Long-term use of nonsteroidal antiinflammatory drugs and other chemopreventors and risk of subsequent colorectal neoplasia.

Our objective was to study the relationship between dispensed aspirin, nonaspirin nonsteroidal antiinflammatory drugs (NSAIDs), steroidal antiinflammatory drugs (SAIDs), acetaminophen, calcium, psyllium, and multivitamin preparations and the risk for subsequent colorectal adenoma and adenocarcinoma. The design was a case-control study. The patient population was from a large municipal teaching hospital in Atlanta, Georgia. In logistic regression models, the risk of colorectal adenoma or adenocarcinoma decreased in the first two years of continuous NSAID use in a linear, time-dependent manner. The risk of colorectal neoplasia after two years of continuous NSAID use was reduced significantly (P < 0.01) as compared to nonusers. Risk reduction appeared greater for adenocarcinoma than adenoma. The use of SAIDs, calcium, multivitamins, and psyllium, as prescribed to our patient population during the mean six-year study period, conferred no measurable risk reduction. These results suggest that in prospective chemoprevention trials, a significant risk reduction can be expected after only two years of aspirin use, in doses similar to those recommended for the prevention of cardiovascular disease, or nonaspirin NSAIDs [correction of nonaspirin. NSAIDs], in doses commonly prescribed for the management of musculoskeletal pain. The results also imply that any short-term reduction in the incidence of colorectal adenoma detected in a phase II trial would underestimate the chemopreventive effect of NSAIDs on the risk of adenocarcinoma.

Topological differences in the expression of collagen type I and collagen type III mRNAs in the rat gingiva.

Collagen mRNA levels in the gingival cells of molars and incisors in rats were measured and correlated with the ratio of interstitial collagen to DNA in these regions. Hybridization of 32P-labeled specific cDNA probes for collagen types I and III with total RNA isolated from gingival tissue of rat molars and incisors showed that the steady-state levels of mRNAs of type I was significantly higher in the molars than in the incisors (molars/incisors = 2.12 +/- 0.12, P < 0.004). However, the ratio of interstitial collagen to DNA in the gingiva of the molars was significantly lower than that found in the incisors (collagen/DNA = 4.13 +/- 0.90 and 12.89 +/- 1.24 respectively, P < 0.001). It is suggested that the difference between the mRNA levels and those of interstitial collagen may reflect an intrinsic characteristic presumably associated with the different modes of mastication between molars and incisors of the rat.

Aspirin and nonsteroidal anti-inflammatory drug use and the risk of subsequent colorectal cancer.

OBJECTIVE: To test the hypothesis that the regular use of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) is negatively associated with the risk of subsequent colorectal cancer. DESIGN: Case-control study with four age- and sex-matched control subjects for each incident colorectal cancer case. POPULATION AND SETTING: Patient population of a large municipal teaching hospital in Atlanta, Ga. MAIN OUTCOME MEASURE: Odds of colorectal cancer as a function of aspirin, nonaspirin NSAIDs, and acetaminophen dispensed to the study population in the 4 years prior to incident colorectal cancer diagnosis. MAIN RESULTS: The risk of colorectal cancer estimated by odds ratios decreased with increasing days of exposure to aspirin linearly in a dose-dependent fashion (likelihood ratio statistic: for cumulative days, P < .001; for cumulative dose, P < .001). The coefficient for days of exposure to aspirin was highly significant even when modeled as a continuous variable (P = .001). There appeared to be a threshold above which nonaspirin NSAIDs afforded protection (likelihood ratio statistic: for cumulative days, P = .021; for cumulative dose, P = .019). Acetaminophen conferred no risk reduction. CONCLUSION: The results of previous experimental animal models, interventional case studies, and some but not all epidemiological investigations and the present data point toward a causal relationship between NSAID use and the prevention of cancer of the large bowel and rectum. Because of the potential gastrointestinal and renal side effects of NSAID use, particularly in the elderly, chemoprevention trials are now needed to allow risk-benefit analysis in populations at high risk for colorectal cancer.

