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A Wilks

Publications and source records attributed to A Wilks.

At least 37 records · Page 2Linked to original sources
PubMed1995

Novel mutation in the first intron of the spontaneously hypertensive rat renin gene upstream of the tandem repeat element.

1. The first 1100 base pairs of the first intron of the renin gene was amplified from the spontaneously hypertensive rat (SHR), Wistar-Kyoto (WKY) and Sprague-Dawley (SD) rats. A Bgl II restriction fragment length polymorphism (RFLP) was identified in this region of the SHR renin gene. 2. Sequence analysis located the Bgl II RFLP between positions 500 and 505 of the reported rat renin gene. The new Bgl II cut site was produced by a single base mutation from G to A. 3. Whether this mutation in the first intron of the SHR renin gene plays a role in the reported overexpression of the renin gene in this strain remains to be elucidated.

Animals↗
PubMed1995

Vascular endothelial growth factor: tissue distribution and size of multiple mRNA splice forms in SHR and WKY.

1. We have determined the optimal polymerase chain reaction (PCR) conditions for the amplification and detection of mRNA for a new vascular growth factor-vascular endothelial growth factor (VEGF)-and determined its size and tissue distribution in genetically normotensive and hypertensive rats. 2. Multiple VEGF mRNA subtypes were obtained which were 625, 520 and 480 base pairs in length. 3. All three species of VEGF mRNA were found in heart, kidney, aorta, adrenal and brainstem and the size and tissue distribution of VEGF mRNA subtypes were not different between spontaneously hypertensive rats (SHR) of the Okamoto strain and normotensive Wistar-Kyoto (WKY) controls. 4. Thus multiple forms of VEGF mRNA can be readily detected by PCR in a variety of tissues. While these preliminary results suggest no difference in size and tissue distribution between SHR and WKY, sequencing and quantitative studies will be required to confirm this.

Animals↗
PubMed1994

Heme oxygenase (HO-1). Evidence for electrophilic oxygen addition to the porphyrin ring in the formation of alpha-meso-hydroxyheme.

Previous studies have established that reaction of the rat heme-heme oxygenase complex with H2O2 proceeds normally to give verdoheme, whereas reaction of the complex with meta-chloroperbenzoic acid yields a ferryl (FeIV = O) species and a protein radical but no verdoheme. The heme-heme oxygenase complex is shown here to react regiospecifically with ethyl hydroperoxide to give alpha-meso-ethoxyheme. Formation of this product exactly parallels the formation of alpha-meso-hydroxyheme in the normal reaction supported by cytochrome P450 reductase/NADPH or H2O2. These results rule out a nucleophilic mechanism for the alpha-meso-hydroxylation catalyzed by heme oxygenase and indicate that it involves electrophilic (or possibly radical) addition of the distal oxygen of iron-bound peroxide (FeIII-OOH) to the porphyrin ring.

Heme↗
PubMed1994

Identification of histidine 25 as the heme ligand in human liver heme oxygenase.

Electronic and resonance Raman spectroscopic studies are reported for the His25Ala mutant of human liver heme oxygenase (HO) and its complex with heme. In the oxidized (ferric) form of the enzyme.substrate complex, the heme is shown to be in a high-spin, five-coordinate state. This is distinct from the same complex in the wild-type enzyme in which the heme is six-coordinate, ligated to a proximal histidine and a water molecule in an environment reminiscent of aquometmyoglobin. The reduced (ferrous) form of the complex of the H25A heme oxygenase mutant has lost the very prominent resonance Raman band at approximately 217 cm-1 seen in the wild-type complex that has been unambiguously assigned to the proximal Fe-N(His) vibrational frequency [Sun et al. (1993) Biochemistry 32, 14151; Takahashi et al. (1994) Biochemistry 33, 1010]. The absence of this band in the spectrum of the mutant protein definitively identifies His 25 as the proximal ligand of the heme substrate. Furthermore, this ferrous heme-H25A HO complex exists as an equilibrium mixture between a five-coordinate, high-spin species and a four-coordinate, intermediate-spin species. Although the H25A mutant protein shows no heme oxygenase activity, the heme is competent to bind carbon monoxide. Studies of the CO adduct of the H25A HO complex show v(CO) and v(Fe-CO) frequencies at 1960 and 529 cm-1, respectively, that are characteristic of a hydrophobic carbon monoxide binding site on a heme with a weak proximal ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Monoxide↗
PubMed1994

