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K Norris

Publications and source records attributed to K Norris.

90 records · Page 5Linked to original sources
PubMed1988

Soluble, prolonged-acting insulin derivatives. III. Degree of protraction, crystallizability and chemical stability of insulins substituted in positions A21, B13, B23, B27 and B30.

It was previously demonstrated that insulins to which positive charge has been added by substituting B13 glutamic acid with a glutamine residue, B27 threonine with an arginine or lysine residue, and by blocking the C-terminal carboxyl group of the B-chain by amidation, featured a prolonged absorption from the subcutis of rabbits and pigs after injection in solution at acidic pH. The phenomenon is ascribed to a low solubility combined with the readiness by which these analogs crystallize as the injectant is being neutralized in the tissue. However, acid solutions of insulin are chemically unstable as A21 asparagine both deamidates to aspartic acid and takes part in formation of covalent dimers via alpha-amino groups of other molecules. In order to circumvent the instability, substitutions were introduced in position A21, in addition to those in B13, B27 and B30, challenging the fact that A21 asparagine has been conserved in this position throughout the evolution. Biological potency was retained when glycine, serine, threonine, aspartic acid, histidine and arginine were introduced in this position, although to a varying degree. In the crystal structure of insulin a hydrogen bond bridges the alpha-nitrogen of A21 with the backbone carbonyl of B23 glycine. In order to investigate the importance of this hydrogen bond for biological activity a gene for the single-chain precursor B-chain(1-29)-Ala-Ala-Lys-A-chain(1-21) featuring an A21 proline was synthesized. However, this single-chain precursor failed to be properly produced by yeast, pointing to the formation of this hydrogen bond as an essential step in the folding process. The stability of the A21-substituted analogs in acid solutions (pH 3-4) with respect to deamidation and formation of dimers was approximately 5-10 times higher than that of human insulin in neutral solution. The rate of absorption of most insulins is decreased by increasing the Zn2+ concentration of the preparation. However, one analog with A21 glycine showed first-order absorption kinetics in pigs with a half-life of approximately 25 h, independent of the Zn2+ concentration. The day-to-day variation of the absorption of this analog was significantly lower than that of the conventional insulin suspensions, a property that might render such an insulin useful in the attempts to improve glucose control in diabetics by a more predictable delivery of basal insulin.

Absorption↗
PubMed1988

Expression of heat stress proteins by human periodontal ligament cells.

The purpose of the present report was to document the stress response produced by physical and chemical abuses to human periodontal ligament cells, and to review some of the known functions of stress response proteins produced as a result of such treatments. For these studies human PDL cells were exposed to sublethal challenges of 43 degrees C heat, sodium arsenite and the amino acid analog L-azetidine-2-carboxylic acid (AZC). The cells were labelled with [35S]-methionine and the proteins produced were examined by autofluorography of SDS-PAGE gels. Heat challenges were shown to induce hsps with an apparent mol. wts. of 90K, 68-72K, 41-47K, and 36 K. Arsenite-treated cells produced similar hsps including a 30k protein not produced by other forms of stress. AZC treatment resulted in the production of apparent functionless hsps with apparent molecular weights of 90,000, 72,000, 68,000 and 36,000. The function of these proteins and their possible role in periodontal disease is discussed.

Arsenic↗
PubMed1988

Tobacco components induce alterations in keratinocyte lipid membrane fluidity and inhibit cell proliferation in culture.

Guinea pig epidermal keratinocytes were harvested and grown in vitro (20). The cells were treated with polyphenolic compounds, acetate/shikimate aromatics, that are ubiquitous in plants. In addition, the cells were exposed to tobacco glycoprotein (TGP), a plant-derived substance prepared from cured tobacco (19). All of the polyphenolic compounds enhanced the attachment of cells to a defined substratum and inhibited cell proliferation. Tobacco glycoprotein (TGP), rutin conjugates and ellagic acid inhibited cell spreading and enhanced the apparent microviscosity of keratinocyte membrane lipids. These data suggest that the use of these compounds may be associated with alterations in cytoskeletal architecture and membrane fluid dynamics that might result in clinical alterations characterized by whitening of mucosae.

Animals↗
PubMed1988

Transfer of murine host protection by using interleukin-2-dependent T-lymphocyte lines.

