The presence of two reduced function variants in CYP2C9 influences the acute response to glipizide.

AIMS: To examine whether the presence of two common missense variants in the CYP2C9 gene (rs1799853, encoding Arg144Cys and denoted as *2, and rs1057910, encoding Ile359Leu and denoted as *3) influences the acute physiological response to a single glipizide dose in individuals naïve to diabetes medications. METHODS: In the Study to Understand the Genetics of the Acute Response to Metformin and Glipizide in Humans (SUGAR-MGH), 786 individuals genotyped for rs1799853/rs41291560 (*2) and rs1057910/rs9332214 (*3) were treated with 5 mg glipizide in the fasting state. Glucose and insulin levels were measured at baseline, 30, 60, 90, 120, 180 and 240 min for calculation of phenotypic endpoints of glipizide response. The challenge was aborted as a result of hypoglycaemia, defined as glucose <2.8 mmol/l or hypoglycaemia-related symptoms. RESULTS: Carriers with two reduced function alleles had a 50% larger insulin area under the curve than carriers with zero or one copy (P=0.037), although this finding was primarily driven by an individual with a robust insulin response. In adjusted analyses, the risk of aborting the glipizide challenge was doubled in two-copy carriers (P=0.034). No significant findings were observed in glucose-based endpoints. CONCLUSIONS: Carriers of two reduced function alleles in CYP2C9 may experience an increased insulin response to glipizide and be predisposed to a higher risk of hypoglycaemia, although no effect of genotype was seen in glucose-based measurements. Further studies are needed to clarify the utility of CYP2C9 genotyping to guide sulfonylurea treatment.

A reference high-pressure CO2 adsorption isotherm for ammonium ZSM-5 zeolite: results of an interlaboratory study.

This paper reports the results of an international interlaboratory study led by the National Institute of Standards and Technology (NIST) on the measurement of high-pressure surface excess carbon dioxide adsorption isotherms on NIST Reference Material RM 8852 (ammonium ZSM-5 zeolite), at 293.15 K (20 °C) from 1 kPa up to 4.5 MPa. Eleven laboratories participated in this exercise and, for the first time, high-pressure adsorption reference data are reported using a reference material. An empirical reference equation n e x = d ( 1 + exp [ - ln ( P ) + a / b ] ) c , [n ex -surface excess uptake (mmol/g), P-equilibrium pressure (MPa), a = -6.22, b = 1.97, c = 4.73, and d = 3.87] along with the 95% uncertainty interval (U k = 2 = 0.075 mmol/g) were determined for the reference isotherm using a Bayesian, Markov Chain Monte Carlo method. Together, this zeolitic reference material and the associated adsorption data provide a means for laboratories to test and validate high-pressure adsorption equipment and measurements. Recommendations are provided for measuring reliable high-pressure adsorption isotherms using this material, including activation procedures, data processing methods to determine surface excess uptake, and the appropriate equation of state to be used.

Abnormal results of dexamethasone suppression tests in nondepressed patients with diabetes mellitus.

To investigate the specificity of the dexamethasone suppression test (DST) for the diagnosis of major depression in patients with diabetes mellitus, we administered 1 mg of dexamethasone to 30 nondepressed diabetics and to 58 normal controls at 11 PM. Diabetic subjects received hemoglobin A1 (Hb A1) determinations, the Hamilton Rating Scale for Depression (HRSD), and five to eight blood glucose determinations during the 48 hours surrounding the DST. Results demonstrated a significantly higher rate of nonsuppression (plasma cortisol level, greater than or equal to 5 micrograms/dL) at 4 PM the following day among diabetics (43%) than among controls (7%) but no difference between these groups in the rate of nonsuppression at 8 AM. Plasma cortisol level at 4 PM correlated with Hb A1 level but not with duration of illness, HRSD score, mean blood glucose level, or maximum blood glucose excursion. These results suggest that the results of the DST used as a diagnostic test for major depression must be interpreted with caution in patients with diabetes.

Cetuximab directly inhibits P-glycoprotein function in vitro independently of EGFR binding.