Growth hormone-binding protein (GH-BP) levels in follicular fluid from human preovulatory follicles: correlation with serum GH-BP levels.

Serum GH-binding protein (GH-BP), which is identical with the extracellular domain of the GH-receptor, has important implications for the distribution and physiological activity of GH and may enable evaluation of GH-receptor function. Recent studies suggest that GH plays an important role in the modulation of ovarian function and GH-receptors/BPs are found in the female reproductive system. The purpose of the present study was to investigate the presence of GH-BP in human follicular fluid (FF) and compare the levels of FF GH-BP with those detectable in serum, in 46 women undergoing in vitro fertilization. Levels of GH-BP were determined by incubation of [125I]hGH with 50 microL FF or serum, in the absence or presence of excess hGH and specific binding was expressed as a percentage of the total counts per min incubated. The mean GH-BP level in patient FF was 12.39 +/- 0.63% (mean +/- SE) and this correlated significantly with serum GH-BP levels (r = 0.82; P < 0.001). The binding of hGH to FF was dose dependent, highly specific, and of high affinity and low capacity. The affinity constants (Ka) obtained by Scatchard analysis for hGH binding to patient's FFs and sera were not significantly different. Furthermore, we have analyzed the sodium dodecyl sulfate gel electrophoretic pattern of GH-BP in FF and serum by both the ligand-blot technique and after cross-linking with [125I]hGH. Our results show that there is close similarity between serum and FF GH-BPs. Significantly lower FF GH-BP levels were measured in patients older than 36 yr compared to younger women (P < 0.05), whereas increased values were obtained both in patients with elevated E2 concentrations in serum (> 7000 pmol/L) and in FF (> 2200 nmol/L), (P < 0.02 and P < 0.05, respectively). This first demonstration of GH-BP in FF is expected to increase our understanding of the possible direct effect of GH on ovarian steroidogenesis, and suggests a possible regulatory role for GH-BP in folliculogenesis.

Type I and type III collagen mRNA levels in kidney regions of old and young rats.

Interstitial fibrosis is a common feature of renal aging. The steady-state levels of type I and type III collagen mRNAs as well as DNA, protein and collagen deposition were investigated in the cortex, inner and outer medulla of aged (22 months old) rats in comparison to young (5 months old) controls. Our data show that the cortex and outer medulla of old rats expressed significantly higher percentage of type I collagen mRNA compared to the respective regions in the young rat kidneys. Moreover, within the group of the old rats, the cortex expressed significantly higher percentage of type I collagen mRNA compared to the inner medulla whereas in the group of the young rats the expression was similar in all kidney regions. The ratio of extracellular collagen to DNA was significantly higher in the cortex, inner and outer medulla of old compared to young rats. The ratio of collagen to total protein, although showing a similar age-related difference, attained statistical significance in the cortex only. Thus, the present study indicates a close relationship between the expression of the mRNA for type I collagen, the major structural constituent of fibrotic tissues, and the deposition of collagen in both the cortex and outer medulla of the kidney. Moreover, the clear differences found between old and young rat kidneys can serve as markers for renal aging and might explain at least some of the kidney impairments caused by fibrosis during senescence.

The effect of human growth hormone therapy on GH binding protein in GH-deficient children.

Previous studies have described the close similarity of the GH binding protein to the liver membrane GH receptor. Since GH regulates its own liver receptors, we examined the effects of short- and long-term hGH therapy on GH binding protein in children with GH deficiency. Six GH-deficient children received their first hGH dose ever, and the pharmacodynamics of serum GH was followed for 12 h, along with measurements of GH binding protein activity. Over the first 6 h, serum GH and GH binding protein activity exhibited a parallel increase, followed by gradual decrease. At 8 h, some of the patients exhibited an apparent second peak in GH binding protein, despite the continuous decrease in serum hGH. During the period of hGH treatment, serum GH binding protein increased progressively over a period of 6 months. In a second uncontrolled group of 7 GH-deficient patients who had been treated with hGH for 30-36 months, GH binding protein activity was also significantly higher than pretreatment values. We suggest that the short-term pharmacodynamic changes probably represent the endogenous turnover of the GH receptor, whereas the elevated GH binding protein activity on hGH treatment may reflect up-regulation of the GH receptor.

