Enzymatic activity of metal-binding proteins in epidermal cells

Summary Enzymatic activity was investigated in metal-binding proteins from rat epidermal cells. Tris-HCl buffer soluble and KSCN solubilized proteins were extracted stepwise from granular and cornified cells of 2-day old rat epidermis. Each extract was separately applied to a Cu²⁺ or Zn^2– chelate Sepharose 6B column and the proteins were eluted with buffers of different pHs and finally with EDTA solution. Metal chelate-binding proteins were found in both soluble and solubilized proteins but there was a larger amount in the latter. Affinity of the proteins to bind with Cu²⁺ chelate was greater than that with Zn²⁺ chelate. In Tris-HCl buffer extract, histidase activity was detected in Cu²⁺ chelate-binding proteins, but not in Zn²⁺ chelate-binding proteins. Acid phosphatase, cysteine proteinase, dipeptidase, cathepsin D, -galactosidase, gelatin hydrolase, and superoxide dismutase did not bind to metal chelates although these enzymes, except acid phosphatase, were inhibited by Cu²⁺, but not by Zn²⁺. In contrast, KSCN solubilized metal chelate-binding proteins showed plasminogen activator, acid phosphatase, and gelatin and casein hydrolases while histone hydrolase did not bind to either chelate column. Since metal-binding proteins in rat epidermal cells have been shown previously to be histidine- and cysteine-rich proteins concentrated in keratohyalin granules, interaction of metals and the structural proteins with certain enzymes may be involved in the regulation of epidermal cell functions.     

Interferon-α, interferon-γ and sizofiran in the adjuvant therapy in ovarian cancer — a preliminary trial

The study included 24 cases of negative second-look laparotomy (SLL) after operation on ovarian cancer. 12 cases were treated with sizofiran and recombinant interferon-gamma before and after SLL and then with human lymphoblastoid interferon-alpha. The remaining 12 cases (controls) were followed up without any drug therapy after SLL. There were no recurrences in the treated group, but in 3 cases of the control group. Also significant difference in survival was noted in the treated group.     

Serotypes and mating types of clinical strains ofCryptococcus neoformans isolated in Taiwan

Twenty-one strains ofCryptococcus neoformans isolated from patients in Taiwan were characterized for serotypes and mating types. Slide agglutination test was performed with 8 factor-specific sera (Iatron Company, Japan) to determine the serotypes. Wheat bran agar (WBA) and malt extract agar (MEA, Wickerham) media were used for the mating tests. Twenty of the isolates were of serotype A, and one was serotype B. Except for 2 strains of serotype A, all of the serotype A strains mated withFilobasidiella neoformans var.neoformans, mating type a. The only serotype B strain mated withF. neoformans var.bacillispora mating type a in MEA medium. These data revealed the low prevalence (1/21; 4.8%) ofC. neoformans var.gattii in Taiwan, a subtropically located island.     

siRNA, miRNA and HIV: promises and challenges

INTRODUCTION The recent discovery of small interfering RNA (siRNA) revealed an important role for small RNAs in regulating gene expression. First described in plants, as “post- trancriptional gene silencing” (PTGS) [1], RNA interfer- ence (RNAi) is a nucleic-acid based immune defense against viruses, transgenes and transposons [2]. Triggered by double-stranded RNA (dsRNA), RNAi leads to the se- quence specific degradation of a target mRNA [3]. In eukaryotic cells, long dsRN…     

Identification of both Gi2 and a novel, immunologically distinct, form of Go in rat myometrial membranes

Immunoblotting of rat myometrial membranes with an antiserum (SG1) which recognizes the alpha-subunits of both Gi1 and Gi2 indicated the presence of detectable levels of an apparently single form of some 40 kDa. A second antiserum (LE2) specific for Gi2 also recognized this protein, confirming its identity. Immunoblotting of the myometrial membranes with a series of antipeptide (OC1, IM1, ON1) and polyclonal (RV3) antisera, all of which recognize rat brain Go, produced a more complex pattern. Antisera OC1 and ON1 recognized a single polypeptide which essentially comigrated with rat brain Go. In contrast, antisera RV3 and IM1 did not recognize the myometrial protein. These data provide evidence for the presence of Gi2 and of a novel G-protein, related immunologically to Go, in rat myometrial membranes.     

The role of salt bridge formation in glucagon: an experimental and theoretical study of glucagon analogs and peptide fragments of glucagon

BACKGROUND: Glucagon is a 29-residue peptide produced in the alpha cells of the pancreas that interacts with hepatic receptors to stimulate glucose production and release, via a cAMP-mediated pathway. Type 2 diabetes patients may have an excess of glucagon and, as such, glucagon antagonists might serve as diabetes drugs. The antagonists that bind to the glucagon receptor but do not exhibit activity could be analogs of glucagon. The presence of salt bridges between some residues of glucagons (such as aspartic acid) and others (such as lysine) might influence both the binding to the receptor and the activity. MATERIALS AND METHODS: Experimental-The solid phase method with 4-methylbenzilhydrilamine resin (p-MBHA resin) was used for the synthesis of glucagon analogs. Rat liver membranes were prepared from male Sprague-Dawley rats by the Neville procedure. The receptor binding essay was performed in 1% BSA, 1 mM dithiothreitol, 25 mM Tris-HCl buffer, pH 7.2. Adenyl cyclase activity was measured in an assay medium containing 1% serum albumin, 25 mM MgCl2, 2 mM dithiothreitol, 0.025 mM GTP, 5 mM ATP, 0.9 mM theophylline, 17.2 mM creatine phosphate, and 1 mg/ml creatine phosphokinase. Theoretical-Quantum chemical calculations using the Titan program with the 6-31G* basis set were performed to calculate the binding energies of salt bridges between aspartic or glutamic acids and lysine. The relative stability of cyclic conformations of glucagon segments versus the extended segments was determined. RESULTS: It was found that the cyclic Glu9-Lys12 amide compound displayed a 20-fold decrease in binding affinity. DesHis1 cyclic compounds Glu20-Lys24 amide and DesHis1Glu9 Glu20-Lys24 amide behave as glucagon antagonists. The calculations show that cyclic conformations of tetrapeptidic and pentapeptidic segments of glucagon are more stable than the extended species. CONCLUSIONS: The biological data and the theoretical calculations show that an intramolecular salt bridge might impart stability to some glucagon antagonists and, when situated at the C-terminus of glucagon, might facilitate induction of an alpha-helix upon initial hormone association with the membrane bilayer. These findings might be a useful tool for the design of new glucagon antagonists.     

Glutamatergic neurotransmission in alcoholism

Substance abuse and dependence is the most common psychiatric problem. Alcohol is the most commonly abused substance and most people who abuse other substance(s) abuse alcohol at the same time. Accumulating evidence suggests that neurophysiological and pathological effects of ethanol are mediated to a considerable extent via the glutamatergic system. Ethanol disrupts glutamatergic neurotransmission by inhibiting the response of the N-methyl-D-aspartate (NMDA) receptor and by promoting neuronal toxicity through upregulation of the NMDA receptor density. Therefore, short-term/acute ethanol treatment results in a blockade of NMDA receptor-mediated neurotransmission and apoptotic cell death by inhibiting the trophic effect mediated by the NMDA receptor whereas chronic ethanol treatment and withdrawal results in an enhanced toxic response toward glutamate. The neurobiology of human alcoholism such as ethanol intoxication, dependence, withdrawal seizures, delirium tremens, Wernicke-Korsakoff syndrome, and fetal alcohol syndrome can be better understood as a spectrum of consequences of ethanol's effect on the NMDA glutamatergic system.