Reduced Activity of Anabolizing Enzymes in 5-Fluorouracil-resistant Human Stomach Cancer Cells

The mechanism of resistance to 5-fluorouracil (5-FU) was studied with NUGC-3/5FU/L, a human stomach cancer cell line which had acquired resistance as a consequence of repeated 5-day exposures to stepwise-increasing concentrations of 5-FU in vitro . NUGC-3/5FU/L was 200-fold and over 16-fold resistant to 96-h and 1-h exposures to 5-FU, respectively. NUGC-3/5FU/L incorporated less 5-FU into RNA, indicating resistance to the RNA-directed action of 5-FU. On the other hand, NUGC-3/5FU/L also showed resistance to in situ thymidylate synthase (TS) inhibition by 5-FU. Polymerase chain reaction-single-strand conformation polymorphism analysis of TS cDNA and a FdUMP ligand binding assay showed that quantitative and qualitative alterations of TS are not responsible for this resistance. In contrast, the ability to metabolize 5-FU to its active metabolites, FUTP and FdUMP, was reduced in NUGC-3/5FU/L. We found that not only the activities of uridine phosphorylase/kinase and orotate phosphoribosyl-transferase (OPRT), but also the level of phosphoribosyl pyrophosphate, a cosubstrate for OPRT, were significantly lower in NUGC-3/5FU/L than in the parent NUGC-3. These results indicated that resistance to 5-FU in NUGC-3/5FU/L is due to reduced activities of 5-FU-anabolizing enzymes, but not to an alteration of TS. 2'-Deoxyinosine effectively enhanced TS inhibition by 5-FU in the resistant cells, thus markedly sensitizing them to 5-FU.     

Effect of toxaphene on isolated hepatocytes of the yellowtail flounder, Pleuronectes ferrugineus storer

The histochemical and enzyme cytochemical effects of Toxaphene were investigated using isolated hepatocytes in suspension culture from laboratory-bred juvenile, female yellowtail flounder (Pleuronectes ferrugineus). Hepatocytes were kept in suspension culture for 4 days and exposed for 3 days to a control medium, to a medium with hexane (the solvent of Toxaphene), or to a medium with Toxaphene in two different concentrations (1 and 10 mocrog/ml). Subsequently, the cultivated cells were examined histochemically (Sudan black B, oil red O, Schmorl's reaction) and enzyme cytochemically (acid phosphatase, NADPH-ferrohemoprotein reductase). Toxaphene decreased the viability of the isolated cells significantly, as compared to the control suspensions. Toxaphene also increased the storage of total and neutral lipids (as demonstrated by Sudan black B and oil red O, respectively) in a dose-dependent manner. In addition, Toxaphene increased the enzymatic activity of acid phosphatase, and increased the storage of lipofuscin pigment (as demonstrated by the Schmorl's reaction) within the hepatocytes, suggesting an increase in the number and/or size of the lysosomes. Hexane did not have a significant toxic effect on the isolated hepatocytes. It is concluded that Toxaphene is potentially toxic to fish in a marine environment and that this in vitro system may provide a model for assessing the direct effect of various toxicants on fish hepatocytes.     

Outcome of Displacement Bone Marrow Transplantation for Inborn Errors of Metabolism

The concept of displacement bone marrow transplantation arose from our work at Westminster 1970–1973, by which time we had extended the donors from matched siblings to other family and unrelated donors. DBMT is not a panacea, but can be applied to about 7% of understood inborn errors, where it is possible to devise in vitro tests to predict in vivo donor effects. Its use to install donor bone marrow as a component factory for the life of the recipient, and the importance of immunoprophylaxis are summarised. Worthwhile correction has been achieved for 48 previously fatal genetic diseases, partial correction for another five, but there has been failure for three diseases. Some 80% of our patients were not found in known families and could not have been prevented.     

Urine Growth Hormone Determinations Compared with Other Methods in the Assessment of Growth Hormone Secretion

Okuno, A., Yano, K., Itoh, Y., Hashida, S., Ishikawa, E., Mohri, Z-I. and Murakami, M. (Department of Paediatrics, Asahikawa Medical College, Hokkaido; Medical College of Miyazaki, Kiyotake, Miyazaki; and Research and Development Division, Sumitomo Pharmaceuticals Co Ltd, Hyogo, Japan). Urine growth hormone determinations compared with other methods in the assessment of growth hormone secretion. Acta Paediatr Scand [Suppl] 337:74, 1987.
Urinary excretion of hGH was studied in children with short stature using a sensitive sandwich enzyme immunoassay technique. Urinary hGH excretion, in terms of hGH: creatinine ratio, showed excellent correlation with the mean and peak hGH values during physiological and pharmacological tests. It seems that the urinary hGH levels reflect serum hGH profiles during the urine collection period. A border zone for the lower limits of normal hGH levels in the urine was 7.5–13.4 ng/g creatinine for the physiological test at night (from 2000 hours to 0600 hours) and 17.4–35.0 ng/g creatinine for the pharmacological tests. Assessment of hGH secretory status by the urinary hGH levels showed good agreement with the serum hGH response. Measurement of urinary hGH could be used as a diagnostic test for impaired hGH secretion, and the multiple blood drawing required in physiological and pharmacological tests might be replaced by urine sampling.     

