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Biomedical subjects

K Norris

Publications and source records attributed to K Norris.

At least 73 records · Page 4Linked to original sources
PubMed1993

Hsp 47 is localized to regions of type I collagen production in developing murine femurs and molars.

To determine whether the proposed molecular chaperone Hsp47 is associated with the production of heterotrimeric procollagen, the distribution of anti-Hsp47 and anti-collagen antibodies were examined in developing murine femurs and molars of 22-23-day CD-1 mice. In addition, the expression of Hsp47, and collagen mRNAs were assessed by in situ hybridization using oligonucleotide probes. These studies revealed that Hsp47 was developmentally expressed and produced in regions that are coincident with type I collagen. Hsp47 was not localized in cartilaginous zones of developing femurs or in the regions of developing molars producing type III collagen. These results support the hypothesis that Hsp47 is necessary for the assurance of type I procollagen and is not expressed with other homotrimeric procollagen molecules.

Animals↗
PubMed1992

Adrenal responsiveness in very-low-birth-weight infants treated with dexamethasone.

This study was designed to investigate the effect of steroid administration in ill premature neonates. Twenty high-risk very-low-birth-weight (VLBW) infants [birth weight (BW) < or = 1,300 g] with a mean BW 948 +/- 220 g, gestational age (GA) 27 +/- 1.7 weeks underwent 1-hour ACTH (Cortrosyn) stimulation tests and determination of 17-hydroxyprogesterone (17OHP)/dehydroepiandrosterone sulfate (DHEAS) at 23.6 +/- 15.9 days poststeroid treatment for bronchopulmonary dysplasia (BPD)/airway obstruction. Metyrapone tests were also obtained in 18 infants. Baseline (nonsteroid-exposed) values for pre-/post-ACTH cortisol, 17OHP, DHEAS, and pre-/post-metyrapone compound S values were obtained in 5 infants. Eight of 18 (44%) infants had evidence of secondary (hypothalamic-pituitary) adrenal suppression based on abnormal metyrapone tests. No difference was found in BW, GA, time on O2 or AV, steroid dose/kg, or neonatal/postneonatal mortality between the suppressed and nonsuppressed groups. Two of 4 infants with borderline ACTH tests had subnormal compound S levels postmetyrapone. No relationship was found between steroid dose/kg and cortisol response post-ACTH. Additionally, corrected GA was not related to change in cortisol, 17OHP, and DHEAS pre-/post-ACTH. Two infants exhibited recovery of adrenal suppression documented by repeated metyrapone testing at 63 and 186 days poststeroid treatment. In conclusion, this study documents the apparent high incidence of secondary adrenal suppression in VLBW infants treated with dexamethasone. Clinical significance of these findings deserves further investigation.

17-alpha-Hydroxyprogesterone↗
PubMed1991

Diverse forms of stress result in changes in cellular levels of osteonectin/SPARC without altering mRNA levels in osteoligament cells.

The osteonectin/SPARC gene has been shown to possess motifs for a heat shock element and metal responsiveness. Also, the expression of the protein has been associated with culture stress in endothelial cells. In the present study, osteoligament (OL) cells derived from the patellar ligament were subjected to diverse forms of stress that included (a) exposure to sodium arsenite, (b) heat shock, (c) cadmium ion, and (d) the amino acid analog, AZC. Osteonectin/SPARC levels in OL cells were determined by Western blot analyses, and immunoprecipitation using antiosteonectin antibodies. Expression of osteonectin/SPARC mRNA was determined by Northern analysis using a 1.5 kb EcoRI restriction fragment of bovine osteonectin cDNA. These studies reveal that osteonectin/SPARC is produced following diverse forms of stress, however, the levels are lower than observed in unchallenged OL cells. In all instances, the mRNA levels were comparable to control cells. These studies indicate that expression of osteonectin/SPARC mRNA is tightly controlled in OL cells and that the protein may be regulated at the level of protein translation.

