to catalyse the conjugation of all acceptor substrates was similar with the exception of the combination of GST 7-7 and gamma-L-Glu-L-Cys-L-Asp, which used CDNB but not ETA as acceptor substrate. In general, GST 7-7 was better than GST 8-8 in utilizing these analogues as substrates, and glycyl analogues were better than gamma-glutamyl analogues as both substrates and inhibitors. These results are compared with those obtained earlier with GSH analogues and GST isoenzymes 1-1, 2-2, 3-3 and 4-4 [Adang, Brussee, Meyer, Coles, Ketterer, van der Gen & Mulder (1988) Biochem. J. 255, 721-724] and the implications with respect to the nature of their active sites are discussed.     

Initiation factor protein modifications and inhibition of protein synthesis.

The protein covalent modification state of eucaryotic initiation factors eIF-2 and eIF-4B in HeLa cells was examined after they were exposed to a variety of conditions or treatments that regulate protein synthesis. A few factors (e.g., variant pH and sodium fluoride) altered the phosphorylation state of the initiation factor proteins, but the majority (hypertonic medium, ethanol, dimethyl sulfoxide sodium selenite, sodium azide, and colchicine) had no effect on either protein. While initiation factor phosphorylation may regulate protein synthesis in response to many physiological situations, other pathways can regulate protein synthesis under nonphysiological circumstances.     

Water stress and protein synthesis: I. Differential inhibition of protein synthesis

Water stress causes both a qualitative change in the types of proteins produced by Avena coleoptile cells as demonstrated by a double-labeling ratio technique, and a quantitative reduction in the rate of incorporation of leucine into proteins. The osmotica mannitol and Carbowax-4000 cause similar changes in the pattern of protein synthesis showing that these effects are due to water stress rather than to a particular osmoticum.     

Design, synthesis and RNase A inhibition activity of catechin and epicatechin and nucleobase chimeric molecules

Several novel catechin/epicatechin and nucleobase chimeric molecules 1-6 have been synthesized via azide-alkyne click chemistry. The structures of these hybrids have been confirmed by NMR and mass spectroscopic data. The synthesized molecules were tested for their RNase A inhibition activities. Gel-based assays showed inhibition in micromolar concentrations. The extent of inhibition was found to be dependent upon the nature of base as well as the configuration at C-3 position of catechin.     

Differential down-regulation of protein kinase C selectively affects IgE-dependent exocytosis and inositol trisphosphate formation.

Recombinant Mu gam gene protein (Mu GAM) synthesized in Escherichia coli accumulates in the form of insoluble inclusion bodies which, after cell lysis and low-speed centrifugation, can be recovered in the pellet fraction. This property was utilized in a purification procedure for Mu GAM based on guanidine hydrochloride denaturation-renaturation followed by a single DEAE-cellulose chromatographic step. The purified Mu GAM was shown by nitrocellulose-filter-binding experiments to bind with high affinity to linear double-stranded DNA and more weakly to supercoiled and single-stranded forms. Mu GAM protects linear DNA from degradation by a variety of exonucleases, but only weakly inhibits endonuclease activity. These results are in accord with a model of Mu GAM conferring protection from exonuclease activity by binding to the ends of DNA.     

Interactions and inhibition of blood coagulation factor Va involving residues 311-325 of activated protein C.

Activated protein C (APC) exerts its physiologic anticoagulant role by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. The synthetic peptide-(311-325) (KRNRTFVLNFIKIPV), derived from the heavy chain sequence of APC, potently inhibited APC anticoagulant activity in activated partial thromboplastin time (APTT) and Xa-1-stage coagulation assays in normal and in protein S-depleted plasma with 50% inhibition at 13 microM peptide. In a system using purified clotting factors, peptide-(311-325) inhibited APC-catalyzed inactivation of factor Va in the presence or absence of phospholipids with 50% inhibition at 6 microM peptide. However, peptide-(311-325) had no effect on APC amidolytic activity or on the reaction of APC with the serpin, recombinant [Arg358]alpha 1-antitrypsin. Peptide-(311-325) surprisingly inhibited factor Xa clotting activity in normal plasma, and in a purified system it inhibited prothrombinase activity in the presence but not in the absence of factor Va with 50% inhibition at 8 microM peptide. The peptide had no significant effect on factor Xa or thrombin amidolytic activity and no effect on the clotting of purified fibrinogen by thrombin, suggesting it does not directly inhibit these enzymes. Factor Va bound in a dose-dependent manner to immobilized peptide-(311-325). Peptide-(311-315) inhibited the binding of factor Va to immobilized APC or factor Xa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of RNase A with VO3- and VO2+ ions. Metal ion binding mode and protein secondary structure