PURPOSE: Cancer chemotherapy typically combines anticancer drugs from different mechanisms of action. However, cancer cells could become resistant to chemotherapy via P-gp or other ATP binding cassette proteins. The objective of this study was to evaluate whether cetuximab, monoclonal antibody directed toward epidermal growth factor receptor, could increase intracellular concentration of conventional chemotherapy by interacting with P-gp. METHODS: Two human ovarian carcinoma (IGROV1) and two human embryonary kidney (HEK) cell lines, overexpressing or weakly expressing P-gp, were used. Their EGFR expressions were compared. Cetuximab effect on P-gp functionality was evaluated by measuring doxorubicin (P-gp fluorescent substrate) intracellular accumulation. Cetuximab ability to increase doxorubicin cytotoxicity was evaluated by MTT test. A quaternary structure model of the P-gp-Cetuximab complex was established. RESULTS: Exposure of cetuximab in therapeutic concentrations range with doxorubicin led to significant doxorubicin accumulation and reversion of doxorubicin resistance in P-gp expressing cells lines. Molecular modeling of P-gp-cetuximab interactions showed that cetuximab is able to bind P-gp extracellular part. CONCLUSIONS: Cetuximab increases a P-gp substrate intracellular accumulation in both P-gp expressing cell lines, independently of their EGFR expression. One hypothesis is that cetuximab binding on P-gp could hamper the conformational changes that occur during drugs efflux. Our results offer new possibilities of research on monoclonal antibodies influence in MDR phenomena.

Phenomenology and family history of affective disorder in Cushing's disease.

Of 16 patients with Cushing's disease, 13 (81%) had lifetime diagnoses of major affective disorder according to DSM-III criteria. However, the rate of familial major affective disorder among these patients was significantly lower than that found among patients with major depression.

Fibromyalgia and major affective disorder: a controlled phenomenology and family history study.

Fibromyalgia is a form of nonarticular rheumatism characterized by diffuse musculoskeletal pain. To investigate the relationship between fibromyalgia and major affective disorder, the authors evaluated 31 patients with fibromyalgia and 14 patients with rheumatoid arthritis for rates of current or past major affective disorder and family history of major affective disorder. Both the rate of major affective disorder and the familial prevalence of major affective disorder were significantly higher in the fibromyalgia patients than the rheumatoid arthritis patients. The results suggest that fibromyalgia may be related to major affective disorder.

Prevalence of anorexia nervosa and bulimia among young diabetic women.

To assess the prevalence of anorexia nervosa and bulimia among young diabetic women, a questionnaire was sent to 264 young women with insulin-dependent diabetes mellitus in two clinical settings. Among the women who responded (30%), none reported a history of anorexia nervosa, but 28 (35%) reported a history of bulimia. These results suggest that bulimia represents a common problem among young women with diabetes.

Hypothalamic-pituitary-adrenal-axis hyperactivity in bulimia.

Hyperactivity of the hypothalamic-pituitary-adrenal axis, demonstrated by nonsuppression of plasma cortisol in the dexamethasone suppression test (DST), has been found in about 50% of patients with major depression. We administered the DST to 47 patients with bulimia and to 22 age- and sex-matched normal controls. Among the bulimics, 47% were nonsuppressors, significantly higher than the 9% prevalence of nonsuppressors in the controls, but similar to the prevalence reported for patients with major depression in other studies. This finding is consistent with evidence from studies of phenomenology, family history, and treatment response which suggest that bulimia may be related to affective disorder.

Free-radical damage by UV or hypoxanthine-xanthine oxidase in cultured human skin fibroblasts: Protective effects of two human plasma fractions.

Two in vitro methods to investigate free radical damage to cultured human skin fibroblasts have been used: irradiation with UVA or UVB, producing intracellular free radicals and DNA damage, or free radical production by the enzymatic system hypoxanthine-xanthine oxidase, releasing free radicals into the culture medium. These methods differ not only in the location of the free radicals generated but also in their nature and kinetics. The antioxidant properties of two human plasma extracts A and B (derived from Cohn's fraction IV and Cohn's fraction I + III) were investigated before, during and/or after the oxidative stress. Protection was observed when the fractions were added concomitantly with the enzymatic system (at 5 g/litre, fractions A and B exhibited, respectively, 77 and 50% activity) or during UVB irradiation (37 and 68% activity for fractions A and B, respectively at 5 g/litre). A small degree of protection was observed against UVA damage. No preventive or restorative effect was observed with the UVB system. The two fractions prevented UVA damage (at 2.5 g/litre, fraction A and B elicited 22 and 23% activity, respectively) but only fraction A also exhibited a restorative effect (at 5 g/litre, activity was 26%). One of the protective mechanisms could be the enhancement of intracellular antioxidant enzyme activity by incubation of cells with fractions A and B (after 24 hr of contact with fraction B, total glutathione peroxidase and glutathione peroxidase Se-dependent activities were significantly increased by 60 and 42%, respectively, compared with control values).

