Wandee Udomuksorn

Influence of mutations associated with Gilbert and Crigler-Najjar type II syndromes on the glucuronidation kinetics of bilirubin and other UDP-glucuronosyltransferase 1A substrates.

Objectives UGT1A1 coding region mutations, including UGT1A1*6 (G71R), UGT1A1*7 (Y486D), UGT1A1*27 (P229Q) and UGT1A1*62 (F83L), have been linked to Gilbert syndrome in Asian populations, whereas homozygosity for UGT1A1*7 is associated with the Crigler–Najjar syndrome type II. This work compared the effects of (a) the individual UGT1A1 mutations on the glucuronidation kinetics bilirubin, β-estradiol, 4-methylumbelliferone (4MU) and 1-naphthol (1NP), and (b) the Y486 mutation, which occurs in the conserved carboxyl terminal domain of UGT1A enzymes, on 4MU, 1NP and naproxen glucuronidation by UGT1A3, UGT1A6 and UGT1A10. Methods Mutant UGT1A cDNAs were generated by site-directed mutagenesis and the encoded proteins were expressed in HEK293 cells. The glucuronidation kinetics of each substrate with each enzyme were characterized using specific high-performance liquid chromatography (HPLC) methods. Results Compared with wild-type UGT1A1, in-vitro clearances for bilirubin, β-estradiol, 4MU and 1NP glucuronidation by UGT1A1*6 and UGT1A1*27 were reduced by 34–74%, most commonly as a result of a reduction in Vmax. However, the magnitude of the decrease in the in-vitro clearances varied from substrate to substrate with each mutant. The glucuronidation activities of UGT1A1*7 and UGT1A1*62 were reduced by >95%. Introduction of the Y486D mutation essentially abolished UGT1A6 and UGT1A10 activities, and resulted in 60–90% reductions in UGT1A3 in-vitro clearances. Conclusions The glucuronidation of all UGT1A1 substrates is likely to be impaired in subjects carrying the UGT1A1*6 and UGT1A1*62 alleles, although the reduction in metabolic clearance might vary with the substrate. The Y486D mutation appears to greatly reduce most, but not all, UGT1A activities.

Effects of alcohol administration during adulthood on parvalbumin and glial fibrillary acidic protein immunoreactivity in the rat cerebral cortex

The pathology of brain atrophy mediated by alcohol was investigated in all parts of the cerebral cortex (the frontal, parietal, temporal lobes and occipital cortex) by using two markers: parvalbumin (PV) and glial fibrillary acidic protein (GFAP). Three-month old male Wistar rats were divided into control (C) and alcohol-exposed groups. The control group received distilled water, whereas the alcohol-exposed groups received either a low dose (2g/kg body wt) or a high dose (5g/kg) of ethanol for periods of 21 days, 3 or 6 months. The brains of the animals were processed for immunohistochemistry using anti-parvalbumin and anti-GFAP antibodies and the number of PV immunoreactive (PV-ir) neurons and GFAP immunoreactive (GFAP-ir) astrocytes were counted per unit area. Results showed that all groups exposed to ethanol had significantly reduced numbers of PV-ir neurons in all parts of the cerebral cortex compared to those of the control group (p<0.05). In contrast, the numbers of GFAP-ir astrocytes were increased in all parts of the cerebral cortex following the exposure to a high dose of ethanol after 21-days (but not a low dose) and both high and low doses of ethanol after 3-months or 6-months treatment compared to those of age-matched control groups (p<0.05). This indicated that in young rats (21-days), PV-ir neurons in all cerebral cortex areas seemed to be more sensitive to alcohol than GFAP-ir astrocytes. Moreover, the change in densities of both PV-ir neurons and GFAP-ir astrocytes became more apparent after exposure to prolonged and high doses of ethanol. The decrease of PV-ir neurons and the increase of GFAP-ir astrocytes indicated that alcohol may induce pathology in broad areas of the cerebral cortex. This may explain the underlying mechanism of brain atrophy and other impairments found in alcoholics. For investigations of the effects of alcohol on mediating brain pathology, we recommend the use of the two markers (PV and GFAP).

