Detection of bladder carcinoma in females by flow cytometry and cytology

The sensitivity of bladder wash flow cytometry (BWFCM), voided urinary cytology (VUC), and cytology of catheterized urine obtained at the time of cystoscopy (CUC) were reviewed on all women evaluated for bladder cancer at Memorial Sloan-Kettering Cancer Center between June 1985 and December 1986. This comprised sixty-four episodes of pathologically proven bladder cancer in 48 women. Considering positive and suspicious results jointly the sensitivities of BWFCM, CUC and 3 VUC were 75%, 64% and 56%, respectively. If only positive results were considered (i.e., suspicious results considered as negative), the sensitivities of BWFCM, CUC and 3 VUC were 64%, 31% and 32%, respectively. The sensitivities of these tests are less than for a predominantly male population, presumably related to the presence of squamous epithelium and greater frequency of pyuria. However, bladder wash flow cytometry and conventional cytology are still a very valuable addition to cystoscopic examination, and the combination of BWFCM with conventional cytology is more sensitive than either procedure alone.     

The SH2- and SH3-containing Nck protein transforms mammalian fibroblasts in the absence of elevated phosphotyrosine levels.

We have established the human nck sequence as a new oncogene. Nck encodes one SH2 and three SH3 domains, the Src homology motifs found in nonreceptor tyrosine kinases, Ras GTPase-activating protein, phosphatidylinositol 3-kinase, and phospholipase C-gamma. Overexpression of human nck in 3Y1 rat fibroblasts results in transformation as judged by alteration of cell morphology, colony formation in soft agar, and tumor formation in nude BALB/c mice. However, overexpression of nck does not induce detectable elevation of the phosphotyrosine content of specific proteins, as is observed for v-crk, another SH2/SH3-containing oncogene. Despite this fact, we demonstrate that Nck retains the ability to bind tyrosine phosphorylated proteins in vitro, using a fusion protein of Nck with glutathione-S-transferase (GST). Moreover, when incubated with lysates prepared from v-src-transformed 3Y1 cells or the nck-overexpressing cell lines, GST-Nck binds to both p60v-src and serine/threonine kinases, respectively. Although phosphotyrosine levels are not elevated in the nck-expressing fibroblasts, vanadate treatment of these cells results in a phosphotyrosine pattern that is altered from the parental 3Y1 pattern, suggestive of a perturbation of indigenous tyrosine kinase pathways. These results suggest the possibility that human nck induces transformation in 3Y1 fibroblasts by virtue of its altered affinity or specificity for the normal substrates of its rat homolog and that Nck may play a role in linking tyrosine and serine/threonine kinase pathways within the cell.     

Effect of chemical sympathectomy on renal hydroelectrolytic handling in dogs with chronic caval constriction

The effect of renal selective chemical sympathectomy by intrarenal infusion of 6-hydroxydopamine (6-OHDA, 5 mg/kg body weight) on the renal excretion of water and electrolytes was studied in 7 dogs in whom a syndrome of sodium and water retention and ascites formation was induced by partial constriction of thoracic inferior vena cava. Propranolol (1 mg) and phentolamine (3 mg) were also injected to obviate acute systemic hemodynamic changes. Sympathectomy was performed once in 4 dogs and three times in 3 dogs. Sympathectomy induced an abrupt and transient increase in urinary flow (from 170 +/- 30 to 890 +/- 60 ml/24 h) and sodium excretion (from 4.5 +/- 1.5 to 178 +/- 21 mEq/24 h). This was accompanied by an important fall in plasma renin activity (from 2.2 +/- 0.2 to 0.5 +/- 0.1 ng angiotensin I/ml/h) and aldosterone, and disappearance of ascites. It is concluded that chemical sympathectomy, by increasing renal sodium and water excretion, mobilizes the ascites induced by chronic caval constriction, a fact that highlights the role of the renal sympathetic system in the pathogenesis of sodium and water retention by the kidney.     

Expression of the v-crk oncogene product in PC12 cells results in rapid differentiation by both nerve growth factor- and epidermal growth factor-dependent pathways.

The transforming gene of the avian sarcoma virus CT10 encodes a fusion protein (p47gag-crk or v-Crk) containing viral Gag sequences fused to cellular sequences consisting primarily of Src homology regions 2 and 3 (SH2 and SH3 sequences). Here we report a novel function of v-Crk in the mammalian pheochromocytoma cell line, PC12, whereby stable expression of v-Crk induces accelerated differentiation, as assessed by induction of neurites following nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) treatment compared with the effect in native PC12 cells. Surprisingly, however, these cells also develop extensive neurite processes after epidermal growth factor (EGF) stimulation, an event which is not observed in native PC12 cells. Following EGF or NGF stimulation of the v-CrkPC12 cells, the v-Crk protein itself became tyrosine phosphorylated within 1 min. Moreover, in A431 cells or TrkA-PC12 cells, which overexpress EGF receptors and TrkA, respectively, a GST-CrkSH2 fusion protein was indeed capable of binding these receptors in a phosphotyrosine-dependent manner, suggesting that v-Crk can directly couple to receptor tyrosine kinase pathways in PC12 cells. In transformed fibroblasts, v-Crk binds to specific tyrosine-phosphorylated proteins of p130 and paxillin. Both of these proteins are also complexed to v-Crk in PC12 cells, as evidenced by their coprecipitation with v-Crk in detergent lysates, suggesting that common effector pathways may occur in both cell types. However, whereas PC12 cellular differentiation can occur solely by overexpression of the v-Src or oncogenic Ras proteins, that induced by v-Crk requires a growth factor stimulatory signal, possibility in a two-step process.     

A 31-amino-acid N-terminal extension regulates c-Crk binding to tyrosine-phosphorylated proteins.