Identification of JAK protein tyrosine kinases as signaling molecules for prolactin. Functional analysis of prolactin receptor and prolactin-erythropoietin receptor chimera expressed in lymphoid cells.

The mechanism of action of prolactin (PRL) was studied in murine lymphoid BAF-3 cells transfected with either the long form of the PRL receptor (PRL-R), or a chimeric receptor consisting of the extracellular domain of the PRL-R and the transmembrane and intracellular domain of the erythropoietin receptor (PRL/EPO-R). PRL sustained normal and long-term proliferation of BAF-3 cells expressing either the PRL-R or the hybrid PRL/EPO-R. Upon [125I]PRL cross-linking, both types of BAF-3 transfectants were shown to express two [125I]PRL cross-linked species differing in size by 20 kDa. These cross-linked complexes, after denaturation, were recognized by antibody against the PRL-R, indicating that they contain the transfected receptor. PRL induced rapid and transient tyrosine phosphorylation of both the PRL-R and the PRL/EPO-R in BAF-3 transfectants. Furthermore, PRL induced rapid tyrosine phosphorylation of the Janus kinase 2 (JAK2) which was already physically associated with the PRL-R or the PRL/EPO-R in the absence of ligand. JAK1 was also associated with PRL-R and PRL/EPO-R in the absence of ligand. However, only in BAF-3 cells expressing the PRL-R does PRL induce rapid and transient tyrosine phosphorylation of JAK1. These results demonstrate that JAK protein tyrosine kinases couple PRL binding to tyrosine phosphorylation and proliferation.

Animals↗
PubMed1994

Proton NMR investigation of substrate-bound heme oxygenase: evidence for electronic and steric contributions to stereoselective heme cleavage.

The substrate-bound form of the enzyme heme oxygenase (HO), which catalyzed the stereospecific alpha-meso bridge cleavage of hemin to yield biliverdin IX alpha, has been investigated by 1H NMR in both its primarily high-spin and its cyanide-inhibited low-spin forms. Both derivatives yield 1H NMR spectra indicative of extensive heterogeneity that is largely resolved when a 2-fold-symmetric hemin substrate is bound. The structural origin of the heterogeneity is shown to result from approximately 1:1 isomeric binding of the native hemin substrate in the binding pocket. The substrate orientational disorder is about the alpha,gamma-meso axis, as established on the basis of 2D NMR experiments that identify characteristic aromatic van der Waals contact in the substrate binding pocket. The isomeric substrate-HO complexes exhibit differential cyanide affinity, and the ratio of isomers is sensitive to the hemin 2,4-substituents. The assignment of hemin signals by isotopic labeling and 2D NMR methods reveals a contact shift pattern that reflects an unusual hemin electronic structure that is characterized by large differences in delocalized spin density for the two positions within a given pyrrole, rather than the more conventional large differences between adjacent pyrroles. This pattern of spin density delocalized primarily to the pyrrole positions adjacent to the alpha,gamma-meso axis can be rationalized by postulating a direct electronic perturbation of the hemin by the protein matrix in the form of an anionic side chain close to the alpha-meso carbon. Similar influences on hemin electronic structure, in the form of chemical substitution of the meso positions, have been observed in iron porphyrin compounds and successfully modeled by simple molecular orbital theory (Tan et al., 1994). This is interpreted as evidence for a direct electronic effect by HO to activate the alpha-meso position for electrophilic rather than nucleophilic attack. The unique contact shift pattern is present to different degrees for the two hemin orientations, is strongly pH dependent, and is largely abolished at acidic pH. Portions of several heme pocket residues are located and it is shown that the pattern of the dipolar shifts for these residues, which likely reflects the distal steric influence on the tilt of the coordinated cyanide, differs significantly for the two substrate orientations.(ABSTRACT TRUNCATED AT 400 WORDS)

