Effect of nutritional rehabilitation on circulating ghrelin and growth hormone levels in patients with anorexia nervosa

Circulating ghrelin and growth hormone (GH) are up-regulated in anorexia nervosa (AN) as a consequence of prolonged starvation. The current study examines the effect of nutritional rehabilitation with improvement of eating behavior on ghrelin and GH levels in AN patients during the course of inpatient treatment. The subjects included 34 female AN patients and 9 age-matched female controls. Fasting blood samples were collected before, during and after treatment. For data analysis, AN subjects were divided into three subtypes. The first group included seven patients with emergent hospitalization (E-AN), who were accompanied by severe emaciation due to their inability for food intake for more than a month. The other two groups included 14 AN with restricting (AN-R) and 13 AN with binge-eating/purging (AN-BP) patients. There were significant correlations between ghrelin, GH and body mass index (BMI) before treatment in all subjects. However, ghrelin levels were not significantly correlated with BMI and GH although there was a relationship between GH and BMI after treatment. Before treatment, E-AN patients had the highest levels of ghrelin and GH with the lowest glucose levels and liver dysfunction. The AN-BP group had a higher level of ghrelin than the AN-R group. During treatment, comparing with the controls group only the AN-R group showed higher level of ghrelin. Contrarily, the ghrelin levels in the E-AN group, who showed improved glucose levels, and the AN-BP group, who stopped vomiting behavior due to our treatment, decreased ghrelin levels. After treatment, only the AN-BP group showed a higher ghrelin level as compared to the controls. Although GH levels of the three AN groups decreased gradually according to our treatment progress, it still showed the higher value than the control group at the end of the treatment because every AN patients could not reach to more than 80% of their ideal body weight at discharge. These findings suggest that (1) severe emaciation with abnormal fasting hypoglycemia in AN patients may cause very high levels of GH and ghrelin, (2) that GH levels in AN patients may relate to nutritional status and (3) that ghrelin may be influenced by not only nutritional status but also the eating behavior of the patients.     

Method for determining monohydroxybenzo[a]pyrene isomers using column-switching high-performance liquid chromatography

A method for determining monohydroxybenzo[a]pyrene (OHBaP) isomers using column-switching high-performance liquid chromatography with fluorescence detection was developed. Eleven of 12 isomers of OHBaP (all except 6-OHBaP) were separated on an alkylamide-type reversed-phase column and, via column-switching, on a beta-cyclodextrin-bonded silica gel column. The detection limits for the OHBaPs were in the range 0.3-8 pg/injection (S/N=3). By using this method, 1-, 3-, and 9-OHBaPs were identified as major metabolites of benzo[a]pyrene in vitro by human recombinant p450 1A1. The method was used to determine OHBaPs in the urine of a nonsmoker subject. After enzymatic hydrolysis of the conjugated metabolites by beta-glucuronidase/aryl sulfatase, the analytes were selectively adsorbed on blue rayon (a cellulose-supported copper phthalocyanine) from the urine matrix. Methanol as the eluting solvent from the rayon gave the best recoveries of OHBaPs and 1-hydroxypyrene (1-OHP) in the range of 91-103%, which was superior to that of the solid-phase extraction method. 1-OHP, a well-known biomarker of the exposure to polycyclic aromatic hydrocarbons, was simultaneously analyzed. Intra- and interday accuracy values for the determination of 3-OHBaP in 200 ml of urine were 95.5 and 100.9%, and those for 1-OHP were 96.4 and 103.6%, respectively. The intra- and interday precision values were 3.9 and 2.4% for 3-OHBaP and 2.4 and 3.2% for 1-OHP, respectively. In 11 kinds of isomers, only 3-OHBaP was detected in the human urine. Urinary concentration of 3-OHBaP was quantified at 0.5 ng/g creatinine concentration and the 3-OHBaP/1-OHP ratio was approximately 1/130.     

A novel diacylglycerol acyltransferase (DGAT2) is decreased in human psoriatic skin and increased in diabetic mice

Psoriasis is a skin disease with epidermal keratinocyte hyperproliferation and altered differentiation. To identify novel psoriasis-related genes, we investigated differentially expressed genes between normal and psoriatic skin. We identified a novel acyl CoA:diacylglycerol acyltransferase 2 (DGAT2) gene, which was decreased in human psoriatic skin. DGAT2 mRNA was expressed in sebaceous glands of normal human skin. DGAT2 protein was detected on endoplasmic reticulum. DGAT2 catalyzes the final step in the production of triglycerides and the accumulation of triglycerides in the tissues is considered to be related to insulin resistance. Therefore, we also investigated the expression of the DGAT2 gene in diabetic mice. DGAT2 mRNA was increased in the adipose, small intestine, and skeletal muscle in diabetic mice.     