Neurosecretory dysfunction of growth hormone secretion in thalassemia major.

The growth retardation of children with thalassemia major is multifactorial. Along the endocrine axis of growth hormone (GH), serum somatomedin has been shown to be deficient and GH response to GH-releasing hormone impaired, while GH response to provocative stimuli is normal. We studied the spontaneous secretion of GH in seven patients with thalassemia major and growth retardation. Three of the patients were hypothyroid, and the other four were euthyroid. Spontaneous secretion of GH in all seven patients was subnormal: the number of pulses, the mean pulse amplitude, and the integrated concentration of GH were all lower than in 14 age- and sex-matched (10 pubertal and 4 prepubertal) control subjects. GH response to provocative stimuli was normal in the euthyroid patients. This pattern of response corresponds with the definition of neurosecretory dysfunction of GH secretion. It is concluded that the growth retardation of patients with thalassemia major is partly due to neurosecretory dysfunction of GH secretion.

Structural and immunological properties of myosin from human platelet external and internal membranes.

Our previous studies indicate that platelets contain two myosin isoforms, one of them localized in the membrane while the other in the cytoplasmic compartment. Structural and functional differences of these myosins have been characterized. In this study two platelet membrane subfractions, the external and the internal membranes, were isolated simultaneously from a crude membrane fraction and their purity was characterized using specific marker enzymes. Myosin was shown to be present in both membrane fractions and its structural and immunological properties were investigated. The electrophoretic mobilities of myosin in both membrane preparations were identical to the mobility of its cytoplasmic counterpart. Two-dimensional peptide mapping of the iodinated tryptic peptides of the myosin heavy chains indicated that at least one peptide is missing in the maps of the myosins from the external and internal membranes as compared to their soluble counterpart. Our data suggest that myosin is located in three distinct platelet compartments: cytosol, external and internal membranes. The same myosin isoform is located in the two membrane compartments, while the isoform found in the cytosol is different. The observed variations in the structure of the two isoforms may reflect differences in their respective physiological functions.

Antigen-induced secretion of histamine and the phosphorylation of myosin by protein kinase C in rat basophilic leukemia cells.

IgE-mediated stimulation of rat basophilic leukemia (RBL-2H3) cells results in the secretion of histamine. Myosin immunoprecipitated from these cells shows an increase in the amount of radioactive phosphate incorporated into its heavy (200 kDa) and light (20 kDa) chains. In unstimulated cells two-dimensional mapping of tryptic peptides of the myosin light chain reveals one phosphopeptide containing the serine residue phosphorylated by myosin light chain kinase. Following stimulation a second phosphopeptide appears containing a serine residue phosphorylated by protein kinase C. Tryptic phosphopeptide maps derived from myosin heavy chains show that unstimulated cells contain three major phosphopeptides. Following stimulation a new tryptic phosphopeptide appears containing a serine site phosphorylated by protein kinase C. The stoichiometry of phosphorylation of the myosin light and heavy chains was determined before and after antigenic stimulation. Before stimulation, myosin light chains contained 0.4 mol of phosphate/mol of light chain all confined to a serine not phosphorylated by protein kinase C. Cells that secreted 44% of their total histamine in 10 min exhibited an increase in phosphate content at sites phosphorylated by protein kinase C from 0 mol of phosphate/mol of myosin subunit to 0.7 mol of phosphate/mol of light chain and to 1 mol of phosphate/mol of heavy chain. When RBL-2H3 cells were made permeable with streptolysin O they still showed a qualitatively similar pattern of secretion and phosphorylation. Our results show that the time course of histamine secretion from stimulated RBL-2H3 cells parallels that of myosin heavy and light chain phosphorylation by protein kinase C.