Cyanides↗
PubMed1994

The lipoxygenase activity of myoglobin. Oxidation of linoleic acid by the ferryl oxygen rather than protein radical.

Linoleic (9(Z),12(Z)-octadecadienoic) acid is oxidized by sperm whale myoglobin and H2O2 to an 84:16 (9S):(9R) enantiomer mixture of 9-hydroperoxy-10(E),12(Z)-octadecadienoic acid. Neither the 9,10- nor 12,13-epoxide of linoleic acid, nor 9-hydroxy-10(E),12(Z)-octadecadienoic nor 13-hydroperoxy-9(Z),11(E)-octadeca-dienoic acids, is detectably formed. Incubations with [(11R)-2H]- and [(11S)-2H]linoleic acids indicate that the pro-R hydrogen is abstracted 76% of the time. An H64V mutant in which access to the heme crevice is increased oxidizes linoleic acid exclusively by abstraction of the pro-R hydrogen to give the (9S)-hydroperoxide. Spectroscopic studies show that the Kd value for binding of linoleic acid to myoglobin is similar to the Km value for its oxidation and indicate that linoleic acid reduces the ferryl species to the ferric state. The stereochemical results, supported by 18O-labeling studies, definitively rule out a significant role for singlet oxygen in the myoglobin-catalyzed, H2O2-dependent oxidation of linoleic acid. The myoglobin protein radical formed with H2O2 also plays no part in the reaction because the Km and Vmax values for the oxidation of linoleic acid are similar for native myoglobin and two mutants (K102Q/Y103F/Y146F/Y151F and H64V/K102Q/Y103F/Y146F/Y151F) with no tyrosine residues. Furthermore, the rate of formation of the 9-hydroperoxide is not changed if the protein radical is allowed to decay before linoleic acid is added. The results establish that linoleic acid is oxidized within the heme crevice by reaction with the ferryl oxygen rather than a protein radical. They indicate, furthermore, that hydrogen abstraction and oxygen addition occur in an antarafacial manner and suggest a specific model for binding of linoleic acid within the myoglobin active site.

Animals↗
PubMed1994

A new restriction fragment length polymorphism in the first intron of the spontaneously hypertensive rat renin gene.

1. Amplification of the entire first intron of the renin gene of spontaneously hypertensive rat (SHR), Wistar-Kyoto (WKY) and Sprague-Dawley rats (SD) followed by Bgl II digestion uncovered a new deletion (approximately 50 bp) which exists upstream of the SHR renin gene first intron tandem repeat element. 2. The SHR tandem repeat element was 600 bp shorter than that in the WKY while the WKY tandem repeat element was 280 bp shorter than that in the SD. 3. Since elements regulating gene expression are known to exist in the first intron of other genes, this new restriction fragment length polymorphism (RFLP) might play a role in the reported overexpression of the SHR renin gene independent of changes in the length of the tandem repeat element.

Animals↗
PubMed1993

Resonance Raman and EPR spectroscopic studies on heme-heme oxygenase complexes.

