Development and performance of npde for the evaluation of time-to-event models.

PURPOSE: Normalised prediction distribution errors (npde) are used to graphically and statistically evaluate mixed-effect models for continuous responses. In this study, our aim was to extend npde to time-to-event (TTE) models and evaluate their performance. METHODS: Let V denote a dataset with censored TTE observations. The null hypothesis (H0) is that observations in V can be described by model M. We extended npde to TTE models using imputations to take into account censoring. We then evaluated their performance in terms of type I error and power to detect model misspecifications for TTE data by means of a simulation study with different sample sizes. RESULTS: Type I error was found to be close to the expected 5% significance level for all sample sizes tested. The npde were able to detect misspecifications in the baseline hazard as well as in the link between the longitudinal variable and the survival function. The ability to detect model misspecifications increased as the difference in the shape of the survival function became more apparent. As expected, the power also increased as the sample size increased. Imputing the censored events tended to decrease the percentage of rejections. CONCLUSIONS: We have shown that npde can be readily extended to TTE data and that they perform well with an adequate type I error.

Exogenous Lipocalin 2 Ameliorates Acute Rejection in a Mouse Model of Renal Transplantation.

Lipocalin 2 (Lcn2) is rapidly produced by damaged nephron epithelia and is one of the most promising new markers of renal injury, delayed graft function and acute allograft rejection (AR); however, the functional importance of Lcn2 in renal transplantation is largely unknown. To understand the role of Lcn2 in renal AR, kidneys from Balb/c mice were transplanted into C57Bl/6 mice and vice versa and analyzed for morphological and physiological outcomes of AR at posttransplantation days 3, 5, and 7. The allografts showed a steady increase in intensity of interstitial infiltration, tubulitis and periarterial aggregation of lymphocytes associated with a substantial elevation in serum levels of creatinine, urea and Lcn2. Perioperative administration of recombinant Lcn2:siderophore:Fe complex (rLcn2) to recipients resulted in functional and morphological amelioration of the allograft at day 7 almost as efficiently as daily immunosuppression with cyclosporine A (CsA). No significant differences were observed in various donor-recipient combinations (C57Bl/6 wild-type and Lcn2(-/-) , Balb/c donors and recipients). Histochemical analyses of the allografts showed reduced cell death in recipients treated with rLcn2 or CsA. These results demonstrate that Lcn2 plays an important role in reducing the extent of kidney AR and indicate the therapeutic potential of Lcn2 in transplantation.

Isolated brain microvessels: a purified, metabolically active preparation from bovine cerebral cortex.

A purified, metabolically active preparation of brain microvessels was isolated from bovine cerebral cortex by using a simple procedure involving mild disruption of the tissue by homogenization and trapping of the vessels on nylon sieves. This preparation permits in vitro metabolic and structural studies of small blood vessels.

The in vitro inhibition of insulin secretion by diphenylhydantoin.

Glucose intolerance has been observed following diphenylhydantoin (DPH) intoxication. Because of this association between DPH and hyperglycemia, the effect of DPH on insulin release in vitro using preparations of isolated islets of Langerhans and pancreatic pieces was examined. In concentrations identical with those considered necessary for adequate anticonvulsant therapy in man, DPH markedly decreases the insulin secretory response of pancreatic pieces to methacholine, 1 mug/ml, tolbutamide, 250 mug/ml, and glucose, 200 mg/100 ml, without any demonstrable alteration in the oxidative conversion of glucose-1-(14)C or glucose-6-(14)C to (14)CO(2) by isolated islets. This DPH-induced inhibition of insulin secretion is not reversed by higher concentrations of glucose (600 mg/100 ml) or by increasing concentrations of extracellular calcium ion (4-6 mmoles/liter). 0.1 mM potassium and 10(-4) M ouabain, however, effectively restore the DPH-induced block of insulin secretion in pancreatic pieces. 60 mM potassium ion, on the other hand, not only restores the insulin secretory response to glucose (200 mg/100 ml) but results in an added stimulation of insulin secretion in the presence of DPH. In the presence of DPH, (22)Na accumulation by isolated islets is decreased by 26-40% as compared with controls. Such evidence is considered to indirectly support the postulate that the electrophysiological properties of DPH on the pancreas are due to a stimulation of the membrane sodium-potassium-magnesium ATPase.

