Tests for renovascular disease.
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Humans↗
Biomedical subjects
A Frankel
Publications and source records attributed to A Frankel.
Structure of a ricin mutant showing rescue of activity by a noncatalytic residue.
Ricin A chain is an N-glycosidase which removes a single adenine base from a conservative loop of 28S rRNA, thereby inactivating eukaryotic ribosomes. The mechanism of action has been proposed to include transition-state stabilization of an oxycarbonium ion on the substrate ribose by interaction with Glu 177. Conversion of Glu 177 to Gln reduces activity nearly 200-fold [Ready, M. P., Kim, Y., & Robertus, J. D. (1991) Proteins: Struct., Funct., Genet. 10, 270-278] while conversion to Ala (E177A) reduces activity only 20-fold [Schlossman, D., Withers, D., Welsh, P., Alexander, A., Robertus, J., & Frankel, A. (1989) Mol. Cell. Biol. 9, 5012-5021]. X-ray analysis of the latter mutant protein shows that a residue at the edge of the active site, Glu 208, rotates into the space left vacant by the mutation. Its rearranged carboxylate partially substitutes for that of Glu 177. This is equivalent to the rescue of enzyme activity by a second-site reversion. Kinetic analysis shows the E177A mutation affects kcat and not Km, consistent with the notion that the carboxylate serves in transition-state stabilization.
Amino Acid Sequence↗
Phase I trial of H65-RTA immunoconjugate in patients with cutaneous T-cell lymphoma.
H65-RTA is an immunoconjugate that consists of the A chain of ricin (RTA), a ribosomal-inhibiting protein, coupled to a murine monoclonal antibody (H65) directed against the pan-T-cell antigen CD5. The CD5 antigen is heterogeneously expressed on cutaneous T-cell lymphoma tumor cells, but is not expressed on normal cells except lymphocytes. A phase I trial was therefore conducted in which 14 patients with cutaneous T-cell lymphoma progressive on other therapies were treated with up to three cycles of H65-RTA. The maximal tolerated dose (MTD) of H65-RTA was 0.33 mg/kg/d administered intravenously for 10 days as defined by dyspnea at rest at higher doses. Other reversible side effects included myalgia, mild hypoalbuminemia with weight gain, pedal edema, fatigue, fevers, and chills. Six patients received more than one cycle of H65-RTA without increased side effects compared with the first cycle. Pharmacokinetic analysis showed that peak serum drug levels were dose-dependent, and ranged from 1.13 to 5.56 micrograms/mL, with a terminal half-life ranging from 1.0 to 2.9 hours. The development of antibodies against the immunoconjugate was associated with a lower peak drug level, but not with enhanced side effects. Partial responses lasting from 3 to 8 months were documented in four patients. Three of the responding patients received more than one cycle of H65-RTA in the presence of anti-immunoconjugate antibodies. The results from this phase I trial suggest that H65-RTA is an active drug in the treatment of cutaneous T-cell lymphoma. The immunoconjugate may be safely administered repeatedly, even in the presence of anti-immunoconjugate antibodies, with responses noted. Additional studies at the MTD are needed to define the response rate in this disease.
Adult↗
Alteration of an amino acid residue outside the active site of the ricin A chain reduces its toxicity towards yeast ribosomes.
Yeast transformants containing integrated copies of a galactose-regulated, ricin toxin A chain (RTA) expression plasmid were constructed and used in an attempt to isolate RTA-resistant yeast mutants. Analysis of RNA from mutant strains demonstrated that approximately half contained ribosomes that had been partially modified by RTA, although all the strains analysed transcribed full-length RTA RNA. The mutant strains could have mutations in yeast genes giving rise to RTA-resistant ribosomes or they could contain alterations within the RTA-encoding DNA causing production of mutant toxin. Ribosomes isolated from mutant strains were shown to be susceptible to RTA modification in vitro suggesting that the strains contain alterations in RTA. This paper describes the detailed analysis of one mutant strain which has a point mutation that changes serine 203 to asparagine in RTA protein. Although serine 203 lies outside the proposed active site of RTA its alteration leads to the production of RTA protein with a greatly reduced level of ribosome modifying activity. This decrease in activity apparently allows yeast cells to survive expression of RTA as only a proportion of the ribosomes become modified. We demonstrate that the mutant RTA preferentially modifies 26S rRNA in free 60S subunits and has lower catalytic activity compared with native RTA when produced in Escherichia coli. Such mutations provide a valuable means of identifying residues important in RTA catalysis and of further understanding the precise mechanism of action of RTA.
