Ribonuclease A variants with potent cytotoxic activity

Select members of the bovine pancreatic ribonuclease A (RNase A) superfamily are potent cytotoxins. These cytotoxic ribonucleases enter the cytosol, where they degrade cellular RNA and cause cell death. Ribonuclease inhibitor (RI), a cytosolic protein, binds to members of the RNase A superfamily with inhibition constants that span 10 orders of magnitude. Here, we show that the affinity of a ribonuclease for RI plays an integral role in defining the potency of a cytotoxic ribonuclease. RNase A is not cytotoxic and binds RI with high affinity. Onconase, a cytotoxic RNase A homolog, binds RI with low affinity. To disrupt the RI-RNase A interaction, three RNase A residues (Asp-38, Gly-88, and Ala-109) that form multiple contacts with RI were replaced with arginine. Replacing Asp-38 and Ala-109 with an arginine residue has no effect on the RI-RNase interaction. In addition, these variants are not cytotoxic. In contrast, replacing Gly-88 with an arginine residue yields a ribonuclease (G88R RNase A) that retains catalytic activity in the presence of RI and is cytotoxic to a transformed cell line. Replacing Gly-88 with aspartate also yields a ribonuclease (G88D RNase A) with a decreased affinity for RI and cytotoxic activity. The cytotoxic potency of onconase, G88R RNase A, and G88D RNase A correlate with RI evasion. We conclude that ribonucleases that retain catalytic activity in the presence of RI are cytotoxins. This finding portends the development of a class of chemotherapeutic agents based on pancreatic ribonucleases.     

Human gallbladder mucosal function: effects on intraluminal fluid and lipid composition in health and disease

OBJECTIVES: Fructose-1,6-diphosphate is a glycolytic intermediate that has been shown experimentally to cross the cell membrane and lead to increased glycolytic flux. Because glycolysis is an important energy source for myocardium during early reperfusion, we sought to determine the effects of fructose-1,6-diphosphate on recovery of postischemic contractile function. METHODS: Langendorff-perfused rabbit hearts were infused with fructose-1,6-diphosphate (5 and 10 mmol/L, n = 5 per group) in a nonischemic model. In a second group of hearts subjected to 35 minutes of ischemia at 37 degrees C followed by reperfusion (n = 6 per group), a 5 mmol/L concentration of fructose-1,6-diphosphate was infused during the first 30 minutes of reperfusion. We measured contractile function, glucose uptake, lactate production, and adenosine triphosphate and phosphocreatine levels by phosphorus 31-nuclear magnetic resonance spectroscopy. RESULTS: In the nonischemic hearts, fructose-1,6-diphosphate resulted in a dose-dependent increase in glucose uptake, adenosine triphosphate, phosphocreatine, and inorganic phosphate levels. During the infusion of fructose-1,6-diphosphate, developed pressure and extracellular calcium levels decreased. Developed pressure was restored to near control values by normalizing extracellular calcium. In the ischemia/reperfusion model, after 60 minutes of reperfusion the hearts that received fructose-1,6-diphosphate during the first 30 minutes of reperfusion had higher developed pressures (83 +/- 2 vs 70 +/- 4 mm Hg, p < 0.05), lower diastolic pressures (7 +/- 1 vs 12 +/- 2 mm Hg, p < 0.05), and higher phosphocreatine levels than control untreated hearts. Glucose uptake was also greater after ischemia in the hearts treated with fructose-1,6-diphosphate. CONCLUSIONS: We conclude that fructose-1,6-diphosphate, when given during early reperfusion, significantly improves recovery of both diastolic and systolic function in association with increased glucose uptake and higher phosphocreatine levels during reperfusion.     