Alcohol administration during adulthood induces alterations of parvalbumin and glial fibrillary acidic protein immunoreactivity in rat hippocampus and cingulate cortex

Alcohol induces impairment of cognition, learning and memory. Neurotoxic effects of alcohol on the pathology of the hippocampus and the cingulate cortex were investigated in experimental rats. Parvalbumin (PV), a calcium-binding protein, is a crucial component of GABAergic neurons and glial fibrillary acidic protein immunoreactive (GFAP-ir) astrocytes have been used as markers. We investigated the effects of ethanol exposure during adulthood on the PV-ir neurons and GFAP-ir astrocytes in the hippocampus and the cingulate cortex of 3-month-old male Wistar rats. The rats were divided into 2 groups: control (C) and alcohol-exposed groups. The control group received distilled water whereas the alcohol-exposed groups received either a low dose (20%w/v, LD) or high dose (40%w/v, HD) of ethanol for periods of 21 days, 3 or 6 months. The brains of the animals were processed for immunohistochemistry using anti-parvalbumin and anti-GFAP antibodies and the numbers of PV immunoreactive (PV-ir) neurons and GFAP-ir astrocytes were counted/unit area. For each period of administration, the number of PV-ir neurons was significantly reduced for groups exposed to both the low and the high doses of ethanol compared to those of control groups in both the hippocampus and the cingulate cortex (p<0.01). In addition, the number of PV-ir neurons was progressively reduced after prolonged ethanol exposure. In contrast, there was a significantly increased number of GFAP-ir astrocytes observed in the hippocampus and the cingulate cortex in all groups exposed to ethanol and this was a function of both the duration and the dose of ethanol exposure, indicating that PV-ir neurons are as sensitive as the GFAP-ir astrocytes to ethanol exposure. Our data indicate that alcohol exposure induced a reduction of PV-ir neurons and an increase of GFAP-ir astrocytes in the hippocampus and the cingulate cortex and this may be associated with the impairment of cognition, learning and memory after chronic alcohol administration.

Estrogen replacement improves skeletal muscle performance by increasing parvalbumin levels in ovariectomized rats.

Muscle weakness is common during menopause. Effective estrogen replacement was hypothesized to prevent sarcopenia. This study aimed to investigate the estrogen level, estrogen receptors (α and β) immunoreactivities, muscle mass and functions, and parvalbumin (PV) levels in the extensor digitorum longus (EDL) and the gastrocnemius muscles of ovariectomized rats. Adult female Wistar rats (12 weeks old) were divided into five groups: sham-operated (SHAM), and ovariectomized (E0) groups that received 10 weeks of estrogen replacements of 0μg/kg (E0), 10μg/kg (E10), 20μg/kg (E20) and 40μg/kg (E40). The estrogen levels, ER α and ER β immunoreactivities, muscle fiber sizes and contractivities and the PV levels were reduced in the E0 group, but increased in all the estrogen replacement groups in both muscles. This study indicated that the reduction of estrogen levels led to a decrease of both ER α and ER β resulting in a decline in muscle mass and PV levels. The decrease of PV levels affected muscle performance, whereas estrogen replacement increased both the ER α and ER β. The increase in the PV levels may result in an improvement of muscle performance. This may explain one mechanism of estrogen on muscle mass and strength in estrogen dependent sarcopenia.

Effects of estrogen via estrogen receptors on parvalbumin levels in cardiac myocytes of ovariectomized rats