Antineoplastic drugs sulindac sulfide and sulfone inhibit cell growth by inducing apoptosis.

The nonsteroidal anti-inflammatory drug sulindac is known to inhibit chemical carcinogenesis in rodent models and cause regression of adenomas in patients with adenomatous polyposis coli. Sulindac is a prodrug that is metabolized to a pharmacologically active sulfide derivative that potently inhibits prostaglandin synthesis. Recent studies, however, have shown that a sulfone derivative of sulindac, which essentially lacks prostaglandin synthesis inhibitory activity, also inhibits chemical carcinogenesis, suggesting that reduction of prostaglandin levels is not necessary for the antineoplastic activity of this class of drugs. Both sulindac sulfide and the sulfone inhibit the growth of cultured tumor cells, although the cellular mechanism(s) responsible for the antineoplastic activity of sulindac derivatives is unknown. In this study, we investigated the effects of sulindac sulfide and sulfone on the proliferation, differentiation, and apoptosis of HT-29 human colon carcinoma cells. Sulindac sulfide and sulfone significantly reduced cell number in both preconfluent and confluent cultures of HT-29 cells with the sulfide showing approximately 4-fold greater potency. In addition to HT-29 cells, both drugs inhibited the growth of a variety of tumor cell lines derived from other tissues, as well as normal epithelial cells and fibroblasts. Neither sulindac sulfide nor sulfone inhibited cell proliferation under conditions where the drugs were growth inhibitory. Only under specific conditions involving mitogenic stimulation did sulindac sulfide and sulfone cause cell cycle arrest. Neither sulindac sulfide nor the sulfone induced differentiation of HT-29 cells, but both drugs strongly induced apoptosis. The apoptotic response to sulindac sulfide and sulfone was both time- and dose-dependent and involved a mechanism independent of their inhibitory effect on cell cycle progression. These data suggest that apoptosis is responsible for the cell growth inhibitory activity of sulindac sulfide and sulfone and represents a potential mechanism for the antineoplastic activity of these drugs.

Sulindac sulfone inhibits azoxymethane-induced colon carcinogenesis in rats without reducing prostaglandin levels.

Nonsteroidal anti-inflammatory drugs (NSAIDs), such as sulindac, have cancer chemopreventive properties by a mechanism that has been suggested to involve cyclooxygenase inhibition and reduction of prostaglandin (PGE2) levels in the target tissue. To test this hypothesis, we studied the effect of dietary sulindac sulfone (500-2000 ppm), a metabolite of sulindac reported to lack cyclooxygenase inhibitory activity, on tumor formation and PGE2 levels in the azoxymethane model of colon carcinogenesis. Rats treated with sulindac at 400 ppm and piroxicam at 150 ppm were used as positive controls. Rats received two s.c. injections of azoxymethane (15 mg/kg) for 2 weeks and were fed either experimental or control diets until necropsy. After 31 weeks of sulfone treatment, a dose-related increase in sulfone levels in both serum and cecal contents was measured; there was no evidence of metabolic conversion to sulindac or other metabolites. Rats treated with sulfone at 1000 and 2000 ppm, sulindac, and piroxicam had significantly fewer colonic adenomas and carcinomas compared with rats fed control diet as measured by tumor incidence, multiplicity, and tumor burden. Sulfone-treated rats also showed a dose-response relationship for inhibiting all tumor parameters. Colons from rats treated with sulindac or piroxicam contained PGE2 levels that ranged from approximately 16-49% of control levels. PGE2 levels in rats treated with sulfone up to 2000 ppm ranged from 78-118% of control levels. Moreover, the effects of sulindac sulfone on various enzymes responsible for regulating prostaglandin levels were evaluated. No significant inhibitory effects were observed for cyclooxygenase, lipoxygenase, or phospholipase A2. These results suggest that reduction of prostaglandin levels in the target tissue may not be necessary for the chemopreventive properties of sulindac.

Metachromatic staining with Coomassie Brilliant Blue R-250 of the proline-rich calf thymus histone, H1.