We have demonstrated in this study that long-term, interleukin-2 (IL-2)-dependent, salmonella antigen-specific T-lymphocyte lines, as well as peritoneal exudate-enriched T cells, could be developed from both the antigen-sensitized inguinal and periaortic lymph nodes. Only those lines (salmonella-specific lymph node cells or peritoneal exudate T cells) were capable of adoptively transferring significant host protection (P less than 0.01) compared with the immune reactions of lethally challenged naive controls or of mice that had ovalbumin-specific T-cell lines transferred. Of particular interest was the finding that IL-2-dependent T-cell lines derived from the lymph nodes could only confer host protection to naive mice when both the transfer and challenge dose were administered via the intravenous route. Likewise, those T-cell lines derived from the peritoneal exudate were only capable of adoptively transferring significant protection when the cells and challenge dose of salmonellae were administered intraperitoneally. These studies indicate that systemic host protection can be transferred to naive mice, but depending on the source, the IL-2-dependent T-cell lines (lymph node or peritoneally isolated) functioned differentially upon challenge. Also, the results of this study indicate that the administration of greater numbers of IL-2-specific T cells may result in decreased, rather than enhanced, host protection. This may be due to the fact that the IL-2-dependent T-cell population consisted of 20 to 25% Lyt-2,3+ cells, indicating that cells of the suppressor/cytotoxic phenotype were present. Thus, increasing the number of cells transferred may result in an abrogation of protection.

Animals↗
PubMed1987

The secretion of glucagon by transformed yeast strains.

Saccharomyces cerevisiae strains were transformed with plasmids coding for modified mating factor alpha 1 leader sequences followed by glucagon. Glucagon-containing peptides which were secreted into the fermentation broth were isolated and their amino acid sequences determined. The yeast strain transformed with the sequence coding for the complete mating factor alpha 1 leader sequence preceding the glucagon gene (MT556) secreted glucagon plus glucagon extended at its N-terminal by parts of the leader sequence. The yeast strain transformed with the sequence coding for a truncated mating factor alpha 1 leader sequence before the glucagon gene (MT615) secreted glucagon. These observations suggest that S. cerevisiae is a suitable vehicle for the efficient expression of plasmids coding for polypeptides similar to glucagon (e.g. VIP, secretin, GIP).

Amino Acid Sequence↗
PubMed1987

Soluble, prolonged-acting insulin derivatives. II. Degree of protraction and crystallizability of insulins substituted in positions A17, B8, B13, B27 and B30.

It has previously been found that insulins, to which positive charge has been added by substitutions in position B30, thus raising the isoelectric point towards pH 7, had a prolonged action when injected as slightly acidic solutions because such derivatives crystallize very readily upon neutralization. Positive charge has now been added by substituting the B13 and A17 glutamic acid residues with glutamines and B27 threonine with lysine or arginine. These substitutions were introduced by site-specific mutagenesis in a gene coding for a single-chain insulin precursor. By tryptic transpeptidation the single-chain precursors were transformed to the double-chain insulin structure, concomitantly with incorporation of residue B30. Thus insulins combining B13 glutamine, A17 glutamine and B27 lysine or arginine with B30 threonine, threonine amide or lysine amide were synthesized. The time course of blood glucose lowering effect and the absorption were studied after subcutaneous injection in rabbits and pigs. The prolonged action of B30-substituted insulins was markedly enhanced by B27 lysine or arginine substitutions and by B13 glutamine. The B27 residue is located on the surface of the hexamer, so a basic residue in this position presumably promotes the packing of hexamers at neutral pH. The B13 residues cluster in the centre of the hexamer. When the electrostatic repulsive forces from six glutamic acid residues are abolished by substitution with glutamine, a stabilization of the hexamer can be envisaged.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Secretion and processing of insulin precursors in yeast.

A series of dibasic insulin precursors including proinsulin was expressed and secreted from Saccharomyces cerevisiae. Recombinant plasmids were constructed to encode fusion proteins consisting of a modified mating factor alpha 1 leader sequence and an insulin precursor. The leader sequence serves to direct the fusion protein into the secretory pathway of the cell and to expose it to the Lys-Arg processing enzyme system. The secreted peptides were purified from the fermentation broth and characterized by sequencing and amino acid analysis. Processing at one or both dibasic sequences was shown in proinsulin and in other insulin precursors containing a short spacer peptide in place of the C peptide. In contrast, no processing was observed in the absence of a spacer peptide in the insulin precursor molecule, e.g., B-Lys-Arg-A (where A and B are the A and B chain of human proinsulin, respectively). This type of single-chain insulin precursors isolated from such constructions could be enzymatically converted into insulin by treatment with trypsin and carboxypeptidase B. The above results suggest that the C-peptide region of proinsulin serves to direct the trypsin-like converting enzyme to process at the two dibasic sequences. We propose that in hormone precursors in general the spacer peptides serve to expose dibasic sequences for processing.

