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Biomedical subjects

R Shapiro

Publications and source records attributed to R Shapiro.

At least 271 records · Page 15Linked to original sources
PubMed1992

Renal transplantation in patients 65 years old or older.

Between January 1982 and August 1989, cadaveric renal transplantation was performed in 22 patients 65 years old or older. Mean recipient age was 68 years (range 65 to 73 years). There were 17 men and 5 women. Additional risk factors included retransplantation (3 patients), high (greater than 30%) panel reactive antibody (4) and diabetes (1). All patients received cyclosporine as part of the immunosuppressive regimen. The 3-year actuarial patient and allograft survival rates were 89% and 71%, respectively. There were 6 graft losses due to chronic rejection (2 patients), renal vein thrombosis (1), myocardial infarction (1), withdrawal of immunosuppression because of sepsis (1) and primary nonfunction (1). Of the 16 patients with a functioning graft 12 currently have a serum creatinine of less than 2.0 mg./dl. These results suggest that cadaveric renal transplantation is an acceptable form of treatment for patients older than 65 years with end stage renal disease.

Age Factors↗
PubMed1992

Pediatric renal transplantation under FK-506 immunosuppression.

Renal transplantation (11 cadaveric and 1 living-related donor) was performed in 12 pediatric recipients (mean age 10.8 years) under FK-506 immunosuppression in combination with prednisone therapy. At a mean followup of 6.1 months, patient and graft survival rates were 100% and 92%, respectively. The only graft loss was due to the recurrent hemolytic uremic syndrome 4 days after transplantation. In the functioning grafts the mean serum creatinine is 1.59 +/- 1.27 mg./dl. and the mean blood urea nitrogen is 36.3 +/- 24.6 mg./dl. Three patients take no prednisone, 5 are receiving 0.15 to 0.25 mg./kg. per day and 3 are taking 0.35 to 0.5 mg./kg. per day. There was a total of 8 rejection episodes in 5 patients. All rejection episodes were successfully reversed. Complications of transplantation included an episode of seizures in 1 patient, cytomegalovirus infection in 1 and steroid-induced diabetes mellitus in 1. Since pediatric transplant recipients are a group in whom the reduction or elimination of steroids is highly desirable, FK-506 immunosuppression may be particularly suited for use in this population.

Adolescent↗
PubMed1992

Milk of calcium in renal cysts: new sonographic features.

Milk of calcium (MOC) in a small renal cyst or calyceal diverticulum is quite common. Ninety-one small renal cysts with MOC in 61 patients were found within a period of 4 years and 7 months. The main sonographic features are echogenic material layering in the dependent portion of a cyst, associated with reverberation echoes without shadowing. Shadowing was seen when MOC was present in larger amounts. Because MOC is usually asymptomatic and requires no treatment, differentiation from other lesions such as calculi or angiomyolipomas is important for proper patient management. MOC is detected more readily by ultrasonography than by radiography or CT.

Adult↗
PubMed1991

Metalloenzymes in DNA repair. Escherichia coli endonuclease IV and Saccharomyces cerevisiae Apn1.

Escherichia coli endonuclease IV and its Saccharomyces cerevisiae homologue Apn1, two DNA repair enzymes for free radical damages, were previously shown to be inactivated by metal-chelating agents. In the present study, atomic absorption spectrometry of endonuclease IV revealed the presence of 2.4 zinc and 0.7 manganese atoms, whereas Apn1 contained 3.3 zinc atoms and no significant manganese. EDTA-inactivated endonuclease IV retained 0.7 zinc atom but little detectable manganese. ZnCl2 reactivated 1,10-phenanthroline-treated Apn1, but was ineffective with endonuclease IV treated with either 1,10-phenanthroline or EDTA. In contrast, enzymatic activity was restored to both enzymes after EDTA treatment by incubation with CoCl2 and to a lesser extent by MnCl2. Endonuclease IV, reactivated with CoCl2 or MnCl2, regained all of the activities characteristic of the native enzyme. MnCl2 was as effective as CoCl2 at restoring activity to the 1,10-phenanthroline-treated enzymes. The results indicate that intrinsic metals play critical roles in both endonuclease IV and Apn1 and that manganese may perform a special function in endonuclease IV. Possible mechanistic roles for the metals in these DNA repair enzymes are discussed.

Amino Acid Sequence↗
PubMed1991

Recombinant mouse leukotriene A4 hydrolase: a zinc metalloenzyme with dual enzymatic activities.