In this report, we show that calf thymus histone 1 stains metachromatically with Coomassie Brilliant Blue R-250. Histone 1 gel bands appear red instead of the more familiar blue color characteristic of the vast majority of proteins. The red color and the spectral properties of histone 1 bands are qualitatively similar to those of collagen and procollagen bands which, as previously shown by others, also stain metachromatically (Micko, S. and Schlaepfer, W.W. (1978) Anal. Biochem. 88, 566-572 and McCormick, P.J., Chandrasekhar, S. and Millis, A.J.T. (1979) Anal. Biochem. 97, 359-366). In contrast to histone 1, histones 2A, 3 and 4 stain blue; histone 2B also stains predominantly blue, but with a faint red tint. Both red and blue bands display an absorption maximum in the vicinity of 560 nm, but red bands display an additional maximum in the vicinity of 530 nm. There are quantitative differences between the red bands; the 530 nm peak is more prominent in the spectrum of the collagen band than in the spectrum of the histone 1 band. The spectra of the blue bands are all very similar except that the spectrum of the histone 2B band is shifted slightly toward lower wavelengths. To account for the spectral differences between protein bands, we propose a model in which closely-spaced proline residues are responsible for the chromotropic effect. Localized concentrations of proline residues are present in both collagens and histone 1 and a small cluster is also present in histone 2B.

Stereochemistry of the hydrogen transfer to NAD catalyzed by D-galactose dehydrogenase from Pseudomonas fluorescens.

The stereochemistry of the hydrogen transfer to NAD catalyzed by D-galactose dehydrogenase (E.C. 1.1.1.48) from P. fluorescens was investigated. The label at C-1 of D-[1--3H] galactose was enzymatically transferred to NAD and the resulting [4--3H]NADH was isolated and its stereochemistry at C-4 investigated. It was found that the label was exclusively located at the 4(S) position in NADH which calls for classification as a B-enzyme. This result was confirmed by an alternate approach in which [4--3H]NAD was reduced by D-galactose as catalyzed by D-galactose dehydrogenase. The sterochemistry at C-4 of the nicotinamide ring would then have to opposite to that in the first experiment. As expected, the label was now exclusively located in the 4(R) position, again confirming the B-calssification of the enzyme.

Stereochemistry of the hydrogen transfer to NAD catalyzed by (S)alanine dehydrogenase from Bacillus subtilis.

The stereochemistry of the hydrogen transfer to NAD catalyzed by (S)alanine dehydrogenase [ (S)alanine: NAD oxidoreductase (EC 1.4.1.1) ] from B. subtilis was investigated. The label at C-2 of (S) [2,3--3H] alanine was enzymatically transferred to NAD, and the [4--3H]NADH produced isolated and the stereochemistry at C-4 investigated. It was found that the label was exclusively located at the (R) position which indicates that (S)alanine dehydrogenase is an A-type enzyme. This result was confirmed in an alternate way by reducing enzymatically [4--3H]NAD with non labeled (S)alanine and (S)alanine dehydrogenase and investigating the stereochemistry of the ]4--3H]NADH produced. As expected, the label was now exclusively located at the (S) position. This proves that (S)alanine dehydrogenase isolated from B. subtilis should be classified as an A-enzyme with regard to the stereochemistry of the hydrogen transfer to NAD.

A symbiotic relationship of energy metabolism between a 'non-glycolytic' mammalian red cell and the liver.

The red cell of newborn pig loses the ability to carry out glycolysis within a month after birth. The metabolic energy source for this 'non-glycolytic' mammalian red cell is unknown. Hepatectomy of an adult pig results in the loss of red cell ATP with a characteristic half-time of 7--8 h which is identical to the rate with which ATP disappears in the pig cells under in vitro substrate-free incubation. Exposure of pig red cells with either normal or depleted levels of ATP to isolated hepatocytes causes a net synthesis of red cell ATP during a 12 h incubation. These findings suggest that a symbiotic relationship of energy metabolism may exist between the red cell and the liver of the pig.

Glucagon structure-function relationships using isolated rat hepatocytes.