G6PD deficiency in newborn infants

Five hundred consecutive newborns were screened for erythrocytic G6PD deficiency in cord blood. The overall incidence of G6PD deficiency was found to be 2.80 percent. The incidence of G6PD deficiency was higher among males (3.77%) compared to females (1.44%). The incidence of erythrocytic G6PD deficiency was higher in Muslims (16.67%) compared to Hindus (2.63%). No definite relationship of erythrocytic G6PD deficiency was observed with consanguinity. Fifty per cent mothers of G6PD deficient newborns were also found to be G6PD deficient. Among brothers and sisters of G6PD deficient children the incidence of G6PD deficiency was 50.00 and 9.10 per cent respectively. There was no significant difference in the incidence of hyperbilirubinemia between erythrocytic G6PD deficient and non deficient newborns.     

Disabling symptoms: what do older women report?

OBJECTIVE: To answer the question, “What do older disabled women report as the main symptoms causing their disability?” DESIGN: Cross-sectional study of 876 women aged 65 and older who participated in the second interview of the Women’s Health and Aging Study, a longitudinal study of community-living women, representing the one third of older women with at least mild to moderate disability. MEASUREMENTS AND MAIN RESULTS: Women were asked to identify the symptom and the condition that was the main cause of disability in basic and instrumental activities of daily living, and lower extremity mobility. Musculoskeletal pain symptoms were reported as the main cause of disability by at least one third of women with each type of disability. Other symptoms that were less frequently reported as main causes of disability were weakness, fatigue, and unsteadiness. Fear of falls was reported by 14% (95% confidence interval, 11.2% to 17.6%) of 472 women with disability in bathing. When asked to report on the main condition causing their disability, many women responded, “old age” or “no specific disease,” but were able to identify symptoms causing their disabilities. CONCLUSIONS: Musculoskeletal pain was the most common cause of disability reported by older women, followed by weakness and balance difficulties. Greater attention to symptoms that interfere with daily activities of older persons may reduce the burden of disability.     

Ecto-diadenosine polyphosphates hydrolase activity on human prostasomes

BACKGROUND: Ecto-diadenosine polyphosphates are ubiquitous compounds with several physiological roles. Ecto-diadenosine polyphosphates hydrolase control their actions by degrading and terminating their signaling. The present work deals with the identification and partial characterization of ecto-diadenosine polyphosphates hydrolase on human prostasomes. METHODS: Reverse-phase and paired-ion HPLC techniques have been used. RESULTS: Prostasomes have an ecto-diadenosine polyphosphates hydrolase that leads to the degradation of several diadenosine compounds. Kinetic parameters of the enzyme show that diadenosine tetraphosphate is the preferred substrate that is further metabolized by the prostasome-ecto-nucleotidases to adenosine. The ecto-enzyme is bound to the prostasome-membranes through a GPI-anchor and is activated by physiological concentration of Ca+2, Mg+2, and Mn+2. Its optimum pH is also in the slightly alkaline physiological range. Human spermatozoa do not possess this hydrolytic activity, but they can acquire it after fusion with prostasomes. CONCLUSIONS: The existence of an enzyme capable of degrading diadenosine compounds and can be transferred to human spermatozoa suggests new physiological implications for the role of prostasomes in fertilization.     

Differences in caffeine and paraxanthine metabolism between human and murine CYP1A2