The binding of ferrous and ferric hemes and manganese(II)- and manganese(III)-substituted hemes to heme oxygenase has been investigated by optical absorption, resonance Raman, and EPR spectroscopy. The results are consistent with the presence of a six-coordinate heme moiety ligated to an essential histidine ligand and a water molecule. The latter ionizes with a pKa approximately 8.0 to give a mixture of high-spin and low-spin six-coordinate hydroxo adducts. Addition of excess cyanide converts the heme to a hexacoordinate low-spin species. The resonance Raman spectrum of the ferrous heme-heme oxygenase complex and that of the Mn(II)protoporphyrin-heme oxygenase complex shows bands at 216 and 212 cm-1, respectively, that are assigned to the metal-histidine stretching mode. The EPR spectrum of the oxidized heme-heme oxygenase complex has a strongly axial signal with g parallel of approximately 6 and g perpendicular approximately 2. 14NO and 15NO adducts of ferrous heme-heme oxygenase exhibit EPR hyperfine splittings of approximately 20 and approximately 25 Gauss, respectively. In addition, both nitrosyl complexes show additional superhyperfine splittings of approximately 7 Gauss from spin-spin interaction with the proximal histidine nitrogen. The heme environment in the heme-heme oxygenase enzyme-substrate complex has spectroscopic properties similar to those of the heme in myoglobin. Hence, there is neither a strongly electron-donating fifth (proximal) ligand nor an electron-withdrawing network on the distal side of the heme moiety comparable to that for cytochromes P-450 and peroxidases. This observation has profound implications about the nature of the oxygen-activating process in the heme-->biliverdin reaction that are discussed in this paper.

Cations, Divalent↗
PubMed1993

Rat liver heme oxygenase. High level expression of a truncated soluble form and nature of the meso-hydroxylating species.

A rat heme oxygenase (HO-1) gene without the sequence coding for the last 23 amino acids has been constructed and expressed behind the pho A promoter in Escherichia coli. The enzyme is expressed at high levels as a soluble catalytically active protein that causes the bacterial cells to accumulate biliverdin. The purified truncated heme-heme oxygenase complex is spectroscopically indistinguishable from the complex with the native enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. Reaction of the recombinant heme-heme oxygenase complex with H2O2 produces a species with the spectroscopic properties of verdoheme. Unidentified products are obtained when this intermediate is directly extracted from the protein, but biliverdin is obtained if the verdoheme-protein complex is exposed to cytochrome P450 reductase and NADPH before the extraction step. In contrast, reaction of the heme-heme oxygenase complex with meta-chloroperbenzoic acid (mCPBA), tert-butylhydroperoxide, or cumene hydroperoxide yields a ferryl (FeIV = O) complex. Reaction of the heme-heme oxygenase complex with mCPBA also produces an EPR-detectable protein radical. In accord with formation of a ferryl intermediate, recombinant heme oxygenase catalyzes the mCPBA- and alkylhydroperoxide-dependent peroxidation of 2-methoxyphenol (guaiacol). Guaiacol oxidation is not observed during turnover of the enzyme by cytochrome P450 reductase/NADPH or H2O2. Conversely, biliverdin is not formed with tert-butylhydroperoxide or mCPBA. H2O2 thus supports the first step of the normal catalytic oxidation of heme by heme oxygenase, but alkyl and acyl hydroperoxides do not. These results suggest that the alpha-meso-hydroxylation required for biliverdin formation is mediated by the distal of the two oxygens in the iron-dioxygen intermediate (Fe-O-O) engendered by reaction with either cytochrome P450 reductase/NADPH or H2O2.

Animals↗
PubMed1993

The roles of His-64, Tyr-103, Tyr-146, and Tyr-151 in the epoxidation of styrene and beta-methylstyrene by recombinant sperm whale myoglobin.