The ability of glucagon and several of its semi-synthetic analogues to stimulate glucose production in isolated rat hepatocytes was measured and compared for relative potencies. The order of decreasing biological activities of glucagon in this assay was as follows: glucagon greater than [HArg12]-glucagon greater than [des-Asn28, Thr29][homoserinehydrazide27]-glucagon approx. equal to [des-His1]-glucagon greater than [des-Asn28, Thr29][homoserinelactone27]-glucagon greater than [des-Asn28, Thr29]-[n-butylhomoserineamide27]-glucagon greater than glucagon1-21. Qualitatively, these results are similar to those obtained previously in the hepatic plasma membrane adenylate cyclase assay. Minor exceptions were noted for the hydrazide derivative and the partial agonist [des-His1]-glucagon, both of which were slightly more potent relative to glucagon in the glycogenolytic assay than in the adenylate cyclase assay. The assay provides important insight into glucagon structure-function relationships.

Antiproliferative effect of nonsteroidal antiinflammatory drugs against human colon cancer cells.

Several lines of evidence suggest that nonsteroidal antiinflammatory drugs may be effective in preventing colorectal cancer. These include animal experiments, case-control studies, and clinical experience with sulindac in promoting the regression of adenomatous colon polyps in adenomatous polyposis coli. We determined the antiproliferative activity of various nonsteroidal antiinflammatory drugs, including two sulindac derivatives, against human colon cancer cells in vitro. Ht-29, SW480, and DLD-1 cells were continuously incubated with serial drug dilutions for 6 days prior to fixation. Cell number was determined using the sulforhodamine B assay, and drug concentrations which inhibited cell growth by 50% were estimated for each agent by interpolation. All drugs exhibited antiproliferative activity against Ht-29 and DLD-1 cells, and most inhibited SW480 cells. For Ht-29 cells, the 50% inhibitory concentration varied from 55 microM for diclofenac to 2100 microM for 5-aminosalicylic acid, with three drug groups of high, intermediate, and low potency evident. Inhibition of cell growth by sulindac sulfide was reversible following drug removal. Nonsteroidal antiinflammatory drugs exert an antiproliferative effect against human colon cancer cells with a wide range of potencies. A cytostatic response was demonstrated with sulindac sulfide. These data further support the potential role of these agents for chemoprevention of colorectal neoplasia.

pH gradient elution of human IgG1, IgG2 and IgG4 from protein A-sepharose.

Pooled human serum having a normal IgG subclass content was chromatographed on a column of protein A-Sepharose. The immunoglobulins that bound to the column at pH 7.0 were eluted with a pH gradient generated by 3 equal volumes of citrate/phosphate buffer at pH 5.0, 4.5 and 2.2. The elution pattern consisted of two major overlapping peaks centered 0.4 pH units apart in the pH gradient; on average the first peak centered at pH 4.7, and the second centered at pH 4.3. Upon second passage of each component, single major peaks centered at the appropriate pH were seen. The subclass distributions of the re-chromatographed peaks were as follows: the high pH-eluting IgG contained less than 1% IgG1, 95% IgG2 and 5% IgG4; the low pH-eluting IgG contained 90% IgG1, 6% Ig2 and 5% IgG4. IgG3 does not bind to protein A and was thus absent from the pH gradient fractions. Chromatography on protein A-Sepharose provides a means for separating normal human IgG1 from IgG2 and may therefore prove useful as an additional tool for studying the relative biological role of these IgG subclasses.

Lipid solubility and affinity for N-demethylation of dansylamides in isolated rat hepatocytes.

Isolated hepatocytes carry out the N-demethylation of dansylamide at near linear rates for up to 8 h. This reaction was measured by following the release of tritium into water on hydroxylation of 3H-labeled methyl groups. The competitive inhibition of dansylamide by dansylated amino acids was studied in this system as an example of competing drug metabolism in a series of compounds which are identical around the site of metabolism and different remote to that site. A correlation between lipid solubility and the Ki was not found over the entire range of substrate analogues. While most of the high Km inhibitors seem to correlate with lipid solubility, the highly lipophilic derivatives of the leucines and phenylalanine are in a separate group. Lipid solubilities of the dansylated amino acids were little affected by changes in pH and thus behaved as "essential nonelectrolytes".

Metabolism-related spectral characterization and subcellular distribution of polychlorinated biphenyl congeners in isolated rat hepatocytes.