Amino Acids↗
Clinical evaluation of intraperitoneal Pseudomonas exotoxin immunoconjugate OVB3-PE in patients with ovarian cancer.
OVB3-PE is an immunotoxin composed of a murine monoclonal antibody reactive with human ovarian cancer and conjugated to Pseudomonas exotoxin (PE). Twenty-three patients with refractory ovarian cancer were treated intraperitoneally (IP) with escalating doses of OVB3-PE to study toxicity, pharmacokinetics, antiimmunotoxin antibody formation, and antitumor response. Dose-limiting CNS toxicity occurred after repeated doses at 5 and 10 micrograms/kg. Other non-dose-limiting toxicities included transient elevation of liver enzymes, fever, and gastrointestinal toxicity. Pharmacokinetics of IP and serum OVB3-PE were determined in 16 patients. Peak peritoneal fluid levels exceeded the in vitro median effective dose at all doses tested. At doses of 1 to 2 micrograms/kg, the immunotoxin concentration in the peritoneal fluid remained constant for up to 8 hours and dropped to negligible levels after 12 hours. At the 5 and 10 micrograms/kg doses, levels remained high for up to 24 hours (greater than 100 ng/mL) and then gradually decreased and became undetectable (less than 4 ng/mL) after 72 hours. Serum levels of OVB3-PE were also analyzed in 16 patients. At doses of 1 micrograms/kg and 2 micrograms/kg, serum levels were not detectable (less than 5 ng/mL). However, after doses of 5 or 10 micrograms/kg, peak serum level occurred at 24 hours after each dose and dropped to negligible levels by 72 hours. Sera from 12 patients were analyzed for anti-PE antibodies and antibodies to mouse immunoglobulin (HAMA). All patients developed antibodies against PE within 14 days of therapy. Domain II of PE appeared to be the most immunogenic portion of the PE molecule. HAMA was detected on day 14 of therapy in nine patients, on day 21 in two, and on day 28 in one patient. No clinical antitumor responses were observed. We conclude that IP OVB3-PE at dose levels of 5 micrograms/kg (x 3) and 10 micrograms/kg (x 2) is accompanied by dose-limiting toxic encephalopathy. Neurologic toxicity is likely to be due to crossreactivity of OVB3 to normal human brain tissue, which was not appreciated during preclinical screening.
ADP Ribose Transferases↗
Radiographic demarcation of the acetabular bone-cement interface. The effect of femoral head size.
Between 1983 and 1988, 182 total hip arthroplasties were inserted using modern cement techniques including metal-backed acetabular components. Femoral head size was 32 mm in 84, 22 mm in 98. Radiographic analysis revealed three-zone demarcation of acetabular bone-cement interface in 56% of the 32 mm group as compared to 5% of the 22 mm group at 19 and 24 months mean follow-up period, respectively. When a subgroup of women under 60 years of age was created to control variables, the high-grade demarcation rates were 67% and 18%, respectively. Although Charnley hip scores remain similar between the two groups, these results emphasize the adverse effects of large femoral head prostheses on cement-bone interface and underline the need for alternative methods of fixation.
Aged↗
Role of arginine 180 and glutamic acid 177 of ricin toxin A chain in enzymatic inactivation of ribosomes.