For the characterisation of murine models of CYP1A2 mediated metabolism in humans we compared the metabolism of caffeine and paraxanthine in human liver microsomes (LM) (two samples) and in LM from CYP1A2-null and wild-type mice. Inhibition experiments were carried out with the quinolones norfloxacin and pefloxacin and the substrate, caffeine. Additionally, in vivo pharmacokinetics of paraxanthine was determined in CYP1A2-null and wild-type mice. All LM produced the primary metabolites of caffeine and paraxanthine. In human LM, the main metabolite of caffeine was paraxanthine (K(M) 0.4 and 0.5 mmol L(-1)). In wild-type and CYP1A2-null mice LM, the main caffeine metabolite was 1,3,7-trimethylurate, but formation was not saturable. Apparent K(M) for paraxanthine formation from caffeine in wild-type and CYP1A2-null murine LM were 0.2 and 4.9 mmol L(-1), respectively. The main metabolite of paraxanthine was 1-methylxanthine in human (K(M) 0.13 and 0.2 mmol L(-1)) and in wild-type mice LM (K(M) 0.53 mmol L(-1)). In CYP1A2-null murine LM, the main paraxanthine metabolite was 7-methylxanthine. The quinolones competitively inhibited caffeine metabolism in human but not in wild-type or CYP1A2-null murine LM. No obvious differences were seen for blood pharmacokinetics and urinary metabolite excretion of paraxanthine between CYP1A2-null and wild-type mice. Thus, for paraxanthine, norfloxacin and pefloxacin interaction with CYP1A2 there were clear differences between mice and man. Our results suggest that an interspecies comparison is required for the metabolism of individual xenobiotics interacting with CYP1A2 prior to the use of mice models to predict its toxicity and/or pharmacological activity in man.     

Quinapril treatment restores the vasodilator action of insulin in fructose-hypertensive rats

1. Angiotensin-converting enzyme (ACE) inhibitors have been shown to improve insulin-resistance both experimentally and clinically. We therefore investigated the effects of quinapril, which has high tissue specificity for ACE, regarding the contribution of insulin to vascular contractions, as well as insulin sensitivity in a dietary rat model of insulin resistance. 2. Male Sprague-Dawley rats were divided into three groups: (i) rats fed normal chow (normal diet group); (ii) rats fed fructose-rich chow containing 40% fructose and 7% lard (fructose diet group); and (iii) rats fed fructose-rich chow plus quinapril (10 mg/kg per day; quinapril-treated group). 3. After 2 weeks, we evaluated systolic blood pressure, insulin sensitivity as assessed by steady state plasma glucose (SSPG) levels, response of aortic rings to phenylephrine (10-9 to 10-6 mol/L) in the presence or absence of insulin and the response of aortic rings to acetylcholine. 4. Feeding rats fructose-rich chow resulted in an elevation of blood pressure (P < 0.01) and SSPG levels (P < 0.01). Quinapril treatment significantly prevented increases in both blood pressure and SSPG, with a return to the levels seen in the normal diet group. 5. In the absence of insulin, the maximal contractile response to phenylephrine did not differ between the three groups. However, in the presence of insulin (100 mU/mL), the contractile response to phenylephrine (10-6 mol/L) was reduced by 22.8 +/- 1.2% in the normal diet group, although no insulin effects were observed in the fructose diet group (P < 0.01). Quinapril restored the inhibitory effect of insulin on phenylephrine-induced contractions. 6. In addition, the reduction in relaxation induced by acetylcholine in the fructose diet group was significantly reversed by quinapril treatment. 7. It is concluded that the fructose diet impairs the vasodilator effects of insulin as well as acetylcholine-induced relaxation in rat thoracic aortas. Quinapril prevented deterioration in the responses of the aortic rings, suggesting that ACE inhibitors may be useful for treating vascular insulin resistance.     

The impact of antihypertensive drug groups on urinary albumin excretion in a non-diabetic population

AIMS: Microalbuminuria (30-300 mg 24 h-1) is recognized to be independently associated with renal and cardiovascular risk. Antihypertensives may lower microalbuminuria. We questioned whether the use of different antihypertensive drug classes in general practice influences microalbuminuria as related to blood pressure in nondiabetic subjects. METHODS: To study this, we used the data from 6836 subjects of an on-going population based study, focused on the meaning of microalbuminuria (PREVEND). Odds ratios, adjusted for age, sex, blood pressure, cholesterol level, smoking and the use of other antihypertensive or cardiovascular drugs, were calculated to determine the association of drug groups with microalbuminuria. Influence of antihypertensives on the relation between blood pressure and (log) urinary albumin excretion was determined by comparing linear regression lines. RESULTS: Microalbuminuria was significantly associated with the use of dihydropyridine calcium channel blockers (odds ratio: 1.76 [1.22-2.54]), but not with other antihypertensive drug groups. The linear regression line of the relation between blood pressure and (log) urinary albumin excretion was significantly steeper (P = 0.0047) for users of calcium channel blockers, but not for other antihypertensives, compared with subjects using no antihypertensive. Users of a combination of renin-angiotensin system inhibitors and diuretics however, had a less steep regression line (P = 0.037). CONCLUSIONS: This study suggests a disadvantageous effect of dihydropyridine calcium channel blockers on microalbuminuria compared with other antihypertensive drug groups. Thus, if microalbuminuria is causally related to an increased risk for cardiovascular morbidity and mortality, dihydropyridines do not seem to be agents of choice to lower blood pressure. Furthermore, the combination of renin-angiotensin system inhibition and diuretics seems to act synergistically.