The disposition and biotransformation of 4,4'-dichlorobiphenyl (4-DCB), 2,2',3,3',6,6'-hexachlorobiphenyl (236-HCB), and 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB) were studied in isolated rat hepatocyte suspensions. The polychlorinated biphenyls (PCBs) were taken up rapidly by the cells but incompletely metabolized. Metabolism followed first-order Michaelis-Menten kinetics for 20 min and plateaued by 60 min, at which point only 32% of 4-DCB (0.005 to 100 microM) and 60% of 236-HCB (0.001 to 100 microM) were metabolized, while metabolism of 245-HCB was not detected (0.1 to 200 microM). Kinetic studies revealed that both 4-DCB and 236-HCB were metabolized by two Michaelis-Menten processes, displaying high- and low-affinity binding. Readdition of congener once metabolism plateaued resulted in a reinitiation of metabolism with the same proportion of metabolites produced. The termination of metabolism was not due to destruction of the mixed-function oxidases or to depletion of cofactors. The metabolism of PCB congeners is influenced by the affinity of the congener for cytochrome P-450 and partitioning of the congener within the hepatocyte. Analysis of absorbance differences (delta absorbance 390-240 nm) of equimolar concentrations of congener (100 microM) revealed that 236-HCB displayed the greatest affinity of binding to cytochrome P-450 followed by 4-DCB, while 245-HCB showed virtually no binding. Microsomal preparations demonstrated equivalent but greater absorbance values. Subcellular distribution of 14C-labeled congener and its metabolites showed that the majority of radioactivity appeared in the cytosolic fraction, representing 70% of the dose added for each congener. Cytosolic binding of congener and metabolites may influence both the availability of congener to cytochrome P-450 and the excretion rate of metabolites from the cell.

Influence of chronic ethanol treatment on alpha1-adrenergic and vasopressin receptor-stimulated phosphatidylinositol synthesis in isolated rat hepatocytes.

Adult male rats were given nutritionally balanced high fat (34% corn oil) diets containing either ethanol or isocalorically substituted sucrose for 4-5 weeks. Phosphatidylinositol-phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine contents of whole hepatocytes were not altered in ethanol-treated rats. However, vasopressin and alpha1-adrenergic stimulation of [32P]-incorporation into phosphatidylinositol of isolated hepatocytes from ethanol-treated rats was increased substantially compared to that of controls. Yet, chronic ethanol treatment had no effect on hepatic alpha1 receptor density or affinity as measured by [3H]prazosin specific binding. These results suggest that the supersensitive phosphatidylinositol response to this alpha1 agonist observed in hepatocytes from ethanol-treated rats occurred distal to cell surface receptors and may be similar for vasopressin.

Interaction of capsaicinoids with drug-metabolizing systems. Relationship to toxicity.

The interaction of capsaicin with microsomal drug-metabolizing systems was assessed to determine the role that bioactivation of capsaicin may play in the induction of hepatotoxicity and neurotoxicity. Capsaicin produced a type I spectral change in rat hepatic microsomes in a high affinity (Ks = 8 microM) concentration-dependent manner and was approximately equipotent with SKF-525A in inhibiting ethylmorphine demethylation. Capsaicin (10 mg/kg, s.c.) inhibited biotransformation in vivo as measured by prolongation of pentobarbital sleep time. Reactive metabolites of capsaicin were studied using [3H]dihydrocapsaicin. [3H]Dihydrocapsaicin bound irreversibly to hepatic microsomal protein after in vitro incubation or in vivo administration. No binding was observed in spinal cord or brain. Although the bioactivation and subsequent covalent binding of capsaicin equivalents may initiate events associated with the hepatotoxicity of capsaicin, it appears that capsaicin-induced neuropathy does not involve covalent interactions with neuroproteins in spinal cord or brain.

Differentiation of the cardiac and pulmonary toxicity of monocrotaline, a pyrrolizidine alkaloid.