Animals↗
PubMed1991

Cell attachment activity of cementum: bone sialoprotein II identified in cementum.

Considerable research effort has been directed at preparing root surfaces in a fashion that would promote cell attachment leading to periodontal regeneration; however, no methods have proven to be clinically predictable. Identification of attachment protein(s) associated with the root surface matrix of cementum may prove valuable for developing effective clinical treatments. In this study cementum proteins were extracted from bovine and human teeth by sequential chaotropic extraction using guanidine followed by guanidine/EDTA. The guanidine/EDTA extract, but not guanidine extract, was found to promote attachment of fibroblasts. This attachment activity was inhibitable with synthetic peptide containing the attachment sequence arginine-glycine-aspartic acid (RGD). Fractionation of the guanidine/EDTA extract revealed several fractions with attachment activity. Immunoblot analysis demonstrated that two of these fractions contain the bone-associated RGD containing attachment protein, bone sialoprotein-II (BSP-II). In addition, attachment activity was also noted in other fractions that could not be attributed to BSP-II or fibronectin. These studies indicate that a component of the attachment activity of cementum is likely to be due to BSP-II and that cementum contains additional, as yet undetermined, attachment proteins.

Amino Acid Sequence↗
PubMed1991

Modulation of neonatal rat myeloid kinetics resulting in peripheral neutrophilia by single pulse administration of Rh granulocyte-macrophage colony-stimulating factor and Rh granulocyte colony-stimulating factor.

Rh granulocyte-macrophage (GM) colony-stimulating factor (CSF) and rh granulocyte (G) CSF have been demonstrated to induce proliferation and maturation of myeloid stem cells and release of mature polymorphonucleocytes (PMNs) from human and animal adult bone marrows. Unfortunately, reduced bone marrow progenitor cells, neutrophil storage pool (NSP) depletion and peripheral neutropenia are characteristic of human and animal newborn bone marrows. We investigated the effect of administering intraperitoneal rhGM-CSF and rhG-CSF to Sprague-Dawley newborn rats (less than 36 h). Newborn rats treated with intraperitoneal CSF (3.0 micrograms/kg) demonstrated significant leukocytosis at 6 and 24 h: rhGM-CSF vs. control, WBC (10(3)/mm3), at 6 h, 8.0 +/- 0.5 vs 4.3 +/- 0.9 (p less than or equal to 0.003), and at 24 h, 7.7 +/- 1.7 vs. 3.8 +/- 0.2 (p less than or equal to 0.008); rhG-CSF vs. control WBC (10(3)/mm3) at 6 h, 6.6 +/- 1.2 vs 4.3 +/- 0.1 (p less than or equal to 0.03), and at 24 h, 8.1 +/- 0.2 vs. 3.75 +/- 0.2 (p less than or equal to 0.003). The absolute neutrophil count was also significantly elevated at 6 h following intraperitoneal CSF (3.0 micrograms/kg): RhGM-CSF vs. control 1,827 +/- 25 vs. 379 +/- 10 (p less than or equal to 0.001); rhG-CSF vs. control, 1,698 +/- 40 vs. 371 +/- 10.1 (p less than or equal to 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Expression of constitutive and inducible HSP70 and HSP47 is enhanced in cells persistently spread on OPN1 or collagen.

Cells persistently spread on OPN or collagen survive heat shock better than cells transiently spread on fibronectin or tissue culture plates. Thus, a central question is whether constitutively or inducible stress proteins are enhanced in cells grown on adhesive proteins that maintain a persistent spread cell shape. Levels of Hsp 72,73, and colligin/Hsp47 were determined by Western blot analyses. The inducible Hsp 72 was prominently expressed following heat shock in cells grown on OPN or collagen, but not in cells plated on fibronectin coated substratum or on tissue culture plates. Colligin/Hsp 47 and Hsp 73 manifested a similar pattern of expression indicating that these adhesive attachment proteins accommodate cell function through organization of cell architecture.