Recombinant mouse leukotriene A4 hydrolase was expressed in Escherichia coli as a fusion protein with ten additional amino acids at the amino terminus and was purified to apparent homogeneity by means of precipitation, anion exchange, hydrophobic interaction and chromatofocusing chromatographies. By atomic absorption spectrometry, the enzyme was shown to contain one mol of zinc/mol of enzyme. Apparent kinetic constants (Km and Vmax) for the conversion of leukotriene A4 to leukotriene B4 (at 0 degree C, pH 8) were 5 microM and 900 nmol/mg per min, respectively. The purified enzyme also exhibited significant peptidase activity towards the synthetic amide alanine-4-nitroanilide. Km and Vmax for this reaction (at 37 degrees C, pH 8) were 680 microM and 365 nmol/mg per min, respectively. Apo-leukotriene A4 hydrolase, prepared by treating the enzyme with 1,10-phenanthroline, was virtually inactive with respect to both enzymatic activities, but could be reactivated by addition of stoichiometric amounts of zinc or cobalt. Exposure of the enzyme to leukotriene A4 resulted in a dose-dependent inactivation of both enzyme activities.

Animals↗
PubMed1991

Leukotriene A4 hydrolase: determination of the three zinc-binding ligands by site-directed mutagenesis and zinc analysis.

Three mutants of recombinant mouse leukotriene A4 (LTA4) hydrolase (3.3.2.6) were produced by site-directed mutagenesis on cDNA. The codons corresponding to His-295, His-299, or Glu-318 were replaced by codons encoding tyrosine, tyrosine, and glutamine, respectively. The mutated cDNAs were expressed in Escherichia coli, and the three mutated proteins were purified to apparent homogeneity. None of these mutants contained significant amounts of zinc, as determined by atomic absorption spectrometry, and all of them were practically devoid of both LTA4 hydrolase and peptidase enzyme activities. Nevertheless, the mutated proteins could be positively identified by their immunoreactivities with an antiserum for human LTA4 hydrolase in immunoblot analysis. Site-directed mutagenesis was also carried out on human LTA4 hydrolase cDNA. Codons encoding His-295, His-299, and Glu-318 were replaced by ones encoding tyrosine, leucine, and alanine, respectively, and the three mutants were expressed in E. coli. The LTA4 hydrolase activities of the total soluble proteins produced in these expressions were less than 10% of that obtained for bacteria harboring nonmutated cDNA. In agreement with earlier predictions, our experimental data demonstrate that His-295, His-299, and Glu-318 constitute the three ligands of the intrinsic zinc atom in LTA4 hydrolase. Additionally, the combined loss of enzyme activities and zinc content in the purified mutated mouse proteins, emphasizes the critical role of the zinc atom for catalysis, whereas the virtually identical chromatographic behaviors of the mutated and nonmutated mouse LTA4 hydrolase proteins suggest that the metal is of limited importance for the maintenance of the enzyme tertiary structure.

Amino Acid Sequence↗
PubMed1991

Dual site model for the organogenic activity of angiogenin.

The residues that are indispensable for the ribonucleolytic activity of angiogenin are also known to be essential for its angiogenic activity. We now demonstrate that residues in another region of the protein, devoid of catalytic residues, are additionally required for angiogenesis. Endoproteinase Lys-C or a baby hamster kidney cell protease cleaves angiogenin at the peptide bond either between Lys-60 and Asn-61 or between Glu-67 and Asn-68, respectively. The two polypeptide fragments resulting from either cleavage remain linked by disulfide bonds. These two derivatives and des-(Asn61-Glu67)-angiogenin--in which both bonds are cleaved--retain their ribonucleolytic activities toward tRNA, 18S and 28S rRNA, and dinucleoside phosphates but are no longer angiogenic on the chicken embryo chorioallantoic membrane. Further, their capacity to elicit a second messenger response in endothelial cells is greatly decreased. Moreover, none of these three derivatives inhibit angiogenin-induced angiogenesis. This contrasts with two active site mutants of angiogenin. These results identify the residues from 60 to 68 as a region of angiogenin that is part of a cell-surface receptor binding site [see accompanying manuscript: Hu, G.-F., Chang, S.-I., Riordan, J.F. & Vallee, B.L. (1991) Proc. Natl. Acad. Sci. USA 88, 2227-2231] and serve as the basis for a dual site model of the organogenic activity of angiogenin.