The gene for ricin toxin A chain was modified by site-specific mutagenesis to change arginine 180 to alanine, glutamine, methionine, lysine, or histidine. Separately, glutamic acid 177 was changed to alanine and glutamic acid 208 was changed to aspartic acid. Both the wild-type and mutant proteins were expressed in Escherichia coli and, when soluble, purified and tested quantitatively for enzyme activity. A positive charge at position 180 was found necessary for solubility of the protein and for enzyme activity. Similarly, a negative charge with a proper geometry in the vicinity of position 177 was critical for ricin toxin A chain catalysis. When glutamic acid 177 was converted to alanine, nearby glutamic acid 208 could largely substitute for it. This observation provided valuable structural information concerning the nature of second-site mutations.
Animals↗
Changing patterns in perforated peptic ulcer disease.
We reviewed our experience with 88 consecutive patients (49 men and 39 women) treated for perforated peptic ulcer between January 1983 and May 1988. The mean age was 61 years (range, 15-89); 63 per cent were more than 60 years of age and 44 per cent were more than 70 years of age. One third of patients had a prior history of peptic ulcer disease. Thirty-nine patients (44%) were taking ulcerogenic drugs (28 were using nonsteroidal anti-inflammatory drugs, 6 were using steroids alone, and 5 were using both). Twenty-eight patients (32%) were taking antacid/H2-blockers, including 15 patients with history of ulcer disease and 11 patients taking ulcerogenic drugs. Concurrent systemic diseases were present in 63 per cent of patients; 12 patients were hospitalized for other illnesses at the time of perforation. Abdominal pain was the chief complaint in 83 patients (94%) and 52 patients (59%) had peritonitis. Leukocytosis was present in 49 patients (56%). Pneumoperitoneum was noted in 65 per cent. The duodenal bulb was the site of perforation in 62 per cent, the pyloric region in 20 per cent, and the gastric body in 18 per cent. A definitive ulcer procedure (V + P, V + A) was performed in 32 patients (38%); 51 patients (58%) had plication, and the remaining five patients did not undergo surgery. A delay in diagnosis and therapy of less than 24 hours occurred in 20 (23%) patients. Mortality was 24 per cent, and correlated significantly with age more than 60 years, but not with treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
Role of glutamic acid 177 of the ricin toxin A chain in enzymatic inactivation of ribosomes.
The gene for the A chain of ricin toxin was fused to a beta-galactosidase marker cistron via a DNA sequence encoding a short collagen linker, and the tripartite fusion protein was expressed in Escherichia coli. Site-specific mutagenesis was used to change glutamic acid residue 177 to aspartic acid or alanine. When the mutant proteins were expressed, purified, and tested quantitatively for enzymatic activity, the carboxylate function at position 177 was found not to be absolutely essential for ricin toxin A-chain catalysis.
Blotting, Western↗
Selection and characterization of ricin toxin A-chain mutations in Saccharomyces cerevisiae.
A DNA sequence encoding the A chain of ricin toxin (RTA) from the castor bean plant, Ricinus communis, was placed under GAL1 promoter control and transformed into Saccharomyces cerevisiae. Induction of expression of RTA was lethal. This lethality was the basis for a selection of mutations in RTA which inactivated the toxin. A number of mutant alleles which encoded cross-reactive material were sequenced. Eight of the first nine mutant RTAs studied showed single-amino-acid changes involving residues located in the proposed active-site cleft.
Amino Acid Sequence↗
Quantitation of renal uptake of technetium-99m DMSA using SPECT.
Quantitative single photon emission computed tomography (SPECT) methodology based on calibration with kidney phantoms has been applied for the assessment of renal uptake of [99mTc]DMSA in 25 normals; 16 patients with a single normal kidney; 30 patients with unilateral nephropathy; and 17 patients with bilateral nephropathy. An excellent correlation (r = 0.99, s.e.e. = 152) was found between SPECT measured concentration and actual concentration in kidney phantoms. Kidney uptake at 6 hr after injection in normals was 20.0% +/- 4.6% for the left and 20.8% +/- 4.4% for the right. Patients with unilateral nephropathy had a statistically significant (p less than 0.001) low uptake in the diseased kidney (7.0% +/- 4.7%), but the contralateral kidney uptake did not differ from the normal group (20.0% +/- 7.0%). The method was especially useful in patients with bilateral nephropathy. Significantly (p less than 0.001) decreased uptake was found in both kidneys (5.1% +/- 3.4% for the left and 6.7% +/- 4.2% for the right). The total kidney uptake (right and left) in this group showed to be inversely correlated (r = 0.83) with serum creatinine. The uptake of [99mTc]DMSA in single normal kidney was higher (p less than 0.001) than in a normal kidney (34.7% +/- 11.9%), however, it was lower than the total absolute uptake (RT + LT = 41.5% +/- 8.8%) in the normal group. The results indicate that SPECT is a reliable and reproducible technique to quantitate absolute kidney uptake of [99mTc]DMSA.