Cell Adhesion↗
PubMed1990

Recombinant human extrinsic pathway inhibitor. Production, isolation, and characterization of its inhibitory activity on tissue factor-initiated coagulation reactions.

Previous studies have shown that extrinsic pathway inhibitor (EPI) is an effective inhibitor of factor Xa alone or factor VIIa-tissue factor complex in the presence of factor Xa. Since tissue factor exposure is implicated in thrombogenesis, we hypothesized that EPI may be valuable in the treatment of some thromboembolic episodes. Furthermore, EPI may be an important factor in bleeding complications in hemophiliacs. In the present study, human EPI was expressed in baby hamster kidney cells using a mammalian expression vector. Transfected cells expressed 1-2 micrograms/ml of recombinant EPI (rEPI) which was purified to homogeneity by heparin-Sepharose chromatography, ion-exchange chromatography, and reverse phase high performance liquid chromatography. Purified rEPI exhibited a specific activity of 30,000 units/mg and migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 42,000. In addition, the NH2-terminal sequence of rEPI was identical to that of HepG2 EPI and HeLa EPI. The ability of rEPI to inhibit factor X activation by a complex of factor VIIa-tissue factor was then examined in the presence and absence of plasma concentrations of human factors VIII and IX. Using relipidated human brain tissue factor apoprotein, rEPI inhibited the factor VIIa-mediated activation of factor X half-maximally at 2.5 and 1 nM in the presence and absence of factors VIII and IX, respectively. Using monolayers of a human bladder carcinoma cell line (J82) as the source of tissue factor, the activation of factor X by cell-bound factor VIIa was inhibited half-maximally by 5 nM rEPI in the presence of factors VIII and IX. The proteolytic activity of J82 cell-bound factor Xa toward prothrombin was inhibited half-maximally at approximately 5 nM rEPI, while the amidolytic activity of factor Xa in solution was inhibited by rEPI with a Ki of 130 pM. Recombinant EPI also inhibited the amidolytic activity of factor VIIa half-maximally at 10 nM rEPI in the presence of relipidated tissue factor apoprotein and calcium. These results indicate that, in the presence of plasma concentrations of factors VIII and IX, at least 10 times the plasma concentration of EPI is required to reduce factor VIIa-dependent factor X activation one order of magnitude in vitro. In the absence of functional factor VIII and IX, rEPI at plasma levels was a potent inhibitor of factor VIIa-mediated factor X activation, and this activity presumably accounts for the inability of hemophiliacs to initiate hemostasis via the extrinsic pathway.

Amino Acid Sequence↗
PubMed1990

Changing the insulin receptor to possess insulin-like growth factor I ligand specificity.

To examine the role of the N-terminal part of the insulin-like growth factor I (IGF-I) receptor and insulin receptor in determining ligand specificity, we prepared an expression vector encoding a hybrid receptor where exon 1 (encoding the signal peptide and seven amino acids of the alpha-subunit), exon 2, and exon 3 of the insulin receptor were replaced with the corresponding IGF-I receptor cDNA (938 nucleotides). To allow direct quantitative comparison of the binding capabilities of this hybrid receptor with those of the human IGF-I receptor and the insulin receptor, all three receptors were expressed in baby hamster kidney (BHK) cells as soluble molecules and partially purified before characterization. The hybrid IGF-I/insulin receptor bound IGF-I with an affinity comparable to that of the wild-type IGF-I receptor. In contrast, the hybrid receptor no longer displayed high-affinity binding of insulin. These results directly demonstrate that it is possible to change the specificity of the insulin receptor to that of the IGF-I receptor and, furthermore, that the binding specificity for IGF-I is encoded within the nucleotide sequence from 135 to 938 of the IGF-I receptor cDNA. Since the hybrid receptor only bound insulin with low affinity, the insulin binding region is likely to be located within exons 2 and 3 of the insulin receptor.

