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Beta(beta)-tubulin isotype variation has recently been implicated in the modulation of resistance to paclitaxel in human lung cancer cells and in primary human ovarian tumour samples. Whether alpha-tubulin is involved in drug resistance has not been reported. We have generated a paclitaxel-resistant cell line (H460/T800) from the sensitive human lung carcinoma parental cell line NCI-H460. The resistant cells are more than 1000-fold resistant to taxol and overexpress P-glycoprotein. Interestingly, H460/T800 cells also overexpress alpha- and beta-tubulin as detected by Western blot analysis. From Northern blot analysis, the mechanism of tubulin overexpression appears to be post-transcriptional. To understand whether alpha-tubulin plays a role in drug resistance, we transfected antisense human kalpha1 cDNA construct into the H460/T800 paclitaxel-resistant cells. The antisense clones displayed a reduced alpha-tubulin expression, and the cells were 45-51% more sensitive to paclitaxel and other known antimitotic drugs, compared with vector transfected controls. Complementary experiments of transfecting the sense kalpha1 cDNA into H460 cells conferred a 1.8- to 3.3-fold increase in the IC(50) of several antimitotic agents. Our study suggests that alpha-tubulin is one of the factors that contributes to drug resistance.

To establish whether the use of proteinase inhibitors in the routine determination of in vitro plasma renin activity overestimates the potency of renin inhibitors in vivo, we examined the effects of phenylmethylsulfonyl fluoride and 8-hydroxyquinoline sulfate on the binding to plasma proteins and the respective IC50 values (50% inhibiting concentrations) of three renin inhibitors. All three renin inhibitors, A-64662, A-65317, and A-74273, bound (> 60%) to plasma proteins at both pH 6.0 and 7.4, with slightly greater binding at pH 7.4. Phenylmethylsulfonyl fluoride (1.45 mmol/L) had no significant effect on the protein binding at either pH 6.0 or 7.4; 8-hydroxyquinoline sulfate (3.4 mmol/L) caused a modest dissociation (10-30%) of the renin inhibitors from plasma proteins at both pH values; and the effects of both proteinase inhibitors together were similar to those of 8-hydroxyquinoline alone. At pH 7.4, phenylmethylsulfonyl fluoride increased the potencies of the three renin inhibitors slightly (< or = 43%), whereas IC50 values determined in the presence of 8-hydroxyquinoline decreased by 1.5- to 3.7-fold. The greatest increase in potency occurred with the most hydrophilic compound, and with both angiotensinase inhibitors the effect was no greater than that of 8-hydroxyquinoline alone. The results show that any dissociation of the hypotensive activity measured in vivo from the plasma renin activity measured in vitro is not simply an artifact in the plasma renin activity assay stemming from the use of these angiotensinase inhibitors, especially if only phenylmethylsulfonyl fluoride is used.

Drug resistance is a prevalent problem in the treatment of neoplastic disease, and the effectiveness of many clinically useful drugs is limited by the fact that they are substrates for the efflux pump, P-glycoprotein. Because there is a need for new compounds that are effective in treating drug-resistant tumors, we tested A-204197 (4-[4-acetyl-4,5-dihydro-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazol-2-yl]-N,N-dimethylbenzeneamine), a novel oxadiazoline derivative with antiproliferative properties, on cell lines that were either sensitive or resistant to known microtubule inhibitors. Cell lines that were resistant to paclitaxel, vinblastine, or colchicine were equally sensitive to A-204197 (proliferation IC50s ranging from 36 to 48 nM) despite their expression levels of P-glycoprotein. The effect of A-204197 on cell growth was associated with cell cycle arrest in G2-M, increased phosphorylation of select G2-M checkpoint proteins, and apoptosis. In competition-binding assays, A-204197 competed with [3H]-labeled colchicine for binding to tubulin (K(i) = 0.75 microM); however, it did not compete with [3H]-labeled paclitaxel. A-204197 prevented tubulin polymerization in a dose-dependent manner (IC50 = 4.5 microM) in vitro and depolymerized microtubules in a time-dependent manner in cultured cells. These findings indicate A-204197 is a promising new tubulin-binding compound with antimitotic activity that has potential for treating neoplastic diseases with greater efficacy than currently used antimitotic agents.