Some of vanadyl complexes have shown potential to inhibit RNase activity by acting as transition state analogue, while at the same time not inhibiting DNase. To gain an insight into the interaction of protein with vanadate (VO3-) and vanadyl (VO2+) ions, the present study was designed to examine the binding of ribonuclase A (RNase A) with NaVO3 and VOSO4 in aqueous solution at physiological pH with metal ion concentrations of 0.001 mM to 1 mM, and protein concentration of 2% w/v. Absorption spectra and Fourier transform infrared (FTIR) spectroscopy with self-deconvolution and second derivative resolution enhancement were used to determine the cation binding mode, association constant and the protein secondary structure in the presence of vanadate and vanadyl ions in aqueous solution. Spectroscopic results show that an indirect metal ion interaction occurs with the polypeptide C = O, C-N (via H2O) with overall binding constants of K(VO3-) = 3.93x10(2) M(-1) and K(VO2+) = 4.20x10(3) M(-1). At high metal ion concentrations, major protein secondary structural changes occur from that of the alpha-helix 29% (free enzyme) to 23-24%; beta-sheet (pleated and anti) 50% (free enzyme) to 64-66% and turn 21% (free enzyme) to 10-12% in the metal-RNase complexes. The observed structural changes indicate a partial protein unfolding in the presence of high metal ion concentration.     

Influence of concanavalin A on protein synthesis and protein release in BHK 21 cells

Addition of concanavalin A to baby-hamster kidney cells (strain BHK 21/C13) grown in Eagle's minimal essential medium devoid of serum but supplemented with insulin as growth-promoting factor, caused a marked reduction of the total protein content of the cells: as early as 1 h after treatment, the amount of protein decreased to about 60–70% of the values found in untreated cultures.Pulse-labelling experiments performed with [³H]leucine demonstrated that the uptake and incorporation of the labelled amino acid was not affected by the lectin up to 6 h after treatment. Pulse-chase experiments gave no evidence for an enhanced degradation of proteins.Examination of the supernatants of concanavalin A-treated cultures as well as their controls, pre-labelled with [³H]fucose and [¹⁴C]leucine revealed that the amount of membrane-derived glycoproteins which were shed into the culture medium was considerably higher in concanavalin A-treated cultures.However, the bulk of protein which accounts for the difference between lectin-treated and untreated cultures consists of intracellular material which was released during the cell harvest procedure. The loss of protein was prevented by α-methyl-d-mannoside (10^?2 M).Scanning electron microscopy of concanavalin A-treated cells showed a change from the smooth surface of the fibroblastic cells to a retracted one as early as 30 min after addition of the lectin. The surface of the altered cells was characterized by the presence of numerous bleb-like protuberances.The viability of the cells was not affected by concanavalin A-treatment during the course of the experiments.Experiments performed with variable concentrations of insulin excluded the possibility that the observed effects might be due to a competition between the lectin and the hormone.     

New bile alcohols - synthesis of (22R)- and (22S)-5beta-cholestane-3alpha,7alpha,12alpha,22,25-pentols.

The synthesis of (22R)- and (22S)-5beta-cholestane-3alpha,7alpha,12alpha,22,25-pentols is described. Bisnorcholyl aldehyde was prepared from cholic acid and converted into the cholestane-pentols by a Grignard reaction with 3-methyl-3-(tetrahydropyran-2-yloxy)-butynylmagnesium bromide followed by hydrogenation and acid hydrolysis. One of the synthetic pentols, the 22R-isomer was identical with a metabolite of 5beta-cholestane-3alpha,7alpha,25-triol formed in the rabbit.     

Differentiated inhibition of DNA, RNA and protein synthesis in L1210 cells by 8-methoxypsoralen

The synthesis of DNA, RNA and protein was measured in L1210 cells following treatment with 8-methoxypsoralen in combination with long wavelength ultraviolet irradiation. The results show that the DNA synthesis is strongly inhibited (approximately 95%) at 200 ng/ml reaching a minimum within 2 hours while RNA synthesis is only weakly affected at this concentration (approximately 40% inhibition). At 2 micrograms/ml the RNA synthesis is inhibited approximately 90%. Even at this concentration only a moderate effect is seen on the protein synthesis. These results strongly indicate that the phototoxic action of 8-methoxypsoralen is primarily due to inhibition of DNA synthesis.