Altered structural and functional properties of myosins, from platelets of idiopathic scoliosis patients.

Platelets of patients with idiopathic scoliosis (IS) have been shown to have decreased capacity to aggregate and secrete in response to certain agonists. Similarities between the contractile protein system of platelets and muscle have made the platelets a popular model for muscle disease. We attempted to characterize the function and structure of myosin in platelets of IS patients. Blood was obtained from seven IS patients and seven matched non-scoliotic healthy controls. The mean Cobb angle measurement of the IS patients was 35.4 degrees with a mean Risser sign of 2.2. Washed platelets were isolated from the blood, and the contractile proteins from the membrane and the cytosol compartments were isolated and analyzed by two-dimensional peptide mapping. As previously reported (J Biol Chem 258:9290, 1983), peptide maps of normal platelets revealed that the heavy chain of myosin located in the platelet membrane lacks one major spot relative to the cytoplasmic myosin. In IS patients the cytoplasmic myosin lacks the same peptide that is missing in the membrane myosin of normal individuals. In addition, the ATPase specific activity of the cytoplasmic myosin from IS platelets was significantly lower compared with the activity of the cytoplasmic myosin from normal platelets. These results suggest the presence of a fundamental abnormality of IS platelet contractile proteins.

The effect of antibodies to human platelet membrane and cytoplasmic myosins on myosin ATPase activity and platelet aggregation.

Myosins were purified from the membrane fraction and the cytoplasm of human platelets. Polyclonal antibodies to the purified myosins were induced in rabbits. Their effects on the ATPase activity of the purified myosins as well as on the process of platelet aggregation were studied. A strong cross reactivity was found between the two myosins and their respective antibodies by the ELISA technique. It was found that the antibodies preferentially bind to the "head" segment of the myosins, since purified myosin "rod" reacted only weakly with the two kinds of antibodies. The two antimyosin antibodies strongly inhibited the K+(EDTA) ATPase activity of both myosins, as well as the activity of the isolated myosin "heads". The amount of antimembrane myosin antibody required to inhibit the above enzymatic activity was smaller than that of the anticytoplasmatic myosin antibody. Similar results were observed with F(ab)2 fragments of the two kinds of antibodies. No effect of these antibodies or their F(ab)2 fragments was observed on platelet aggregation induced by various agonists, although their inhibitory effect on the platelet myosin ATPase activity was strong.

Immunohistochemical studies with antibodies to myosins from the cytoplasm and membrane fraction of human blood platelets.

Antibodies were raised to myosins extracted from the cytoplasm and solubilized membranes of human blood platelets. Both antibodies had similar titers as shown by enzyme-immunoassay and bound to the same sites as shown by immunohistochemistry. They were specific for cytoplasmic myosins (e.g., in human white blood cells, platelets and fibroblasts and rat endothelial cells). They showed no crossreaction with human or rat smooth muscle.

Is serum selenium a risk factor for cancer?

A narrow band of counties extending along the southeastern Atlantic coast from Jacksonville, Florida to Charleston, South Carolina were found to have excessively high incidence rates for esophageal cancer in non-white males. White males in the same areas have a 30% higher incidence rate for lung cancer but only average incidence rates were found for non-white males. Selenium is considered to decrease cancer risk in the animal model. In this coastal region, a study of 130 cancer patients who developed a malignancy 2-12 years after baseline examination showed no dose response relationship between baseline serum selenium levels and risk of subsequent cancer.

Serum retinol and risk of subsequent cancer: extension of the Evans County, Georgia, study.