The study investigated the effects of estrogen on parvalbumin (PV) levels in cardiac myocytes of ovariectomized rats, which is a model system for postmenopausal woman. Parvalbumin acts as a relaxing factor in cardiac myocytes. Adult female Wistar rats, 12 weeks old, were randomly divided into 5 groups of 10: sham-operated (SHAM), ovariectomized (OVX), and OVX receiving estrogen replacement of 10 μg/kg (Es10), 20 μg/kg (Es20) and 40 μg/kg (Es40). After 10 weeks, serum estrogen levels were measured and the α and β estrogen receptors in cardiac myocytes were investigated by immunohistochemistry. PV levels were examined by immunohistochemistry and Western blot analysis. Cardiac myocytes of all animals showed strong staining intensities for α immunoreactive (Es α-ir), but weak staining for β immunoreactive (Es β-ir) estrogen receptors. The Es α-ir was reduced in the cardiac myocytes of the OVX groups, but increased in the Es10, Es20 and Es40 groups. We therefore suggest that estrogen effects are mediated via Es α receptors rather than Es β receptors in female rat hearts. Estrogen and PV immunoreactive (PV-ir) levels and the intensity of the PV band observed in the OVX group were less than those of the SHAM group. In the Es10, Es20 and Es40 groups, the increased intensity of the PV-ir and PV bands correlated with the increased estrogen levels. The low PV levels in cardiac myocytes induced by low estrogen were restored by estrogen replacement therapy. Therefore a reduction of PV may lead to diastolic dysfunction in menopause.

Effects of alcohol on the levels of parvalbumin in rat hearts.

Chronic excessive alcohol administration has been reported to be associated with diastolic dysfunction. Parvalbumin (PV) is a calcium-binding protein present in cardiac myocytes and involved in mediating relaxation. Therefore, alteration of PV levels may affect relaxation in cardiac myocytes. This study investigated the effects of alcohol administration on the levels of PV in the rat heart. Male Wistar rats weighing 200-250 g were divided into 2 groups: control (C) and alcohol-treated groups. The control group was provided with distilled water and the alcohol groups were provided with either a low dose (LD, 2g/kg) or high dose of ethanol (HD, 5 g/kg) once daily for 21 days, 3 months or 6 months. The PV levels in the ventricles were investigated by immunohistochemistry and Western blot analysis. In the 21-day ethanol-treated groups, parvalbumin immunoreactivity (PV-ir) and protein levels were not different when compared to the C, LD and HD groups. In the 3-month ethanol-treated groups, PV-ir and PV protein levels were decreased in both the LD and HD groups compared to that of the control group. In the 6-month ethanol-treated groups, PV-ir and PV protein levels decreased significantly in both the LD and HD groups (P<0.05). This indicates that short-term ethanol treatment may not affect PV levels, whereas, long-term ethanol treatment clearly reduced PV levels. The decrease of PV was predominantly due to the direct toxic effects of alcohol rather than malabsorption caused by pathological changes in the duodenum and liver. The toxic effects of alcohol leading to a reduction of PV levels may lead to diastolic impairment.

Potential wound healing activity of Quercus infectoria formulation in diabetic rats

Background Quercus infectoria G. Olivier (Fagaceae) nutgalls have been widely employed in traditional Asian medicine for several treatments, especially wounds and skin disorders. However, the effects of this plant on wound healing have not yet been clearly elucidated. This present work was focused on utilization of Quercus infectoria (Qi) as a topical agent for chronic wound treatment. Methods Twenty Qi formulations (QiFs) were pharmaceutically formulated and antibacterial activity of all formulations was performed. The best formulation based on an antibacterial activity was selected for evaluation of wound healing property. Total phenolics, total flavonoids, and an anti-oxidant activity of the selected formulation were also investigated. Wound healing activity was assessed in streptozotocin-induced diabetic rats and control rats. Streptozotocin injection (50 mg/kg) was found to induce marked hyperglycaemia, compared with citrate-injected controls. Two wounds were created on the upper back of each animal. QiF was topically applied three days after wounding to one of the duplicate wounds on each animal and physiological saline (control) was applied to the other. All wounds were cleaned once a day until wound closure. Results QiF10, which exhibited antibacterial and anti-oxidant activities, had the ability to enhance the wound healing process in diabetic rats with abundant cellular infiltration, collagen deposition, and re-epithelialization when compared with the control. Discussion This study suggested that QiF10 could be a novel alternative treatment for diabetic wounds.