Effect of biophysical changes on propidium iodide access to DNA during oxidative stress of cultured human skin cells.

The sensitive single-cell analytical techniques of flow cytometry and propidium iodide-binding have been used to examine the molecular effect of oxidative stress on cultured human skin fibroblasts. Cells synchronized by limited time attachment were exposed to a hypoxanthine-xanthine oxidase (HX/XO) system at different intervals after subculture. The characteristic feature of the treated population was a variation of the amount of nuclear DNA propidium iodide (PI)-fluorescence staining. Increased fluorescence intensity was observed with a shift of the G1/G0 and G2/M peak, which is dependent on both cell cycle stage and treatment level. When scavenger molecules (catalase, silybin) were added to the oxidative reaction, the nuclear DNA histogram of HX/XO-treated cells was similar to that obtained from untreated cells. In parallel, UV absorbance studies in vitro have shown that PI is capable of binding extensively to DNA when isolated from HX/XO-exposed cells, compared with control cells or HX/XO-exposed cells in the presence of scavengers. These results indicate that free radicals are responsible for the increase in fluorescence intensity in the HX/XO-exposed cells. This change in DNA stainability would be due to an opening of the DNA strands in situ, leading to an unmasking of new PI-binding sites on DNA. The strand separation may facilitate access on the fluorochrome to DNA, thereby enhancing dye binding. This flow cytometric assay based on DNA biophysical changes caused by free radicals is a useful means of measuring pro- and/or anti-oxidant potential.

Comparison of six different methods to assess UVA cytotoxicity on reconstructed epidermis. Relevance of a fluorimetric assay (the calcein-AM) to evaluate the photoprotective effects of alpha-tocopherol.

A three-dimensional culture of human keratinocytes exposed at the air-liquid interface has been developed and used in conjunction with fluorimetric, colorimetric and radioligand incorporation assays to assess the in vitro toxicity of UVA. The aims of the study were: (1) to compare the relevance of the neutral red uptake (NR), MTT metabolism, (35)S-methionine incorporation, IL1-alpha release and calcein-AM esterification assays for the evaluation of UVA injury; (2) to test the preventive protective effect of an emulsion containing 3% of tocopherol applied on the reconstructed epidermis, in comparison with an application of tocopherol 3% diluted in culture medium either on the apical compartment or in the underneath compartment of the skin culture insert. Viability measurement methods are based on different endpoints. None of the five endpoints measured produced LD(50) values (40 J/cm(2)) that differed significantly from the others. However, calcein-AM assay was relatively more reproducible and easier to handle than the others, and seemed to be a better choice for the evaluation of the protective effects of the tocopherol emulsion. Tocopherol diluted in culture medium under the epidermis 24 hr before irradiation failed to protect the epidermis against UVA damage, whereas diluted in culture medium or in oily emulsion and applied to the epidermis reduced cellular death (cellular recovery values are, respectively, 24% and 21%). Since cosmetic or pharmaceutical formulations can be directly applied on the reconstructed epidermis as in vivo, this model in combination with a fluorescent viability assay appears to be a suitable approach for pharmaco-toxicological evaluations.

In vitro cytotoxic effects of enzymatically induced oxygen radicals in human fibroblasts: Experimental procedures and protection by radical scavengers.

Introduction of hypoxanthine and xanthine oxidase into human fibroblast cultures induces a dose-dependent cytotoxicity as a result of free-radical formation. The influence of medium, cell density and the power of recovery after free-radical attack were investigated. It appears that toxicity is higher in physiological Dulbecco phosphate buffer or Hanks' balanced salt solution than in modified Eagle medium, is inversely proportional to cell density and that damage is most often irreversible. Using this model, we studied the protective effects of a hydrosoluble flavonoid, silybin, and of a well known antioxidant, BHT (butylated hydroxytoluene). These molecules were administered before and during free-radical attack. With BHT significant protection was observed when it was added before free-radical attack (24% protection at a concentration of 10(-4)m) and before and during exposure (20% protection at a concentration of 10(-5)m). When silybin is applied during radical attack maximal activity is recorded at a concentration of 8 x 10(-4)m (45%), but the most interesting results are observed when 1 x 10(-4) and 8 x 10(-4)m are used, respectively, before and during radical exposure (63% of activity).