Amino Acid Sequence↗
PubMed1984

Harvey murine sarcoma virus p21 ras protein: biological and biochemical significance of the cysteine nearest the carboxy terminus.

Previous studies of premature chain termination mutants and in frame deletion mutants of the p21 ras transforming protein encoded by the transforming gene of Harvey murine sarcoma virus (Ha-MuSV) have suggested that the C terminus is required for cellular transformation, lipid binding, and membrane localization. We have now further characterized the post-translational processing of these mutants and have also studied two C-terminal v-rasH point mutants: one encodes serine in place of cysteine-186, the other threonine for valine-187. The Thr-187 mutant was transformation-competent, and its p21 protein was processed normally, as was the p21 encoded by a transformation-competent deletion mutant from which amino acids 166-175 had been deleted. The Ser-186 mutant was defective for transformation. The p21s encoded by the Ser-186 mutant and by the previously described transformation-defective mutants did not undergo the posttranslational processing common to biologically active ras proteins: their electrophoretic migration rate did not change, they remained in the cytosol, and they failed to bind lipid. Since the cell-encoded ras proteins also contain this cysteine, we conclude that this amino acid residue is required for all ras proteins.

Animals↗
PubMed1984

Structure of a human gastrin gene.

A gastrin gene was isolated from a genomic library of human DNA. The human gastrin gene is about 4100 base pairs long and contains two intervening sequences. Thus, a 3500-base-pair intervening sequence is located 5 base pairs proximal to the ATG initiator codon, while a 129-base-pair intervening sequence separates the region coding for the principal hormonal form of gastrin, the heptadecapeptide, from the region coding for the major amino-terminal portion of the gastrin precursor. The 5' flanking region of the gene contains the conserved sequences, T-A-T-A-A and G-A-C-T-C-A-T-A-T, in positions similar to those of other eukaryotic genes.

Amino Acid Sequence↗
PubMed1983

Efficient site-directed mutagenesis by simultaneous use of two primers.

A rapid and efficient procedure for site specific mutagenesis is described. A double primed synthesis with a 17-mer mismatch primer and a "universal" 15-mer M13 sequencing primer was used to introduce a T to A transversion into an ompF signal peptide gene cloned in the M13mp8 vector. The two primers were annealed to the circular single stranded M13 template. After a short extension and ligation reaction, a double stranded restriction fragment containing the mismatch (ompF*/EcoR1-SalI) was cut out of the partly single stranded circular DNA and inserted into pBR322. 42% of the E.coli transformants harboured plasmid with the desired mutation, which could be detected by the appearance of a new restriction site (MboII) and by dot blot hybridization of plasmid DNA with the 32P-labeled 17-mer.

Bacterial Outer Membrane Proteins↗
PubMed1983

Molecular cloning of human gastrin cDNA: evidence for evolution of gastrin by gene duplication.

An oligo(dT)-primed cDNA copy of the mRNA coding for the human gastrin precursor was constructed from poly(A)-containing RNA from a human pancreatic, gastrin-producing tumor (a gastrinoma). The cDNA was inserted into the Pst I endonuclease site of plasmid pBR322 by the use of the poly(dC) and poly(dG) tailing procedure. Clones containing gastrin sequences were selected by hybridization to a purified single-stranded 32P-labeled gastrin cDNA probe. This probe was constructed with gastrinoma mRNA as template. As primer for the cDNA synthesis, we used a synthetic oligonucleotide mixture, d(AG-A-A-AG-T-C-C-A-T-C-C-A), corresponding to the gastrin-specific amino acid sequence Trp-Met-Asp-Phe. In this way we determined the nucleotide sequence of the entire coding region (303 nucleotides), the entire 3' untranslated region (102 nucleotides), and 8 nucleotides of the 5' untranslated region. A striking homology between parts of the coding region suggests that evolution of the gastrin gene has involved a gene duplication.

Amino Acid Sequence↗
PubMedDate not supplied

Non-natural CBP2 binding peptides and peptomers modulate carcinoma cell adhesion and invasion.