A previous report from the Evans County, Ga., cohort correlated low base-line retinol levels in 1960-62 to an increased risk of cancer developing in the following 12-14 years. So that this inverse association could be retested, retinol determinations were performed on sera in 135 incident cancer cases, identified during a vital status follow-up in 1981, and in 237 controls matched by race, sex, age, and date of venipuncture. The serum was drawn during the second cohort examination between 1967 and 1969 and stored at -20 degrees C until analysis by high-pressure liquid chromatography in January 1982. Eligible for the study were patients whose initial cancer diagnosis was established more than 24 months after venipuncture. Base-line retinol levels of subsequent cancer cases and their matched controls were similar in the 4 race-sex groups. The risk of cancer at all sites associated with the lowest retinol quintile was similar to that associated with the highest quintile. In multivariate analysis, with control for age, cholesterol, body mass, smoking status, and social class, no significant relationship of serum retinol and case status was found. In summary, these data fail to confirm the strong dose-response relationship between baseline retinol levels and subsequent cancer reported in the previous study.

Characterization of the ATPase activities of myosins isolated from the membrane and the cytoplasmic fractions of human platelets.

Myosin was purified from the membrane fraction and the cytoplasm of human platelets, and the K+(EDTA)- and Ca2+-dependent ATPase activities were studied under various experimental conditions. The ATPase activity of the myosin from the membrane fraction was slightly lower than that of its cytoplasmic counterpart, regardless of the different assay conditions (pH, ionic strength, and temperature). Both myosins showed the same pH optima and a similar ionic strength dependence for the two ATPase activities measured. In addition, they exhibited the same substrate specificity using ATP, CTP, and GTP as substrates. The activation energy of the Ca2+-dependent ATPase activity was essentially the same for the two myosins, while the activation energy of the K+(EDTA)-dependent ATPase activity of the membrane myosin was higher than that of the cytoplasmic myosin. The ATPase activity of the membrane myosin was found to be more sensitive to freezing and thawing than the cytoplasmic myosin. The alkylation of the thiol groups by N-ethylmaleimide or N-iodoacetyl-N-(5-sulfo-1-naphtyl)ethylenediamine, and the trinitrophenylation of the lysyl residues by 2,4,6-trinitrobenzenesulfonate caused a significant decrease in the K+(EDTA)-dependent ATPase activity of the two myosins. However, the membrane myosin was much less affected than the cytoplasmic myosin. Actin induced inhibition of the K+ (EDTA) ATPase of both myosins, and much smaller quantities of actin were needed to inhibit the cytoplasmic myosin ATPase compared to quantities needed to inhibit the myosin ATPase from the membrane fraction. This indicates that the membrane myosin has a lower affinity toward actin. The observed variations in the ATPase activity of the myosins isolated from the membrane and the cytoplasm fractions of human platelets may reflect differences in their respective physiological functions.

Structural properties of myosin from the particulate fraction of human blood platelets.

Fractionation of human blood platelets has revealed that myosin, a contractile and mechanochemical protein, is present in both the soluble and particulate fraction. The aim of this study was to elucidate whether platelets contain more than one myosin isoform, especially in view of the fact that in other cellular systems (cardiac muscle, amoeba) several myosin isoenzymes were found. The particulate fraction was solubilized by Triton X-100, and the myosin was purified by the same procedure used for the cytoplasmic myosin. The final preparation contained, in addition to myosin, a 130-kDa polypeptide, which was observed also in myosin preparations obtained from the soluble fraction. The electrophoretic mobilities of the two myosins were identical under both dissociating and nondissociating conditions. Comparison of the molecular structure of the heavy chain of the two myosins by limited proteolysis with Staphylococcus aureus V8 protease showed that the proteolytic fragments of the two myosins were rather similar, with only minor alterations in the quantitative distribution of the products. Two-dimensional peptide mapping of the iodinated tryptic peptides of the myosin heavy chains indicated that at least one peptide is missing in the map of the particulate myosin, as compared to its soluble counterpart. According to the two-dimensional peptide map, the 130-kDa polypeptide seems to be a proteolytic fragment of the myosin heavy chain and most probably the rod portion of the molecule. The observed minor variations in the structure of myosins isolated from the soluble and the fractions of human platelets may reflect differences in their respective physiological functions.