To establish whether the use of proteinase inhibitors in the routine determination of in vitro plasma renin activity overestimates the potency of renin inhibitors in vivo, we examined the effects of phenylmethylsulfonyl fluoride and 8-hydroxyquinoline sulfate on the binding to plasma proteins and the respective IC50 values (50% inhibiting concentrations) of three renin inhibitors. All three renin inhibitors, A-64662, A-65317, and A-74273, bound (60%) to plasma proteins at both pH 6.0 and 7.4, with slightly greater binding at pH 7.4. Phenylmethylsulfonyl fluoride (1.45 mmol/L) had no significant effect on the protein binding at either pH 6.0 or 7.4; 8-hydroxyquinoline sulfate (3.4 mmol/L) caused a modest dissociation (10-30%) of the renin inhibitors from plasma proteins at both pH values; and the effects of both proteinase inhibitors together were similar to those of 8-hydroxyquinoline alone. At pH 7.4, phenylmethylsulfonyl fluoride increased the potencies of the three renin inhibitors slightly (or = 43%), whereas IC50 values determined in the presence of 8-hydroxyquinoline decreased by 1.5- to 3.7-fold. The greatest increase in potency occurred with the most hydrophilic compound, and with both angiotensinase inhibitors the effect was no greater than that of 8-hydroxyquinoline alone. The results show that any dissociation of the hypotensive activity measured in vivo from the plasma renin activity measured in vitro is not simply an artifact in the plasma renin activity assay stemming from the use of these angiotensinase inhibitors, especially if only phenylmethylsulfonyl fluoride is used.

In 150 retinoblastomas the authors found a uniform thickness of the cuff of viable retinoblastoma cells that surrounds blood vessels. The mean thickness was 98.7 microns with a standard deviation of 11.9 microns. The cross-sectional area of the cuff was negatively correlated with the mitotic activity in the cuff and positively correlated with the diameter of the central vessel. The mitotic activity in the cuff of cells was inversely related to the distance from the central blood vessel. When the cuff was divided into three concentric rings, the inner ring contained a mean of 6.2 mitotic figures, the middle ring contained a mean of 2.9 mitotic figures, and the outer ring contained a mean of 0.6 mitotic figures. This pattern of growth is similar to that observed in other rapidly growing neoplasms in humans and experimental animals. In these tumors this pattern results from reduction in oxygen tension with increased distance from the central blood vessel.

This study was designed to investigate in sodium-depleted monkeys the renal hemodynamic and excretory effects resulting from blockade of the renin-angiotensin system induced by intrarenal infusion of the primate-selective renin inhibitor A-65317. Intrarenal infusion of A-65317 (n = 6) at a dose of 0.01 micrograms.kg-1.min-1 elicited an increase (P less than 0.05) in renal blood flow (RBF) from 43.5 +/- 2.7 to 49.4 +/- 4.4 ml/min and glomerular filtration rate (GFR) from 6.3 +/- 0.3 to 6.9 +/- 0.4 ml/min, with no significant changes in mean arterial pressure (MAP) or plasma renin activity (PRA). Increases (P less than 0.05) in the urine flow rate (0.18 +/- 0.04 to 0.28 +/- 0.04 ml/min) and the fractional excretion of sodium (0.18 +/- 0.06 to 0.35 +/- 0.13%) were also observed. After a recovery period, the intrarenal infusion dose of A-65317 was increased to 0.1 microgram.kg-1.min-1 and RBF increased (P less than 0.05) from 42.9 +/- 3.9 to 53.0 +/- 3.7 ml/min in conjunction with a significant 85 +/- 4% inhibition of PRA and a 14 +/- 4 mmHg reduction in MAP. GFR and electrolyte excretion remained at control levels. Intrarenal infusion of vehicle (n = 6) had no significant effect on any of the variables studied. In a separate group of monkeys, intravenous (iv) infusion of A-65317 at 0.01 microgram.kg-1.min-1 (n = 5) did not result in significant changes from control.(ABSTRACT TRUNCATED AT 250 WORDS)

A series of low-nanomolar renin inhibitors containing novel C-terminal heterocycles has been designed by formally cyclizing the C-terminus of a glycol-based inhibitor to the second hydroxyl. Molecular modeling suggests that the heterocyclic oxygen hydrogen bonds as an acceptor to the flap region of renin and that the second hydroxyl in the glycol-based inhibitors behaves similarly.