Adolescent↗
Small colon polyps.
One thousand forty-eight small (up to 6 mm) colorectal polyps, removed during colonoscopy, have been analyzed. Sixty-one percent of these small polyps were neoplastic, the remainder being equally divided between hyperplastic polyps and polypoid mucosa with normal-appearing glands. The number of polyps was evenly distributed throughout the colon. Proximally, neoplastic polyps predominated, accounting for 73% of all polyps in the right colon. This was reversed in the distal colon where non-neoplastic polyps comprised 65% of all polyps in the rectum. The incidence of carcinoma was extremely low in small colon polyps, 0.1%. All polyps should be removed when encountered during colonoscopy due to the high prevalence of adenomas among small colon polyps.
Adult↗
The value of right atrial emptying rate in predicting reduction in pulmonary artery pressure in patients with chronic obstructive pulmonary disease.
We used an improved noninvasive radionuclide method, recently developed by us, to evaluate changes in pulmonary artery pressure induced by sublingual nifedipine in patients with chronic obstructive pulmonary disease and pulmonary hypertension. The new method enhances the predictive power of right ventricular ejection fraction by using the right atrial emptying rate as an index of reduced right ventricular compliance. The results were compared to those of invasively measured pulmonary arterial pressure. In the 21 patients studied 20 mg of nifedipine sublingually reduced pulmonary arterial pressure by 13.35% from 36.95 +/- 13.95/12.71 +/- 6.24 (mean 20.79 +/- 8.19) mmHg to 32.67 +/- 12.17/10.9 +/- 6.2 (mean 18.16 +/- 7.3) mmHg (p less than 0.05 for all pressures). Cardiac index increased and the pulmonary and systemic resistances decreased. The percent changes in right atrial emptying rate showed an excellent correlation with the percent change in pulmonary pressure. An increase of 12% or more in right atrial emptying rate predicted in all patients a reduction in pulmonary arterial pressure of at least 8%, the specificity and positive predictive accuracy being 100%. The sensitivity and the predictive accuracy of a negative test were 93% and 80%, respectively. The new method is useful for long-term evaluation of drug therapy in patients with pulmonary hypertension.
Blood Pressure↗
Decrease in global ejection fraction after volume challenge in long-standing hypertension.
An easy noninvasive volume challenge method which takes advantage of the diastolic filling differences existing between patients with recent onset and long-standing hypertension is presented. By passively elevating the patients' legs at 45 degrees for 5 minutes, a sudden increase in venous return was induced in 14 healthy and in 42 hypertensive subjects. The global ejection fraction measured by radionuclide ventriculography increased markedly in both 14 normal subjects and in 18 patients with recent onset hypertension (67 +/- 9% to 75 +/- 6%, p less than 0.001 and 64 +/- 10% to 71 +/- 11%, p less than 0.001, respectively). In contrast, a decrease in global ejection fraction was found in all patients with long-standing hypertension (66 +/- 4% to 58 +/- 10%, p less than 0.001). Similar results were obtained when echocardiographic measurements were made after the legs-up procedure in 24 patients. The correlation with the radionuclide measurements done in the same patients was excellent, r = 0.89. In 20 patients with long-standing hypertension in whom exercise radionuclide ventriculography was done, a marked elevation in global left (LVEF) and right ventricular ejection fraction (RVEF) was found (65 +/- 5% to 71.5 +/- 11% LVEF, and 42 +/- 5% to 45 +/- 7% RVEF, p less than 0.01), whereas the legs-up procedure induced a significant reduction in both global LVEF and RVEF (65 +/- 5% to 57 +/- 6% LVEF, and 44 +/- 7% to 37 +/- 5% RVEF, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Early detection of diastolic impairment in long-standing hypertension by a noninvasive volume challenge method.