Human epidermis reconstructed on synthetic membrane: influence of experimental conditions on terminal differentiation.

Cell suspensions of human keratinocytes seeded onto cell culture inserts may undergo terminal differentiation in the absence of fibroblasts. Among the parameters that control these morphogenic events, exposure to air and the composition of the culture medium were investigated. In the latter case, three media were considered DMEM:Ham's F12, MCDB 153, and keratinocyte SFM medium at equivalent calcium (1.5 mM) and fetal calf serum (5%) concentrations. Immunochemical methods and transmission electron microscopy show that cells cultured in DMEM:Ham's F12 medium, and then raised at the air-liquid interface, form a basal layer plus suprabasal cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasal keratinocyte layers show morphologies that resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-filaggrin granules. When the cultures are kept submerged, the keratinocytes show occasional keratohyalin granules and are connected by fewer desmosomes. Additionally, no proper stratum corneum is formed. In keratinocyte SFM medium and MCDB 153, cultures raised at the air-liquid interface are not able to form an epithelium of normal architecture and do not express terminal differentiation markers. Differentiation is initiated, however, since desmosomes and bundles of keratin filaments appear; on the other hand, filaggrin is not expressed even after 28 d in culture. Membrane-bound transglutaminase is expressed throughout the entire suprabasal compartment in MCDB153 and DMEM:Ham's F12 media but never appears in keratinocyte SFM medium. These studies show the relative independence of epidermal differentiation program to the composition (including the calcium concentration) of the media contacting the dermis and filling the extracellular space.(ABSTRACT TRUNCATED AT 250 WORDS)

Disposition of everolimus in mdr1a-/1b- mice and after a pre-treatment of lapatinib in Swiss mice.

The aim of this study was to document the in vivo transport of everolimus (inhibitor of mTOR) by P-glycoprotein (P-gp), and to investigate the influence of lapatinib (inhibitor of P-gp) on everolimus disposition. Pharmacokinetics of everolimus (0.25mg/kg) has been investigated after oral administration in mdr1a-/1b- mice compared to the wild type. Also, everolimus pharmacokinetics was characterized after oral administration on Swiss mice either alone or after 2 days of pre-treatment of lapatinib (200mg/kg). The influence of lapatinib pre-treatment on intestinal P-gp expression was investigated by Western blot analysis. The non-compartimental analysis was performed using Winonlin professional version 4.1 software (Pharsight, Mountain View, CA). The areas under the plasma concentration-time curve (AUC) were compared using Bailer's method. A significant 1.3-fold increase of everolimus AUC observed in mdr1a-/1b- mice suggested that everolimus is transported in vivo by intestinal P-gp in mice. In addition, a 2.6-fold significant increase of everolimus AUC with lapatinib pre-treatment as compared with the everolimus alone group was noticed. The elimination half-life was comparable (t(1/2)=5.3h vs. t(1/2)=4h). A 38.5% significant decrease of P-gp expression was observed in duodenum segment in lapatinib pre-treated group as compared with control group. In conclusion, lapatinib enhanced everolimus absorption by decreasing intestinal P-gp expression. An inhibition of CYP 450 could not be excluded. These results confirm the necessity of a therapeutic monitoring of everolimus combined with an inhibitor of the P-gp and CYP 450 like lapatinib in a future anti-tumor treatment.

Endocrine dysfunction in anorexia nervosa and bulimia: comparison with abnormalities in other psychiatric disorders and disturbances due to metabolic factors.

The eating disorders of anorexia nervosa and bulimia are associated with marked disturbances in endocrine function. Studies of the hypothalamic-pituitary-adrenal,-thyroid, and -ovarian axes are reviewed, in order to ascertain whether and to what extent alteration in endocrine response is the consequence of abnormal eating, or whether the endocrine abnormalities are primary. Many, if not all, of the disturbances which have been documented can be accounted for the metabolic consequences of disturbed eating behavior, including especially the effects of weight loss. However, it is possible that primary hypothalamic dysfunction may account for or contribute to the following abnormalities: hypercortisolism, blunted TSH response to TRH, and hypogonadism. Given the possibility that primary hypothalamic disturbances are present, the specificity of these disturbances with respect to eating disorders as opposed to other psychiatric disorders, is investigated. It is concluded that, with the possible exception of alterations in the hypothalamic-pituitary-ovarian axis, such disturbances appear not to be specific to eating disorders; rather, they are also present in other psychiatric disorders. In particular, the data reviewed are consistent with the hypothesis that the eating disorders have psychobiological features in common with major affective disorder.