Expression of adrenomedullin and its receptor by chondrocyte phenotype cells

For clarifying a process of de-differentiation in culturing chondrocytes, the present study was undertaken to investigate the secretion of adrenomedullin (AM) by chondrocyte phenotype cells and whether or not AM effects this proliferation in a cAMP-dependent fashion. Chondrocyte phenotype cells expressed AM and the AM receptor, and secreted high concentration of AM into the culture medium. When added to cultures, AM increased the intracellular cAMP level and decreased the number of these cells in a similar concentration-dependent fashion. Addition of forskolin and dibutyryl-cAMP caused a significant decrease in the number of these cells. Furthermore, the effect of AM was inhibited by a cAMP-dependent protein kinase A inhibitor (H89). The present findings indicate that AM has an autocrine/paracrine type of anti-proliferative effect on these cells mediated via a cAMP-dependent pathway and raise the possibility that AM plays a role in the local modulation of a process of de-differentiation by culturing chondrocyte phenotype cells.     

Gamma-secretase activity is present in rafts but is not cholesterol-dependent

Cholesterol has been claimed to be involved in the generation and/or accumulation of amyloid beta protein (Abeta). However, the underlying molecular mechanisms have not been fully elucidated yet. Here, we have investigated the effect of membrane cholesterol content on gamma-secretase activity using Chinese hamster ovary cells stably expressing beta-amyloid precursor protein (APP) and either wild-type or N141I mutant-type presenilin 2. Cholesterol was acutely depleted from the isolated membrane by methyl-beta-cyclodextrin, and Abeta production was assessed in a cell-free assay system. Reduced cholesterol did not significantly alter the amounts of Abeta produced by either total cell membranes or cholesterol-rich low-density membrane domains. Even its extremely low levels in the latter domains did not affect Abeta production. This indicates that the membrane cholesterol content does not directly modulate the activity of gamma-secretase. To ascertain that gamma-secretase resides in cholesterol-rich membrane domains, low-density membrane domains were further fractionated with BCtheta (biotinylated theta-toxin nicked with subtilisin Carlsberg protease), which has recently been shown to bind selectively to rafts of intact cells. The membrane domains purified with BCtheta did indeed produce Abeta. These observations indicate that the gamma-cleavage required for generating Abeta occurs in rafts, but its activity is virtually cholesterol-independent.     

DNA-protein cross-link formation mediated by oxanine. A novel genotoxic mechanism of nitric oxide-induced DNA damage

Chronic inflammation is a risk factor for many human cancers, and nitric oxide (NO) produced in inflamed tissues has been proposed to cause DNA damage via nitrosation or oxidation of base moieties. Thus, NO-induced DNA damage could be relevant to carcinogenesis associated with chronic inflammation. In this report, we report a novel genotoxic mechanism of NO that involves DNA-protein cross-links (DPCs) induced by oxanine (Oxa), a major NO-induced guanine lesion. When a duplex DNA containing Oxa at the site-specific position was incubated with DNA-binding proteins such as histone, high mobility group (HMG) protein, and DNA glycosylases, DPCs were formed between Oxa and protein. The rate of DPC formation with DNA glycosylases was approximately two orders of magnitude higher than that with histone and HMG protein. Analysis of the reactivity of individual amino acids to Oxa suggested that DPC formation occurred between Oxa and side chains of lysine or arginine in the protein. A HeLa cell extract also gave rise to two major DPCs when incubated with DNA-containing Oxa. These results reveal a dual aspect of Oxa as causal damage of DPC formation and as a suicide substrate of DNA repair enzymes, both of which could pose a threat to the genetic and structural integrity of DNA, hence potentially leading to carcinogenesis.     

The cyclin-dependent kinase inhibitors p27Kip1 and p21Cip1 cooperate to restrict proliferative life span in differentiating ovarian cells

The timing of cellular exit from the cell cycle during differentiation is specific for each cell type or lineage. Granulosa cells in the ovary establish quiescence within several hours after the ovulation-inducing luteinizing hormone surge, whereas they undergo differentiation into corpora lutea. The expression of Cdk inhibitors p21(Cip1/Waf1) and p27(Kip1) is up-regulated during this process, suggesting that these cell cycle inhibitors are involved in restricting proliferative capacity of differentiating granulosa cells. Here we demonstrate that the lack of p27(Kip1) and p21(Cip1) synergistically renders granulosa cells extended an proliferative life span. Immunohistochemical analyses demonstrated that corpora lutea of p27(Kip1), p21(Cip1) double-null mice showed large numbers of cells with bromodeoxyuridine incorporation and high proliferative cell nuclear antigen expression, which were more remarkable than those in p27(Kip1) single-deficient mice showing modest hyperproliferation. In contrast, differentiating granulosa cells in p21(Cip1)-deficient mice ceased proliferation similarly to those in wild-type mice. Interestingly, granulosa cells isolated from p27(Kip1), p21(Cip1) double-null mice exhibited markedly prolonged proliferative life span in culture, unlike cells with other genotypes. Cultured p27(Kip1), p21(Cip1) double-null granulosa cells maintained expression of steroidogenic enzymes and gonadotropin receptors through 8-10 passages and could undergo further differentiation in responses to cAMP accumulation. Thus, the cooperation of p27(Kip1) and p21(Cip1) is critical for withdrawal of granulosa cells from the cell cycle, in concert with luteal differentiation and possibly culture-induced senescence.