Monocrotaline, given to rats as a 20 mg/l solution in drinking water for 3 weeks, doubled the mass of the right heart and lung. The rise in lung mass preceded that of the heart. These increases were accompanied by increases in the absolute protein content of the two organs, together with increases in the rates of both protein and RNA syntheses. The increase in lung mass was not accompanied by a change in total collagen content, as measured by two independent methods: 4-hydroxyproline content and detergent fractionation. In contrast, the right ventricle showed more than a 4-fold increase in total collagen content. Total pulmonary lipids increased by 86%, but the lipid: protein ratio was unchanged. Right ventricular lipids were unchanged in amount but the lipid: protein ratio fell by 29%. Lung DNA:RNA ratio decreased 49% and right ventricle DNA:RNA ratio decreased 69%, indicating that both of these organs were responding to monocrotaline with hypertrophy. These results suggest that the processes of hypertrophy differ in the two organs: in the lung, there was no fibrosis despite a marked increase in dry weight, while right ventricular hypertrophy was characterized by increased collagen deposition. There was no alteration in the left ventricle in any of the parameters investigated.

Sulindac derivatives inhibit growth and induce apoptosis in human prostate cancer cell lines.

We examined the activity of two metabolites of sulindac (a nonsteroidal anti-inflammatory drug), sulindac sulfide and sulindac sulfone (exisulind, Prevatec), and a novel highly potent analog of exisulind (CP248) on a series of human prostate epithelial cell lines. Marked growth inhibition was seen with the BPH-1, LNCaP, and PC3 cell lines with IC50 values of about 66 microM, 137 microM, and 64 nM for sulindac sulfide, exisulind, and CP248, respectively. DNA flow cytometry and 4',6'-diamido-2-phenylindole (DAPI) staining indicated that these three compounds also induced apoptosis in all of these cell lines. Similar growth inhibition also was seen with the PrEC normal human prostate epithelial cell line, but these cells were resistant to induction of apoptosis at concentrations up to 300 microM, 1 mM, and 750 nM of sulindac sulfide, exisulind, and CP248, respectively. Derivatives of LNCaP cells that stably overexpress bcl-2 remained sensitive to growth inhibition and induction of apoptosis by these compounds. In vitro enzyme assays indicated that despite its high potency in inhibiting growth and inducing apoptosis, CP248, like exisulind, lacked cyclooxygenase (COX-1 and COX-2) inhibitory activity even at concentrations up to 10 mM. Moreover, despite variations of COX-1 and COX-2 expression, the three benign and malignant prostate cell lines showed similar sensitivity to growth inhibition and induction of apoptosis by these three compounds. Therefore, sulindac derivatives can cause growth inhibition and induce apoptosis in human prostate cancer cells by a COX-1 and -2 independent mechanism, and this occurs irrespective of androgen sensitivity or increased expression of bcl-2. These compounds may be useful in the prevention and treatment of human prostate cancer.

Glutathione effects on toxicity and uptake of mercuric chloride and sodium arsenite in rabbit renal cortical slices.

The mechanism of renal uptake of nephrotoxic heavy metals such as HgCl2 and NaAsO2 is not clear. The metals are known to react with endogenous sulfhydryls such as glutathione (GSH), so metal-GSH conjugates may be delivered to the kidney. To study this possibility, renal cortical slices from male New Zealand white rabbits were incubated with 10(-4) M HgCl2 or 10(-3) M NaAsO2 +/- stoichiometric amounts (1-3x) of GSH; or synthetic metal-GSH conjugates [10(-4) M Hg(SG)2 or 10(-3) M As(SG)3]. Incubations were performed at 37 degrees C in DME-F12 buffer (95/5 O2/CO2) for 8 hr. Hg(SG)2 reduced slice K+/DNA content, as an indicator of viability, significantly less than HgCl2. As(SG)3 exhibited a 2-hr delay in K+/DNA content reduction compared to NaAsO2. This delay in toxicity was not correlated to changes in uptake. Arsenic and mercury accumulation, determined by proton-induced X-ray emission, were also identical between the metal salts and the metal-GSH conjugates. Exogenous GSH decreased HgCl2 cytotoxicity and was correlated to a decrease in Hg accumulation in the slice. Exogenous GSH had limited if any protective effects against cytotoxicity by NaAsO2 and a decrease in As accumulation was not observed. Complex metal-GSH interactions appear to exist and impact on the uptake and toxicity of these metals.