Goldfish (Carassius auratus) were exposed to outboard exhaust products in water or to toluene (a constituent of outboard motor exhaust water) via a continuous flow bioassay dosing apparatus. Various physiologic and pathologic changes were noted. In the blood a consistent decrease (p less than 0.05) in the partial pressure of oxygen, a significant increase (p less than 0.05) in the partial pressures of carbon dioxide, and significant decreases (p less than 0.05) in pH and oxygen saturation were found in many of the blood gas experiments. Laboratory experiments also indicated that these fish are capable of metabolizing toluene to hippuric acid (p less than 0.05). Exposure up to 30 days to these exhaust products produced gross and microscopic lesions in the high-, intermediate-, and low-dose fish. Grossly, livers were smaller and pale; intestines were empty of ingesta and feces; and gills were coated excessively with mucus. Microscopically, the livers of the exposed fish had a decreased cytoplasmic:nuclear ratio, gill filaments were fused, and some kidneys had tubular vacuolization.

The Collaborative Group for the Study of Stroke in Young Women and other similar studies linked oral contraceptives with increased risk of cardiovascular diseases. It is hypothesized that an increased risk for stroke should also be seen among postmenopausal women using estrogens as compared with nonusers. To test this hypothesis, a total of 198 postmenopausal subjects, most of whom were between 50 to 80 years of age, and with a diagnosis of stroke during the period 1972 to 1974, were compared with 396 controls (those who had not had strokes) chosen randomly from the data bank of the Kaiser Foundation Health Plan. The 198 subjects were from the Northern California Kaiser Foundation Hospitals. Both groups were studied for estrogen use and for the associated risk factors of diabetes, hypertension, and coronary artery disease. Of those who had had strokes, 20.7 percent had been taking estrogens, compared to 18.4% in the control group (the difference was insignificant at Chi-Square=0.4396). Relative risk of stroke was calculated by the relative odds method to be 1.16 times as great in estrogen users as nonusers, with 95% confidence limits of 0.75 and 1.77. Estrogen replacement therapy is beneficial for some postmenopausal women. Its risks and benefits must be carefully weighed. This study refutes the association between estrogen use in physiological replacement doses and increased risk of stroke in postmenopausal women.

A study was conducted at two beaches on Lake Erie to evaluate the water sampling design for the collection of several microbiological indicator organisms in relation to day, time, and location of collection. The concentrations of these organisms were generally found to vary significantly (P less than 0.05) by the specific time of day and day of weekend that collection took place. However, the concentrations of these organisms did not vary significantly (P greater than 0.05) at various locations in the bathing area. Future studies investigating the health effects of recreational water as related to microbiological variables should be designed to collect water samples at the specific time of day and day of weekend that an individual was exposed. In addition, sampling at various locations in the bathing area should probably be considered for those beaches having poor dispersion of fecal waste sources.

A method is given for analyzing a slope ratio assay in which a test drug is compared with a standard drug, two or more response variates being measured on each subject at each of several successively increased drug doses. The method requires all subjects to receive the same number of doses, all subjects on the same drug to receive the same doses, the ratio of corresponding doses of the two drugs to be constant over the successive increases, and response variables to be measured only once on each subject at each dose with no missing data allowed. The technique is also applicable when doses are randomly assigned, provided there is no carry-over effect between doses. For each of the J response variates, the relative potency of the test drug with respect to the standard is defined and estimated in the usual way; a 100(1-alpha)% confidence region is then obtained for the vector of the J relative potencies. A procedure is given for testing the equality of some or all of the J relative potencies; an estimator of a common relative potency is obtained by a standard multivariate least squares method. A common relative potency is of interest because the multiple outcome variables are often different indicators of a general physiologic response. The procedures in the paper are illustrated by a simple example concerning the effects of two anesthetics on children.

In response to the November 1972 ruling of the California Supreme Court requiring complete, detailed disclosure of risks for procedures and treatments, a survey was made of the reactions of one hundred patients to this decision. A fictional man was described with a clinical diagnosis of brain tumor, and then the procedure for cerebral angiography and all of that procedure's potential complications were described. The majority of the patients surveyed responded that this complete disclosure of risks helped them in making an intelligent decision about giving consent for the procedure. However, 50 percent of these patients would have withheld consent for the procedure on learning of the complications. Although physicians would have reacted differently, it is certainly the patients' right to make the choices they did.