Amino Acid Sequence↗
PubMed1990

The expression of colligin/hsp47 after stress in human periodontal fibroblasts in vitro.

Fibroblasts from human periodontal ligaments were grown in vitro. The levels of collagen and total protein in these cells were compared with subject- and passage-matched gingival fibroblasts. In 3 subjects the levels of both were greater in ligament than in gingival fibroblasts. These increased levels were also associated with increased levels of proteins reacting with anti-colligin/hsp47 antibodies on SDS-PAGE. Ligament and gingival fibroblasts were subjected to heat shock, sodium arsenite and the amino acid analogue AZC. These studies showed that (a) sodium arsenite and AZC enhanced the cellular levels of hsp47 in both types of fibroblast, (b) the colligin/hsp47 levels expressed were associated with elevated levels of protein and collagen production and (c) the presence of colligin/hsp47 was decreased under conditions of serum deprivation.

Arsenic↗
PubMed1990

Persistent spreading of ligament cells on osteopontin/bone sialoprotein-I or collagen enhances tolerance to heat shock.

Fibronectin (FN), bone sialoprotein-I (BSP-I), Type I collagen, and a number of synthetic peptides containing the integrin attachment sequence (RDG) were evaluated for their ability to affect stress tolerance in osteo-ligament cells (OL). The attachment and spreading of OL cells was determined by the method of Klebe (1974) and Akiyama et al. (1986). Survival from heat shock was evaluated after the methods of Gerner et al. (1976). These studies showed that FN, BSP-I, and synthetic RGD peptides enhance attachment of OL cells. Increased survival from heat was limited to cells spread on fibronectin, BSP-I, and Type I collagen. OL cells that persistently spread on BSP-I and Type I collagen had more survivors than cells demonstrating transient spreading on FN. These studies indicate that (a) cell spreading is a prerequisite for stress tolerance and (b) enhanced stress tolerance is mediated by protein sequences other than those immediately surrounding the RGD sites in native proteins.

Animals↗
PubMed1990

Prophylactic or simultaneous administration of recombinant human granulocyte colony stimulating factor in the treatment of group B streptococcal sepsis in neonatal rats.

Despite the emergence of newer antibiotic treatments, group B streptococcal infection still carries a high mortality rate in the newborn and is characterized by reduced neutrophil proliferative pools, neutrophil storage pools, neutropenia, and polymorphonuclear cell dysfunction. Recombinant human granulocyte-colony stimulating factor (rhG-CSF) has recently been demonstrated to induce neutrophilia and modulate neutrophil proliferative pools and neutrophil storage pools in the newborn rat. We therefore investigated the adjuvant effect of rhG-CSF given to group B streptococcus (GBS) septic Sprague-Dawley newborn (less than 36 h) rats treated with and without antibiotic therapy. After inoculation of GBS, a GBS survival curve established the LD50 at 50 h to be approximately 3 X 10(6) organisms/gm. Newborn rats were divided into four treatment groups after GBS inoculation. rhG-CSF was administered at the same time as GBS inoculation. At 24 h, there was approximately 100% survival in all groups. However, by 72 h after GBS inoculation, there was a significant difference in survival. Group 1, PBS/Alb, had a survival rate of 4%; group 2, rhG-CSF, 9%; group 3, antibiotics, 28%; and group 4, antibiotics plus rhG-CSF, 91% (p less than or equal to 0.001). Additionally, when rhG-CSF was administered prophylactically (6 h before GBS), a similar significant synergistic effect in survival was demonstrated with granulocyte colony stimulating factor plus antibiotics versus antibiotics alone (70 versus 10%) (p less than or equal to 0.01). These preliminary data suggest that either simultaneous or prophylactic pulse administration of rhG-CSF may have a synergistic and protective effect on survival in antibiotic-treated experimental GBS in the neonatal rat.

