Biomedical subjects

R W Tarnuzzer

Publications and source records attributed to R W Tarnuzzer.

24 records · Page 2Linked to original sources

PubMed1992

Catalysis by mutants of human carbonic anhydrase II: effects of replacing hydrophobic residues 198 and 204.

Previous studies shows that the replacement of Phe-198 in carbonic anhydrase III to the corresponding Leu residue found in carbonic anhydrase II caused the appearance of isozyme II-like activity (LoGrasso et al. (1991) Biochemistry 30, 8463-8470). Carbonic anhydrase II is more efficient in the catalysis of CO2 hydration by 500-fold and has an apparent pKa for this catalysis about two pKa units above that of carbonic anhydrase III. Moreover, isozyme II catalyzes the hydrolysis of 4-nitrophenyl acetate, whereas isozyme III shows no appreciable catalysis. The purpose of this work was to test the hypothesis that making the converse replacement Leu-198-->Phe as well as Leu-204-->Glu and the double replacement in carbonic anhydrase II would give the resulting mutants of isozyme II properties of isozyme III. The catalytic activities of these mutants in CO2 hydration and 4-nitrophenyl acetate hydrolysis were smaller by at most 5-fold and the pKa values for these catalyses were identical compared with wild-type isozyme II. The different effects of converse mutants of HCA II and III indicate complexity in structure not evident from their similar backbone conformations.

Base Sequence↗

PubMed1990

Expression of the mammalian system A neutral amino acid transporter in Xenopus oocytes.

In this report, we demonstrate the expression of the mammalian System A neutral amino acid transporter in Xenopus laevis oocytes following microinjection of mRNA from rat liver, Chinese hamster ovary (CHO) cells, and human placenta. Stage 6 oocytes were injected with poly(A+) mRNA from one of these three sources and incubated for 24 h prior to assaying Na(+)-dependent 2-aminoisobutyric acid transport to monitor the increase in System A activity. The endogenous 2-aminoisobutyric acid uptake rates in oocytes were sufficiently slow so as to provide a low background value that was subtracted to obtain transport rates for the mammalian carrier alone. The degree of expression of the mammalian System A activity in Xenopus oocytes corresponded to the known transport rates in the tissue from which the mRNA was prepared. For example, hepatic mRNA from glucagon-treated rats produced greater System A activity than mRNA from control animals, and the mRNA from the CHO transport mutant cell line alar4-H3.9, which overproduces System A, resulted in higher transport rates than mRNA from the parental cell line (CHO-K1). Fractionation of total mRNA poly(A+) by nondenaturing agarose gel electrophoresis revealed transport activity associated with a 2.0-2.5-kilobase mRNA fraction common to each of the three tissues tested.

Aminoisobutyric Acids↗

PubMedDate not supplied

Horse complement protein C9: primary structure and cytotoxic activity.

Lack of hemolytic activity of horse serum is an inherent property of horse C9. To understand the molecular reasons for this deficiency we have cloned C9 cDNA from a horse liver cDNA library and have sequenced the cDNA yielding the complete coding sequence for horse C9. Purification of C9 from horse plasma and microsequencing established the N-terminus of the mature protein and verified that the correct horse C9 cDNA clone had been isolated. The deduced amino acid sequence corresponds to a mature protein of 526 amino acids that is 77% identical to human C9. It has the same domain structure as human C9 and contains 22 cysteines and four invariant tryptophans. The few differences include the N-terminus, which is an unblocked glycine in horse C9 but pyroglutamine in human C9, and three potential N-glycosylation sites compared to two in human C9. The N-terminal difference is unimportant since microsequencing of bovine C9, which is strongly hemolytic, established that it also has an unblocked glycine identical to horse C9. There are no obvious structural differences apparent that could resolve the differences in hemolytic potency between the two molecules. Aside from a few conservative replacements, both C9 sequences are identical between positions 250 and 360. This region includes the membrane interaction domain in C9 and the postulated transmembrane segment that is thought to constitute the wall of a putative transmembrane pore and, therefore, should be required for cytotoxicity. In agreement with this prediction we have observed that, in contrast to the marked decrease in hemolytic activity, horse C9 is very efficient in killing a variety of Gram-negative bacteria. These results demonstrate that horse C9 is a structurally competent molecule with efficient cytotoxic activity. Its inability to lyse erythrocytes may be related to the action of control proteins on target cell membranes.