A combinatorial approach that utilized a repertoire of bacteriophage-peptides has identified a number of non-natural CBP2 binding peptides. Moreover, co-localization of some of these peptides with CBP2 in a number of tumor cell lines demonstrated that the peptides were directed to an intracellular location spatially coincident with the normal distribution of CBP2 [Sauk et al., 2000]. From among these sequences WHYPWFQNWAMA and LDSRYSLQAAMY were the most effective CBP2 binding peptides and best fulfilled the combinatorial motif containing deep hydrophobic pockets. When the hydropathic profiles of collagen alpha1(IV) and alpha2 (IV) were compared with these dodecapeptides, the hydropathic profiles of WHYPWFQNWAMA and LDSRYSLQAAMY closely matched those of alpha1(IV) 414-452 and alpha1(IV)531-543. These peptides were shown to be functional peptidomimics and possessed the ability to alter cell adhesion and invasion of human squamous cell carcinoma cell lines. Peptomers were formed of these non-natural peptides to explore the role that a repetitive peptide may have on cell adhesion. The enhanced cell adhesion observed with the peptomers required both CBP2 antibodies and integrin antibodies for inhibition. The enhanced adhesion observed even in the face of combined antibody inhibition was consistent with such complexes possessing correspondingly slower dissociation rates. Thus, suggesting that peptomers may function in a like manner to multimeric peptide MHC complexes (tetramers) binding more than one cell receptor on a specific cell. These findings evoke both peptidomimics of native ligands and their peptomers as potential reagents by which to target tumor cells for chemotherapy, imaging, or retargeting viral vectors for gene therapy.

Bacteriophages↗
PubMedDate not supplied

Estrogen replacement therapy use in minority postmenopausal women.

OBJECTIVE: To determine the use of estrogen replacement therapy (ERT), and awareness of relative benefits and risks of ERT in under-served minority postmenopausal women. DESIGN: Survey. SETTING: General medical and geriatric clinics of an acute care teaching hospital in south central Los Angeles. PARTICIPANTS: Convenience sample of 155 postmenopausal women. MEASUREMENTS: Current and past ERT use, awareness of risks and benefits, reasons for not using ERT. RESULTS: Of the 143 African-American and Hispanic postmenopausal women, only 16% reported current ERT use. Of the 111 women who have never taken ERT, 86% responded that their physicians have not discussed ERT with them. Fifteen percent of the women were aware of the coronary artery disease (CAD) benefit, and 22% were aware of the osteoporosis benefit. Eighteen percent were aware of the relative risk of endometrial cancer. CONCLUSION: Over 80% of the under-served postmenopausal women surveyed in our clinics were not receiving ERT, and most were unaware of the benefits and risks of ERT. Lack of physician discussion was cited as the primary reason. Special educational efforts to improve physician counseling practices for ERT in this and similar populations need to be developed.

Black or African American↗
PubMedDate not supplied

Comparative efficacy of exosurf and survanta surfactants on early clinical course of respiratory distress syndrome and complications of prematurity.

OBJECTIVES: To determine the comparative efficacy of Exosurf Neonatal and Survanta surfactants on the early course of respiratory distress syndrome (RDS), arterial blood gases, ventilatory support, outcome morbidity rate, and complications of prematurity and RDS. STUDY DESIGN: Medical records from 203 premature newborn infants undergoing mechanical ventilation for respiratory distress syndrome, and who received up to four rescue doses of either Exosurf or Survanta, were retrospectively reviewed. RESULTS: All groups were comparable for birth weight and gestational age. Although the two randomized groups were similar in severity of RDS based on fraction of inspired oxygen (FIO2) and ventilatory support, a significantly greater improvement in respiratory function as evidenced by FIO2, mean airway pressure, alveolar-arterial partial pressure of oxygen difference, and oxygen index, was observed in the Survanta group from 12 hours (p < 0.05) through 48 hours (p < 0.01). Comparison of outcome morbidity rate by gestational age showed a higher occurrence of retinopathy of prematurity (p < 0.02) among the older infants (28 to 32 weeks) who were treated with Exosurf. CONCLUSION: Survanta exerted a significantly faster response in the early clinical course of RDS compared with Exosurf. However, no difference in the impact on eventual respiratory outcome was observed. We therefore conclude that both surfactants are effective for the treatment of RDS.

Biological Products↗