A simple, easily reproducible, noninvasive, provocative test for the assessment of early diastolic impairment in long-standing hypertension is presented. Volume challenge of the heart was produced by elevating the subject's legs to 45 degrees for 5 minutes, thus increasing the venous return to the right heart. Using gated radionuclide ventriculography and Fourier analysis, the atrial structures were delineated and time-activity curves were generated. Early diastolic emptying slope of the left atrium was used to assess left ventricular compliance changes in hypertensives. The left atrial early diastolic emptying rate was markedly reduced in 38 hypertensive patients (45.5 +/- 15.4 counts/s) when compared with 14 healthy subjects (78 +/- 16 counts/s). The legs-up procedure induced subsequent decrease in left atrial emptying rate in patients with long-standing hypertension and left ventricular hypertrophy (-27.4 +/- 11%). In patients with recent onset hypertension there was a depressed early diastolic emptying rate (55.79 +/- 10 counts/s), but a "normal" response to the legs-up procedure (an increase in left atrial emptying rate: 12.84 +/- 7%). Global ejection fraction was normal in all subjects studied, decreasing after induction of augmented venous return in long-standing hypertension. Reduction in left atrial early diastolic emptying rate, caused by the legs-up manoeuvre appears to be an early and sensitive indicator of the left ventricular diastolic impairment in essential hypertension.
Blood Volume↗
Evaluation of monoclonal antibodies for the development of breast cancer immunotoxins.
Eighty-five antibodies recognizing breast cancer-selective antigens were conjugated to ricin toxin A-chain using a disulfide linkage. The cytotoxicities of the resulting immunotoxins were determined on breast cancer cells and normal human fibroblasts. Twenty-four antibodies formed immunotoxins that were toxic to at least one breast cancer cell line at concentrations of 10 nM or less but were nontoxic to human fibroblast lines used as negative controls. Some of the breast tumor-selective immunotoxins were as toxic as a conjugate between monoclonal anti-transferrin receptor and ricin toxin A-chain (50% inhibition of cellular protein synthesis at approximately 0.1 nM). Another set of four immunotoxins were indiscriminately toxic to human breast tumor cell lines, two human fibroblast cell lines, and a human lymphoblastoid line. Several of the antibodies the toxin conjugates of which specifically killed breast cancer cell lines may be useful in cancer therapy, since they show a wide range of binding to individual breast tumors and cell lines and a limited range of binding to normal tissue types.
Antibodies, Monoclonal↗
Bone metastases and bone pain in breast cancer. Are they closely associated?
One hundred ninety patients with breast cancer were prospectively evaluated for bone pain and had technetium Tc 99m-methylene diphosphonate bone scintigraphy for bone metastases. Of the 66 patients showing evidence for bone metastases, 21 (32%) did not have bone pain. There were 155 sites of skeletal metastases, but pain was found only in 50 sites. The age of the patient or involvement of weight-bearing bones did not seem to affect the association between bone metastases and pain. We discuss the need for periodic bone scintigraphy, even when the clinical state does not seem to warrant it.
Adult↗
Chronic pseudo-obstruction secondary to side-to-side intestinal anastomosis.
An unusual late complication of side-to-side intestinal anastomosis, chronic small-bowel obstruction with massive proximal ileal dilation despite a widely patent anastomosis, occurred in a patient. The classic blind loop syndrome was not present. Several potential mechanisms are suggested, including regional absence of normal peristalsis on a mechanical basis and bacterial overgrowth. This report adds support to the concept that side-to-side intestinal anastomosis should be avoided whenever possible.
Blind Loop Syndrome↗