Previous studies demonstrated that at least two mechanisms are involved in the epoxidation of styrene and stilbene by myoglobin and H2O2 (Ortiz de Montellano, P. R., and Catalano, C. E. (1985) J. Biol. Chem. 260, 9265-9271). One mechanism, reaction of the olefin with the ferryl oxygen, preserves the olefin stereochemistry and incorporates an oxygen from H2O2 into the epoxide. The second mechanism, proposed to be a protein-mediated co-oxidation process, results in loss of stereochemistry and incorporation of an atom of molecular oxygen. To examine the role of individual residues in olefin epoxidation, we have examined the catalytic activities of the possible Tyr-->Phe mutants, the His-64-->Val mutant, and a protein combining all the tyrosine and histidine mutations. The latter protein is less stable than the other mutants and is the only one for which a protein radical is not detected in the reaction with H2O2. The Km and Vmax for styrene epoxidation range, respectively, from 0.3-8 mM and 12-35 pmol/min/nmol of protein. Incubation with H(2)18O2 results in 20-30% incorporation of labeled oxygen into the epoxide with all the mutants except Y103F/Y151F, Y146F/Y151F, H64V, and H64V/Y103F/Y146F/Y151F, for which 52, 58, 89, and 96% of the epoxide oxygen, respectively, is labeled. Oxidation of cis-beta-methylstyrene by wild-type myoglobin yielded a 54:46 ratio of cis- and trans-beta-methylstyrene oxides. The cis-isomer accounts for 47-100% of the epoxide produced by the mutant hemoproteins, with the two H64V mutants yielding almost exclusively the cis-epoxide. The oxygen in the cis-epoxide derives primarily or exclusively from H2O2 and that in the trans-epoxide from an alternative source. These results indicate that tyrosine residues may participate in, but are not essential for, protein-mediated epoxidation. In contrast, His-64 appears to be essential for co-oxidative epoxidation because in its absence olefin epoxidation is mediated almost exclusively by ferryl oxygen transfer.

Amino Acid Sequence↗
PubMed1993

Accurate determination of the size of the first intron deletion in the renin gene of SHR from an Australian colony.

1. It has been reported that the first intron of the renin gene of the spontaneously hypertensive rat (SHR) is shorter than that of the Wistar-Kyoto (WKY) rat, suggesting a role for this difference in the development of hypertension. 2. The genealogy of some Australian SHR colonies has raised doubts about its similarity to the Japanese, American and European colonies. 3. The polymerase chain reaction (PCR) technique has been used with primers identical to the flanking region of the first intron of the renin gene to determine its length in SHR, WKY and Sprague-Dawley (SD) from an Australian colony. 4. The size of the first intron of the renin gene in SHR, WKY and SD was found to be approximately 3870, 4550 and 5000 base pairs, respectively, results which agree with those previously published. 5. It was concluded that the first intron deletion, and its size, has been conserved in an Australian SHR colony.

Animals↗
PubMed1992

Intramolecular translocation of the protein radical formed in the reaction of recombinant sperm whale myoglobin with H2O2.

A sperm whale myoglobin gene containing multiple unique restriction sites has been constructed in pUC 18 by sequential assembly of chemically synthesized oligonucleotide fragments. Expression of the gene in Escherichia coli DH5 alpha cells yields protein that is identical to native sperm whale myoglobin except that it retains the terminal methionine. Site-specific mutagenesis has been used to prepare all the possible tyrosine----phenylalanine mutants of the recombinant myoglobin, including the three single mutants at Tyr-103, -146, and -151, the three double mutants, and the triple mutant. All of the mutant proteins are stable except the Tyr-103 mutant. Introduction of a second mutation (Lys-102----Gln) stabilizes the Tyr-103 mutant. Absorption spectroscopy suggests that the active sites of the mutant proteins are intact. EPR and absorption spectroscopy show that all the proteins, including the triple mutant devoid of tyrosine residues, react with H2O2 to give a ferryl species and a protein radical. The presence of a protein radical in all the mutants suggests that the radical center is readily transferred from one amino acid to another. Cross-linking studies show, however, that protein dimers are only formed when Tyr-151 is present. Tyr-103, shown earlier to be the residue that primarily cross-links to Tyr-151 (Tew, D., and Ortiz de Montellano, P. R. (1988) J. Biol. Chem. 263, 17880-17886), is not essential for cross-linking. Electron transfer from Tyr-151 to the heme, which are 12 A apart, occurs in the absence of the intervening tyrosines at positions 103 and 146. The present studies show that the peroxide-generated myoglobin radical readily exchanges between remote loci, including non-tyrosine residues, but protein cross-linking only occurs when radical density is located on Tyr-151.