Shelf-life of prepeeled potato cultivated, stored, and processed by various methods

The present study was aimed to investigate the effects of indigestible dextrin and polydextrose, soluble dietary fibers with low molecular weight, on lipid metabolism and disaccharidase activities of intestinal mucosa in rats fed a high sucrose diet. Their effects were compared with those of well-known soluble fibers, pectin, and guar gum, and also with an insoluble fiber, cellulose. Dietary fibers added to diets at the 5% (w/w) level were alpha-cellulose, pectin, guar gum, indigestible dextrin, and polydextrose. Male Sprague-Dawley rats were given free access to test diets for 6 weeks. Body weight gain was the lowest in rats fed guar gum, the highest in rats fed cellulose, and in-between in rats fed other diets. Although guar gum, pectin, and indigestible feeding dextrin had lower plasma lipid values than cellulose feeding did, the differences were statistically insignificant. Liver triglyceride of the guar gum-fed group was about a third that of the cellulose-fed group, but although those of rats fed polydextrose, indigestible dextrin, and pectin were lower than that of cellulose, the differences were insignificant. Liver cholesterol and phospholipid concentrations were similar among groups. Daily fecal excretion of total lipid, cholesterol, and bile acids were highest in rats fed guar gum, followed by pectin-fed and cellulose-fed rats, and the lowest in rats fed indigestible dextrin and polydextrose. Jejunal sucrase activity was low in the order of guar-gum, polydextrose, indigestible dextrin, pectin, and cellulose. The results indicate that the hypolipidemic effect of soluble dietary fibers would be lessened with reduction in molecular weight, but that the lower sucrase activity by soluble fibers with low molecular weight might be beneficial for hypoglycemic effect.     

Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae

We present here the first insights into the organization of proteins on the RNA in the U5 snRNP of Saccharomyces cerevisiae. Photo-crosslinking with uniformly labeled U5 RNA in snRNPs reconstituted in vitro revealed five contacting proteins, Prp8p, Snu114p, p30, p16, and p10, contact by the three smaller proteins requiring an intact Sm site. Site-specific crosslinking showed that Snu114p contacts the 5' side of internal loop 1, whereas Prp8p interacts with five different regions of the 5' stem-loop, but not with the Sm site or 3' stem-loop. Both internal loops in the 5' domain are essential for Prp8p to associate with the snRNP, but the conserved loop 1 is not, although this is the region to which Prp8p crosslinks most strongly. The extensive contacts between Prp8p and the 5' stem-loop of U5 RNA support the hypothesis that, in spliceosomes, Prp8p stabilizes loop 1-exon interactions. Moreover, data showing that Prp8p contacts the exons even in the absence of loop 1 indicate that Prp8p may be the principal anchoring factor for exons in the spliceosome. This and the close proximity of the spliceosomal translocase, Snu114p, to U5 loop 1 and Prp8p support and extend the proposal that Snu114p mimics U5 loop 1 during a translocation event in the spliceosome.     

The fibroblast-populated collagen microsphere assay of cell traction force--Part 2: Measurement of the cell traction parameter

In Part 1 of this work, we formulated and analyzed a mathematical model for our fibroblast-populated collagen microsphere (FPCM) assay of cell traction forces (Moon and Tranquillo, 1993). In this assay, the FPCM diameter decreases with time as the cells compact the gel by exerting traction on collagen fibrils. In Part 1 we demonstrated that the diameter reduction profiles for varied initial cell concentration and varied initial FPCM diameter are qualitatively consistent with the model predictions. We show here in Part 2 how predictions of a model similar to that of Part 1, along with the determination of the growth parameters of the cells and the viscoelastic parameters of the gel, allow us to estimate the magnitude of a cell traction parameter, the desired objective index of cell traction forces. The model is based on a monophasic continuum-mechanical theory of cell-extracellular matrix (ECM) mechanical interactions, with a species conservation equation for cells (1), a mass conservation equation for ECM (2), and a mechanical force balance for the cell/ECM composite (3). Using a constant-stress rheometer and a fluids spectrometer in creep and oscillatory shear modes, respectively, we establish and characterize the linear viscoelastic regime for the reconstituted type 1 collagen gel used in our FPCM traction assay and in other assays of cell-collagen mechanical interactions. Creep tests are performed on collagen gel specimens in a state resembling that in our FPCM traction assay (initially uncompacted, and therefore nearly isotropic and at a relatively low collagen concentration of 2.1 mg/ml), yielding measurements of the zero shear viscosity, mu 0 7.4 x 10(6) Poise), and the steady-state creep compliance, J0e. The shear modulus, G (155 dynes/cm2), is then determined from the inverse of J0e in the linear viscoelastic regime. Oscillatory shear tests are performed in strain sweep mode, indicating linear viscoelastic behavior up to shear strains of approximately 10 percent. We discuss the estimation of Poisson's ratio, v, which along with G and mu 0 specifies the assumed isotropic, linear viscoelastic stress tensor for the cell/collagen gel composite which appears in (3). The proliferation rate of fibroblasts in free floating collagen gel (appearing in (1)) is characterized by direct cell counting, yielding an estimate of the first-order growth rate constant, k (5.3 x 10(-6) s-1). These independently measured and estimated parameter values allow us to estimate that the cell traction parameter, tau 0, defined in the active stress tensor which also appears in (3), is in the range of 0.00007-0.0002 dyne.cm4/mg collagen.cell.(ABSTRACT TRUNCATED AT 400 WORDS)     