Amino Acid Sequence↗
PubMed1991

Interaction of human placental ribonuclease with placental ribonuclease inhibitor.

The interactions of human placental ribonuclease inhibitor (PRI) with bovine pancreatic ribonuclease (RNase) A and human angiogenin, a plasma protein that induces blood vessel formation, have been characterized in detail in earlier studies. However, studies on the interaction of PRI with the RNase(s) indigenous to placenta have not been performed previously, nor have any placental RNases been identified. In the present work, the major human placental RNase (PR) was purified to homogeneity by a five-step procedure and was obtained in a yield of 110 micrograms/kg of tissue. The placental content of angiogenin was also examined and was found to be at least 10-fold lower than that of PR. On the basis of its amino acid composition, amino-terminal sequence, and catalytic properties, PR appears to be identical with an RNase previously isolated from eosinophils (eosinophil-derived neurotoxin), liver, and urine. The apparent second-order rate constant of association for the PR.PRI complex, measured by examining the competition between PR and angiogenin for PRI, is 1.9 X 10(8) M-1 s-1. The rate constant for dissociation of the complex, determined by HPLC measurement of the rate of release of PR from its complex with PRI in the presence of a scavenger for free PRI, is 1.8 X 10(-7) s-1. Thus the Ki value for the PR.PRI complex is 9 X 10(-16) M, similar to that obtained with angiogenin, and 40-fold lower than that measured with RNase A. Complex formation causes a small red shift in the protein fluorescence emission spectrum, with no significant change in overall intensity. The fluorescence quantum yield of PR and the Stern-Volmer constant for fluorescence quenching by acrylamide are both high, possibly due to the presence of an unusual posttranslationally modified tryptophan residue at position 7 in the primary sequence.

Amino Acids↗
PubMed1991

Effects of in utero and postnatal sodium saccharin exposure on the nutritional status of the young rat. I. Effects at 30 days post-birth.

The incidence of sodium saccharin (NaS)-associated bladder tumours in male rats increases when exposure to high doses begins in utero or at birth compared with treatment after weaning. The present experiment evaluated the effect of NaS exposure on selected physiological parameters in young second generation rats. 6-wk-old male and female Sprague-Dawley rats were placed on either a diet supplemented with 7.5% NaS or an untreated diet, and mated 4-6 wk later. Treatment was continued through lactation and the offspring were weaned on to the same diet. Body weights were significantly depressed in NaS-treated litters by 4 days after birth, and were 35% lower than controls by 30 days when the animals were killed. NaS treatment of the offspring was associated with an increase in faecal moisture content and caecal content weight, changes in several urinary analytes, a 50% increase in serum cholesterol a 10-fold increase in serum triglycerides and decreases in serum and hepatic vitamins. In addition, NaS-treated dams and pups were anaemic. Relatively few differences between males and females were noted, but significant inter-litter differences existed. The numerous physiological changes indicate that 7.5% dietary NaS exceeds the maximum tolerated dose for weanling rats.

Animals↗
PubMed1991

Effects of in utero and postnatal sodium saccharin exposure on the nutritional status of the young rat. II. Dose response and reversibility.

A previous study in our laboratory demonstrated that 30-day-old Sprague-Dawley rats exposed to 7.5% sodium saccharin (NaS) since conception differ from untreated rats in several physiological parameters. In the present study, to determine the dose response of the changes associated with NaS treatment, animals were evaluated at 30 days post-birth, after treatment with dietary levels of 0, 1, 3 or 7.5% NaS since conception. Most physiological consequences of NaS treatment in the weanling rat, including anaemia and reductions in serum folate and vitamin A concentrations, were dose dependent. Serum vitamin E, cholesterol and triglyceride concentrations were decreased at the two lower doses of NaS but were significantly increased with 7.5% NaS. The no-effect level (NOEL) was similar for physiological effects and for bladder tumour production in two-generation studies (1% NaS in the diet). The reversibility of the effects of 7.5% NaS was examined in 90-day-old rats. The increases in lipids and vitamin E were reversible. Although values for haematological parameters and serum vitamin A remained significantly reduced at 90 days, changes were less severe than at 30 days. Histological examinations revealed that the effects of 7.5% dietary NaS on the bladder were negligible, indicating that the physiological changes observed in the young rat are probably not directly related to the production of bladder tumours.

Animals↗