Animals↗
PubMed1992

Isolation and characterization of a novel receptor-type protein tyrosine kinase (hek) from a human pre-B cell line.

In this report we describe the identification and characterization of a novel tumor-associated receptor-type tyrosine kinase (hek). We produced a monoclonal antibody (III.A4) that detected a novel glycoprotein on the immunizing pre-B cell acute lymphoblastic leukemia cell line (LK63). This antigen was shown to be expressed sporadically on hemopoietic tumor cell lines and on ex vivo tumors. However, using antibody staining, the molecule was undetectable on normal tissues. Further biochemical characterization showed this molecule (hek) to be a phosphoroprotein. This observation taken together with the tumor-associated nature of hek expression suggested that hek might be a receptor-type protein tyrosine kinase. This was demonstrated by affinity purification of hek. In in vitro kinase experiments the purified hek protein was autophosphorylated on tyrosine and also mediated tyrosine phosphorylation of casein. Purified hek was subjected to N-terminal amino acid sequence analysis which showed that hek had a unique N terminus. Amino acid sequence determination of peptides from a V8 protease digest of hek yielded one 21-amino acid stretch of sequence which showed close homology with the eph subfamily of protein tyrosine kinases. These studies show hek to be a novel human tumor-associated protein tyrosine kinase, which by analogy with previously characterized protein tyrosine kinase proto-oncogenes, may have a role in tumorigenesis.

Amino Acid Sequence↗
PubMed1989

Does indomethacin affect the control of breathing in premature infants?

The effect of indomethacin on the control of breathing was simultaneously evaluated in 10 premature infants who had significant patent ductus arteriosus and received indomethacin therapy. In an attempt to maintain high plasma level in these infants of advanced postnatal age (greater than or equal to 6 weeks), indomethacin was administered intravenously at a dosage of 0.3 mg/kg, at 8-hour intervals, for a total of three doses. Following indomethacin therapy, there was a significant increase in tidal volume, minute ventilation, tidal volume/inspiratory time and in airway pressure (P0.1, Pmax) generated during airway occlusion. Seven infants required lower ventilatory rates after study. In spite of desirable plasma indomethacin level, there was no significant improvement in echo left atrium/aortic root dimension ratio, cardiovascular dysfunction score and in blood pH, PO2 and PCO2. None of the infants showed clinical evidence of ductus closure. The results of the study suggested that indomethacin may stimulate respiration and that endogenous prostaglandin may play a role in the regulation of breathing.

Ductus Arteriosus, Patent↗
PubMed1987

Identical mRNA for preproglucagon in pancreas and gut.

The structure of the human glucagon gene and the sequence around the putative promoter region and transcriptional start site are shown. Cloning and sequencing of the cDNAs, coding for preproglucagon from human pancreas and colon, as well as primer extension analysis of RNA from pancreas, ileum and colon point to an identical transcriptional start site in all tissues, about 3 X 10(3) base pairs upstream from the first coding exon as defined by Bell et al.

Animals↗
PubMed1986

Indomethacin therapy in premature infants with patent ductus arteriosus and oliguria.

Simultaneous administration of one dose of indomethacin (0.3 mg/kg, i.v.) and furosemide (1 mg/kg, i.v.) was given to 8 consecutive premature infants who had patent ductus arteriosus (PDA), and oliguria because of prerenal failure. Four infants responded with ductus closure and 2 infants showed improvement in echocardiogram and clinical distress. There was a significant increase in U/O, GFR, FENa and FECl following drug administration. This study suggests that simultaneous administration of indomethacin and furosemide can be safely used in infants with PDA and oliguria.

Anuria↗