Familial association of primary pulmonary hypertension and a new low-oxygen affinity beta-chain hemoglobinopathy, Hb Washtenaw

A Hungarian-American kindred with familial primary pulmonary hypertension (PPH) and a new, low-oxygen affinity beta-chain variant hemoglobin, Hb Washtenaw, is described. The index case presented with severe PPH and was found to have the abnormal hemoglobin. Two siblings with the abnormal hemoglobin also demonstrated increased pulmonary artery pressures on exercise echocardiography suggestive of early PPH. The occurrence of PPH and the abnormal hemoglobin could be due to genetic or biochemical factors or simply coincidental. A previous study had described a possible association of an abnormal beta-chain variant hemoglobin, Hb Warsaw, and PPH. It was suggested that the putative gene for familial PPH may be located near the beta-globin gene on chromosome 11. The association of PPH and the beta-chain variant hemoglobin in this kindred adds further support to this hypothesis.     

Unstable angina: good long-term outcome after a complicated early course

OBJECTIVES: This study was performed to investigate the long-term outcome of patients with unstable angina within subgroups of the Braunwald classification. BACKGROUND: Long-term follow-up studies of patients with unstable angina are rare and date from more than two decades ago. This study was performed to establish the prognosis of different subgroups of patients with unstable angina (Braunwald criteria) during a 7-year follow-up period. METHODS: We registered a well defined group of 417 consecutive patients, admitted to the hospital for suspected unstable angina. The definite diagnosis was unstable angina in 282 patients (68%) and evolving myocardial infarction in 26; in 109 patients (26%), the symptoms were attributed to other or nonspecific causes. Patients with definite unstable angina were subclassified according to the Braunwald classification. Survival, survival without infarction and survival without infarction or intervention were determined for each class. RESULTS: After a median follow-up period of 94 months, the mortality rate in the first year was 6% and 2% to 3% in the following years. The frequency of revascularization was 47% in the first year, and that for myocardial infarction was 11% in the first year and 1% to 3% thereafter. The Braunwald classification appeared to be appropriate for risk stratification in the first year. However, at 7 years the event rates in all classes were similar. In particular, the Braunwald classification had no long-term impact on mortality or infarction rates. However, patients with acute angina at rest or postinfarction angina and patients with extensive anginal treatment had high intervention rates. CONCLUSIONS: To our knowledge, this study is the first to demonstrate that despite a complicated course during the first year, current management results in good long-term outcome in patients with unstable angina.     

Inequivalence of the two tyrosine ligands in the N-lobe of human serum transferrin

Human serum transferrin N-lobe (hTF/2N) has four iron-binding ligands, including one histidine, one aspartate, and two tyrosines. The present report elucidates the inequivalence of the two tyrosine ligands (Tyr 95 and Tyr 188) on the metal-binding properties of hTF/2N by means of site-directed mutagenesis, metal release kinetics, and absorption and electron paramagnetic resonance (EPR) spectroscopies. When the liganding tyrosines were mutated individually to phenylalanine, the resulting mutant Y95F showed a weak binding affinity for iron and no affinity for copper, whereas, mutant Y188F completely lost the ability to bind iron but formed a stable complex with copper. Since other studies have demonstrated that mutations of the other two ligands, histidine and aspartate, did not completely abolish iron binding, the present findings suggest that the tyrosine ligand at position 188 is essential for binding of iron to occur. Replacement of Tyr 188 with phenylalanine created a favorable chemical environment for copper coordination but a fatal situation for iron binding. The positions of the two liganding tyrosines in the metal-binding cleft suggest a reason for the inequivalence.