Tubulosine: an antitumor constituent of Pogonopus speciosus.

From the antitumor-bioactive sap of Pogonopus speciosus, tubulosine [1] was isolated, by activity-directed fractionation using the brine shrimp lethality test, as the major antitumor constituent. 1H-nmr assignments, obtained from HETCOR and COSY, and X-ray crystallographic results are reported for the first time. Psychotrine [2] was also isolated, and its spectral data are also reported.

Influence of fibroblasts on epidermization by keratinocytes cultured on synthetic porous membrane (insert) at the air-liquid interface.

Culture of keratinocytes on a noncoated porous synthetic membrane maintained at the air-liquid interface allows the establishment of a fibroblast/keratinocyte co-culture, without direct cell-cell contact between the two cellular layers. The influence of fibroblasts (proliferating, confluent or blocked by mitomycin C) on epidermization (i.e., expression of integrins and markers of epidermal differentiation) was studied by immunohistochemistry in two culture media. In the medium supplemented with FCS or Ultroser G and in the absence of fibroblasts, alpha 2, alpha 3, alpha 5 and alpha 6 subunits of integrins are expressed by the basal keratinocytes, except alpha 5 which does not appear with the medium supplemented with Ultroser G. During stratification, the alpha 3 subunit is the only one to persist on suprabasal cells and all the markers of epidermal differentiation studied (filaggrin, involucrin, transglutaminase, keratins K1/K10) are expressed at the 14th day of emerged culture. The presence of fibroblasts modifies the expression profile of integrins: when they are proliferative, the expression of alpha 2 and alpha 6 chains is delayed in the medium supplemented with FCS, and the alpha 6 chain is absent in the medium supplemented with Ultroser G; when they are confluent or blocked by mitomycin C, greater changes are observed only in the medium supplemented with Ultroser G and lead to inhibition or delay of the expression of alpha 2 and alpha 6.(ABSTRACT TRUNCATED AT 250 WORDS)

Activity of Vittel water on proliferation of human fibroblasts, proliferation and differentiation of human keratinocytes.

Synopsis The effects of Vittel water on the proliferation of fibroblasts, proliferation and differentiation of keratinocytes from human origin were studied. To determine the relative importance of calcium and other elements of the mineral water, cultures were raised in a low-calcium medium (low-Ca medium), in a medium prepared with Vittel water (Vittel medium) and in a medium containing an identical calcium concentration to that of Vittel medium (Ca medium). The fibroblasts and keratinocytes were cultured in immersion for proliferation assays and on a reconstructed epidermis at the air-liquid interface to evaluate keratinocyte differentiation. Vittel medium decreased proliferation of keratinocytes when compared to low-Ca medium. The effect was similar to that of Ca medium at the beginning of the experiment, but significantly higher at day 7. A stratified epithelium appeared with the three types of media when keratinocytes were incubated at the air-liquid interface; however the number of sheets was more regular and greater in Vittel medium and Ca medium than in low-Ca medium. Filaggrin and transglutaminase expression appeared earlier with Vittel medium than with the other media. After 2 weeks, expression of markers was similar in the three media. After 3 weeks culture in Vittel medium, there was a greater expression of filaggrin. Proliferation of young fibroblasts was significantly higher in Vittel medium than in Ca medium. It was lower in low- Ca medium. With old fibroblasts the degree of proliferation was lower than with young fibroblasts. The augmentation of proliferation happen earlier in Vittel medium than in low-Ca medium and Ca medium. Vittel medium regulated the growth rate of old fibroblasts, rendering it identical to that of young fibroblasts in low-Ca medium. The effects of Vittel water were not linked to the sole presence of calcium since, with medium at an equimolecular concentration in calcium, the medium containing Vittel water had a better activity. One explanation of these effects of Vittel water might be the presence of magnesium.

Protective effect of curcuminoids on epidermal skin cells under free oxygen radical stress.

Curcuminoids from Curcuma longa L. (Zingiberaceae) protected normal human keratinocytes from hypoxanthine/ xanthine oxidase injury. Since curcuminoids synergistically inhibited nitroblue tetrazolium reduction, a decrease in superoxide radical formation leading to lower levels of cytotoxic hydrogen peroxide was proposed as an explanation for this protective effect.