Influence of aging and long-term swimming exercise on parvalbumin distribution in rat hearts

Parvalbumin (PV), which is a small (12kDa) cytoplasmic calcium-binding protein, has been implicated in mediating relaxation in cardiac myocytes. The influence of aging and exercise on the distribution of PV in rat heart was investigated. Male Wistar rats aged 3, 6, 12 and 18-months were divided into sedentary and exercise groups. The exercise group underwent exercise in the form of regular swimming for 6 months. The hearts were processed for immunohistochemistry and Western blotting. The intensity of PV immunoreactivity was strong in the 9 and 12-month hearts and decreased in the 18-month hearts. The smallest amount was detected in the 24-month rat hearts when compared to those of the 9, 12 and 18-month rat hearts. Significantly less PV was detected in the 18 and 24-month hearts compared to the 12-month rat hearts (P<0.05). The intensity of PV immunoreactivity was considerably stronger in hearts of the 9, 12 and 18-months exercised rats than in hearts of age-matched sedentary rats. However, in the hearts of 24-month rats, immunoreactivity was only slightly stronger in the exercised rats in comparison with those of sedentary rats. A significant increase of PV detection in hearts was found in the exercised rats in comparison with sedentary rats in the 9 (P<0.05) and 18-month samples (P<0.01). Our data indicate that PV is down-regulated in the rat heart during aging. In addition, our data indicate that long-term swimming exercise could induce an increase of PV expression.

Age-related changes in parvalbumin in the heart of female rats.

Changes of parvalbumin protein levels and immunolocalisation during the postnatal development of the female rat heart were investigated in order to determine if they were correlated with age-related changes in cardiac function. Hearts from newborn, 3-month-old (young), 6-month-old (young adult) and 12-month-old (adult) female Wistar rats were processed for immunohistochemical localization of parvalbumin and for Western blotting assay. Parvalbumin was detected by both methods in all age groups from newborn to 12-month-old rats. In the newborn rat heart, parvalbumin immunoreactivity did not fully fill the sarcoplasm of the cardiac myocytes and the amount of parvalbumin was low compared to the adult levels. In contrast, in 3-12-month-old rats, strong parvalbumin immunoreactivity was detected throughout the sarcoplasm of all cardiac myocytes and the amount of parvalbumin increased with increasing age (from newborn to adult). Our study indicates that an increase of parvalbumin levels in the female rat heart with increasing age may be associated with maintenance of proper relaxation of the cardiac myocytes needed to cope with the increasing workload of the heart during postnatal growth.

Molecular cloning and distribution of oxytocin/vasopressin-like mRNA in the blue swimming crab, Portunus pelagicus, and its inhibitory effect on ovarian steroid release

This study was aimed to characterize the full length of mRNA of oxytocin/vasopressin (OT/VP)-like mRNA in female Portunus pelagicus (PpelOT/VP-like mRNA) using a partial PpelOT/VP-like sequence obtained previously in our transcriptome analysis (Saetan, 2014) to construct the primers. The PpelOT/VP-like mRNA was 626 bp long and it encoded the preprohormones containing 158 amino acids. This preprohormone consisted of a signal peptide, an active nonapeptide (CFITNCPPG) followed by the dibasic cleavage site (GKR), and the neurophysin domain. Sequence alignment of the PpelOT/VP-like peptide with those of other animals revealed strong molecular conservation. Phylogenetic analysis of encoded proteins revealed that the PpelOT/VP-like peptide was clustered within the group of crustacean OT/VP-like peptide. Analysis by RT-PCR revealed the expression of mRNA transcripts in the eyestalk, brain, ventral nerve cord (VNC), ovary, intestine and gill. The in situ hybridization demonstrated the cellular localizations of the transcripts in the central nervous system (CNS) and ovary tissues. In the eyestalk, the mRNA expression was observed in the neuronal clusters 1-5 but not in the sinus gland complex. In the brain and the VNC, the transcripts were detected in all neuronal clusters but not in the glial cell. In the ovary, the transcripts were found in all stages of oocytes (Oc1, Oc2, Oc3, and Oc4). In addition, synthetic PpelOT/VP-like peptide could inhibit steroid release from the ovary. The knowledge gained from this study will provide more understanding on neuro-endocrinological controls in this crab species.