Amino Acid Sequence↗

PubMedDate not supplied

Biochemical analysis of acute and chronic wound environments.

The process of wound healing involves a complex interaction between numerous cell types, extracellular matrix molecules, and soluble mediators including growth factors and cytokines. This complex milieu is under active investigation for the purposes of beginning to understand how this environment regulates tissue repair. Quantitation of growth factors, cytokines, and matrix metalloproteinases within surgical wound fluids may help to elucidate this regulatory network, not only in noncomplicated wound healing but also in pathologic lesions such as chronic ulcers.

Journal Article↗

PubMedDate not supplied

Alpha-smooth muscle actin expression in rat and mouse mesenteric wounds after transforming growth factor-beta1 treatment.

Rat mesenteric perforations heal by contraction within 5 to 7 days, whereas mouse mesenteric perforations seldom close within 3 weeks unless stimulated by transforming growth factor-beta1. In this article, we quantified the expression of alpha-smooth muscle actin by quantitative-reverse transcription-polymerase chain reaction and the orientation of actin filaments at the wound margin by Fourier transformation image analysis after treatment with transforming growth factor-beta1. The expression of transforming growth factor-beta1 and its type II receptor was also assessed. Actin filaments were shown to increase with time at the wound margin in both species and the expression of alpha-smooth muscle actin mRNA increased simultaneously. Transforming growth factor-beta1 enhanced the alpha-smooth muscle actin expression four to five times in rats and three to four times in mice on day 5, but the number of copies expressed per cell was 15-fold higher in rats than in mice. Transforming growth factor-beta1 was down-regulated after wounding in free peritoneal cells of rats, but maintained until day 5 in transforming growth factor-beta1-treated mice. The main finding of this study was that untreated, normal rats expressed substantially more alpha-smooth muscle actin than mice. After treatment with transforming growth factor-beta1, this expression increased similarly in both species. It can be hypothesized that normal closure of mesenteric perforations requires a minimum level of actin expression. This level is not reached in normal mice, but is exceeded after stimulation. Perforations in the rat always close, because the alpha-smooth muscle actin expression is always above this level.

Journal Article↗

PubMedDate not supplied

Expression of matrix metalloproteinases and tissue inhibitor of matrix metalloproteinases in mesothelial cells and their regulation by transforming growth factor-beta1.

Tissue injury and pelvic inflammation often results in peritoneal scar tissue formation. The objective of this study was to determine whether mesothelial cells which line the peritoneal cavity express matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs), and if their expression is regulated by transforming growth factor-beta1, a key regulator of tissue fibrosis. For this purpose we used Met-5A cells, a cell line derived from human normal mesothelial cells, and for comparative analysis we used U-937 cells, a human monocytic/macrophage cell line. The cells were treated with transforming growth factor-beta1 (1 ng/ml) for various time periods and the levels of MMP and TIMP mRNA and protein expression were determined using quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The results indicate that the mesothelial cells and macrophages express MMP-1 (collagenase-1), MMP-3 (stromelysin-1), TIMP-1 and TIMP-2 mRNA and protein at various levels, with significantly higher TIMPs than MMPs, and higher MMP-1 than MMP-3 (p < 0.001). The mesothelial cells express significantly less MMP-1, higher MMP- 3 and similar levels of TIMP mRNA compared to macrophages. In a time-dependent manner, treatment of the mesothelial cells with transforming growth factor-beta1 resulted in a significant decrease in the expression of MMP-1, while increasing the expression of TIMP-1 mRNA (p = 0.05). In contrast, MMP-3 and TIMP-2 expression was unaffected in mesothelial cells and in macrophages, compared to untreated controls. There was a significant increase in secreted MMP-1 and TIMP-2 by mesothelial cells following transforming growth factor-beta1 treatments in a time-dependent manner (p = 0.05 and p = 0.01), without affecting the secretion of these proteins by macrophages. A major portion of MMP-1 in the culture conditioned media of both cell types was found in complex with TIMP-1. The ratios of MMP-1/TIMPs production were significantly higher than MMP-3/TIMPs in mesothelial cells and macrophages, and progressively decreased following transforming growth factor-beta1 treatments (p < 0.05). In conclusion, these results indicate that mesothelial cells express MMP and TIMP mRNA and protein, and their expression is differentially regulated by transforming growth factor-beta1, a mechanism that in part may influence the outcome of peritoneal tissue repair and adhesion formation.

Analysis of Variance↗