Animals↗
PubMed1990

Aprotinin and aprotinin analogues expressed in yeast.

Synthetic genes encoding aprotinin and aprotinin analogues were constructed and fused in frame to the S. cerevisiae mating factor alpha 1 signal-leader (1-85) sequence. Expression in yeast resulted in secretion into the culture medium of a moderate yield of correctly processed aprotinin (1-58) together with two N-terminally extended forms. Des-Arg1, Pro2-aprotinin was expressed in a higher yield. In this case only the correct N-terminal amino acid sequence was found. Substitution of Ser42 for Arg42 in the potential internal KEX2 processing site improved the secretion yield. The aprotinins are characterized by an inhibition profile similar to that of native aprotinin. Des-Arg1, Pro2-[Arg15, Ser42] aprotinin has a strongly increased plasma kallikrein inhibition profile.

Aprotinin↗
PubMed1989

Genetic linkage analysis of hereditary arthro-ophthalmopathy (Stickler syndrome) and the type II procollagen gene.

Hereditary arthro-ophthalmopathy (AO), or Stickler syndrome, is a dominantly inherited disorder characterized by vitreo-retinal degeneration and frequently accompanied by epiphyseal dysplasia and premature degenerative joint disease. Three large families with AO were analyzed for clinical manifestations of the disease and for coinheritance of the genetic defect with RFLPs in the type II procollagen gene (COL2A1). Genetic linkage between AO and COL2A1 was demonstrated in the largest family, with a maximum LOD score of 3.52 at a recombination distance of zero. Data from a second family also supported linkage of AO and COL2A1, with a LOD score of 1.20 at a recombination distance of zero. These results are consistent with the conclusion that mutations in the COL2A1 gene are responsible for AO in these two families. In a third AO family, however, recombination between AO and COL2A1 occurred in at least one meiosis, and the data were inconclusive with respect to linkage.

Chromosomes, Human, Pair 12↗
PubMed1988

Competitive expression of two heterologous genes inserted into one plasmid in Saccharomyces cerevisiae.

Plasmids were constructed which contained two expression units encoding single-chain insulin precursors. Surprisingly, the total amount of insulin precursor produced was similar to that produced from plasmids containing a single expression unit. In this system, therefore, two expression cassettes can be brought to compete for the limited ability of the yeast cell for synthesis and secretion. Using genes encoding B(1-29)-A(1-21) and B(1-29)-Ala-Ala-Lys-A-(1-21), the slightly different precursors could be quantified individually after separation by high-performance liquid chromatography from the culture supernatant. The two-cassette system allowed a sensitive and well controlled comparison of parameters important for optimal expression of a heterologous gene in Saccharomyces cerevisiae. The system was used to compare two promoter constructions and also to evaluate the position of expression cassettes in the plasmid. Finally the codon usage in the gene to be expressed was found to influence its ability to compete for expression.

Amino Acid Sequence↗
PubMed1988

Monomeric insulins obtained by protein engineering and their medical implications.

The use of insulin as an injected therapeutic agent for the treatment of diabetes has been one of the outstanding successes of modern medicine. The therapy has, however, had its associated problems, not least because injection of insulin does not lead to normal diurnal concentrations of insulin in the blood. This is especially true at meal times when absorption from subcutaneous tissue is too slow to mimic the normal rapid increments of insulin in the blood. In the neutral solutions used for therapy, insulin is mostly assembled as zinc-containing hexamers and this self-association, which under normal physiological circumstances functions to facilitate proinsulin transport, conversion and intracellular storage, may limit the rate of absorption. We now report that it is possible, by single amino-acid substitutions, to make insulins which are essentially monomeric at pharmaceutical concentrations (0.6 mM) and which have largely preserved their biological activity. These monomeric insulins are absorbed two to three times faster after subcutaneous injection than the present rapid-acting insulins. They are therefore capable of giving diabetic patients a more physiological plasma insulin profile at the time of meal consumption.

Animals↗