Overproduction of v-Crk, but not of c-Crk, in chicken embryo fibroblasts results in cell transformation. The transforming activity of v-Crk mutants correlates with their ability to cause increased tyrosine phosphorylation of specific cellular proteins, a property that depends on the binding of v-Crk to phosphotyrosine residues via its SH2 domain. In this study, proteins translated in rabbit reticulocyte lysates were used to analyze interactions between Crk derivatives and tyrosine-phosphorylated proteins, particularly the epidermal growth factor (EGF) receptor. The results demonstrate that the binding affinity of c-Crk is much lower than that of v-Crk, despite the fact that both proteins contain identical SH2 domains. Moreover, a 31-amino-acid N-terminal extension of c-Crk, resulting from upstream translational initiation at a CUG codon, significantly increases the ability of the resulting protein to bind to phosphotyrosine-containing proteins. Of those 31 amino acids, 24 can be found in the 27-amino-acid region between Gag and Crk sequences in v-Crk, and removal of this region results in a protein with lower affinity toward the EGF receptor. In addition, fusion of Gag to the amino terminus of c-Crk yields a protein with a binding activity that is lower than that of v-Crk but significantly higher than that of c-Crk without the fusion. These data suggest that sequences N terminal to the Crk SH2 regulate binding activity to tyrosine-phosphorylated proteins and that the amino acids encoded immediately 5' to the c-Crk initiator AUG specifically increase binding affinity. In contrast, deletion of one or two SH3 domains of c-Crk proteins did not change their affinity for the EGF receptor. These results were confirmed in vivo by using A431-derived cell lines overproducing either the chicken c-Crk protein or c-Crk with the 31-amino-acid N-terminal extension. Furthermore, the in vivo experiments suggest that binding of Crk proteins to the stimulated EGF receptor results in Crk phosphorylation and subsequent loss of binding affinity.     

Gibberellic acid production using different solid-state fermentation systems

Gibberellic acid production in liquid fermentation was compared with production of this compound in solid-state fermentation systems using cassava flour, sugar cane bagasse and low density polyurethane. Gibberella fujikuroi produced 23 mg of gibberellin/ml in 120h of liquid fermentation. Solid-state fermentation on bagasse showed excellent growth but presented gibberellin extraction problems. Very low production and growth was observed in solid-state fermentation with low density polyurethane as an inert support. Solid-state fermentation on cassava flour showed high production (250 mg/kg of dry solid medium) in a very short time (36h).     

Influence of exercise on the fiber composition of skeletal muscle

Summary Biopsy samples from the vastus lateralis muscle (VLM) of man were examined for fiber composition at rest and at selected intervals during prolonged exercise ranging in intensity from 40% to 75% of the total body maximal oxygen uptake (VO _2max) and one-min bouts of exercise at 150%VO _2max. Because of the heterogeneity of fibers in human VLM, studies were also completed where the effect of exercise on the fiber composition of the rat soleus muscle (SM) was examined. In some animals the SM from one hindlimb was removed 9 days prior to their being exercised after which the remaining SM was removed. Exercise reduced muscle glycogen in all experiments. In the studies with man, blood lactate exceeded 17 mmoles/l after the heavy exercise but was largely unchanged by endurance exercise. Colonic temperature of the exercised rats exceeded 40° C. In studies where fibers were identified only as type I and type II, type II fibers in the VLM of all samples (16) taken at rest averaged 61.2±12.5% as compared to 59.0±12.0% after exercise (54 biopsy samples). In a second series of studies with man where the subtypes of type II fibers were identified, there were also no differences in fiber composition of the VLM after varying periods of exercise. Glycogen content and percent fiber composition were the same in right and left SM obtained from rested rats. Exercise (30 to 40 min) did not alter the fiber composition of the rat SM. These data demonstrate that the histochemically demonstratable myofibrillar actomyosin ATPase of skeletal muscle is not altered by a single exercise bout.     

The kinetic mechanism of sheep liver sorbitol dehydrogenase.

The relations between the kinetic parameters for both sorbitol oxidation and fructose reduction by sheep liver sorbitol dehydrogenase show that a Theorell-Chance compulsory order mechanism operates from pH 7.4 to 9.9. This is supported by many parallels with the kinetics of horse liver alcohol dehydrogenase, which operates by this classical mechanism. An isotope-exchange study using D-(2H8)sorbitol confirmed the existence of ternary complexes and that, under maximum velocity conditions, their interconversion is not rate-determining. Substrate inhibition at high concentrations of D-sorbitol or D-fructose confirmed rate-determining enzyme--coenzyme product dissociation, slowed by the existence of more stable abortive ternary enzyme-coenzyme product complexes with substrate. The effect of the inhibitor/activator 2,2,2-tribromoethanol showed the existence of enzyme-NAD-CBr3CH2OH complexes inhibiting the first phase of reaction and enzyme-NADH-CBr3CH2OH complexes dissociating more rapidly than the usual rate-determining enzyme-NADH coenzyme product dissociation in the final phase. Inhibition studies with dithiothreitol also confirmed an ordered binding of coenzymes and second substrates to sorbitol dehydrogenase. Neither D-sorbitol nor D-fructose had any effect on enzyme inactivation by the affinity labelling reagent DL-2-bromo-3-(5-imidazolyl)propionic acid, thus giving no evidence for their existence as binary enzyme-substrate complexes. Several alternative polyol substrates for sorbitol dehydrogenase gave the same maximum velocity as sorbitol. This indicated a common rate-limiting binary enzyme-NADH product dissociation and a similarity of mechanism. An enzyme assay for pH 7.0 and 9.9 is given which enables the concentration of sorbitol dehydrogenase to be determined from initial rate measurements of enzyme activity.