A novel, 4-h DNA extraction method for STR typing of casework bone samples.

Bones are often found in mass grave crime scene. To increase DNA identification success rates, a highly efficient DNA extraction method should be selected. Several DNA extraction methods for human bones have been published yet never been systematically compared, and some are time-consuming or complex. As such, a quick and highly efficient DNA extraction method was developed and compared with three published methods (Hi-Flow silica-based, total demineralization (TD) and PrepFiler BTA) using 70 fresh and 22 casework bones from different body parts. The highest median DNA concentrations were obtained from developed method (135.85 ng/µL and 0.224 ng/µL for fresh and casework bones, respectively). For residual PCR inhibitors, the threshold cycle (Ct) of the internal positive control (IPC) showed that developed method and PrepFiler BTA removed most PCR inhibitors. Similarly, 95.45% of casework STR profiles obtained using the developed protocol meet the standard requirements for Australian National Criminal Investigative DNA Database (NCIDD) entry, followed by 86.35% using TD, 81.82% using PrepFiler BTA, and 45.45% using Hi-Flow. Additionally, DNA extracts from seven different bones revealed that the 1st distal phalange of the hand contained the highest DNA concentration of 338.43 ng/µL, which was three times higher than the tibia and femur. Our findings suggest that developed method was highly efficient for casework bone analysis. It significantly reduced the extraction processing time down to 4 h and is two to four times cheaper compared with other methods. In practice, both the extraction method and the bone sampling must be considered by a forensic DNA analyst to increase the chances of successful identification.

Effect of glabridin on collagen deposition in liver and amelioration of hepatocyte destruction in diabetes rats.

Abnormalities in insulin hormone levels leads to a hyperglycemic condition of diabetic mellitus. Hyperglycemia seriously induces organ and system destructions. The excessive accumulation of collagen fiber deposits occurs in inflammatory and reorganization processes of chronic liver diseases in type I insulin-dependent diabetes. Regarding the research objective, glabridin (GLB), an active compound of licorice, was used as a daily supplement (40 mg/kg) in order to decrease hepatocyte destruction and collagen deposition in liver tissue of diabetic animals induced by streptozotocin. A total of 40 were randomly allocated to five groups (each, n=10), control, control treated with GLB (GLB), diabetic rats (DM) injected with single dose of streptozotocin (60 mg/kg) to induce a diabetic condition, diabetic rats receiving GLB (DM+GLB; 40 mg/kg) and diabetic rats treated with glibenclamide (DM+GL; 4 mg/kg). Characteristic histopathological changes in liver cells and tissues of rats were determined by Masson's trichrome staining and transmission electron microscopy (TEM). Western blotting was used to detect the expression of the key markers, collagen type I and fibronectin proteins. The histological investigation of liver tissue of the DM group revealed that the collagen fiber deposition was increased in the periportal, pericentral and perisinusoidal spaces compared with controls. Hepatocytes appeared as small and fragmented cells in TEM examination. Collagenization of the perisinusoidal space was recently demonstrated to represent a new aspect of the microvascular abnormalities and liver fibrosis. Healthy hepatocytes with round nucleus were observed following supplementation of glabridin. In addition, collagen fiber deposition was reduced in the area adjacent to the perisinusoidal space. The expression of collagen type I and fibronectin decreased strongly following glabridin supplementation in DM+GLB rats compared with DM rats, indicating that the hepatic tissue reorganization regained its normal morphology. These findings suggest that it may be beneficial to examine the role of glabridin as a therapeutic agent in diabetes treatment in future research.

The presence of abalone egg-laying hormone-like peptide in the central nervous system and ovary of the Spotted Babylon, Babylonia areolata.

Recently, the neuronal classification of the ivory shell Spotted Babylon, Babylonia areolata, was readily demonstrated. Regarding its importance as marine economic molluscan species, the attempt to understand the neuroendocrine regulation is necessary. This study firstly demonstrated the neurosecretory cells as well as the existence and distribution of the egg-laying hormone (ELH)-like peptide in the central nervous system (CNS) and ovary of the B. areolata. The neurosecretory cell was characterized by the cytoplasmic purple dot-like structure as stained by the Gomori's paraldehyde fuchsin. Using the anti-abalone (a) ELH, we detected the aELH-like-peptide in neurons (Nr) and neurosecretory cells (Ns) of all ganglia including the cerebral, pleural, parietal, pedal and buccal ganglia. The aELH-like peptide was also present in the neuropil of each. It was noted that not all Ns presented the aELH-like peptide. In the ovary, the aELH-like peptide was slightly detected in early developing oocytes and strongly detected in late developing oocytes and follicular cells. This study firstly reported the evidence of ELH-like peptide in the CNS and ovary of the B. areolata. The molecular cloning as well as to investigate the function of ELH in this species is needed as it will be beneficial for future applications in aquaculture.

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 ß) 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 ß 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 ß 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 ß. 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.

Histological studies of neuroprotective effects of Curcuma longa Linn. on neuronal loss induced by dexamethasone treatment in the rat hippocampus.

Long term exposure to dexamethasone (Dx) is associated with brain damage especially in the hippocampus via the oxidative stress pathway. Previously, an ethanolic extract from Curcuma longa Linn. (CL) containing the curcumin constituent has been reported to produce antioxidant effects. However, its neuroprotective property on brain histology has remained unexplored. This study has examined the effects of a CL extract on the densities of cresyl violet positive neurons and glial fibrillary acidic protein immunoreactive (GFAP-ir) astrocytes in the hippocampus of Dx treated male rats. It showed that 21 days of Dx treatment (0.5mg/kg, i.p. once daily) significantly reduced the densities of cresyl violet positive neurons in the sub-areas CA1, CA3 and the dentate gyrus, but not in the CA2 area. However, CL pretreatment (100mg/kg, p.o.) was found to significantly restore neuronal densities in the CA1 and dentate gyrus. In addition, Dx treatment also significantly decreased the densities of the GFAP-ir astrocytes in the sub-areas CA1, CA3 and the dentate gyrus. However, CL pretreatment (100mg/kg, p.o.) failed to protect the loss of astrocytes in these sub-areas. These findings confirm the neuroprotective effects of the CL extract and indicate that the cause of astrocyte loss might be partially reduced by a non-oxidative mechanism. Moreover, the detection of neuronal and glial densities was suitable method to study brain damage and the effects of treatment.

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 ß 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 ß immunoreactive (Es ß-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 ß 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 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).

Young coconut juice, a potential therapeutic agent that could significantly reduce some pathologies associated with Alzheimer's disease: novel findings.

Brains from ovariectomised (ovx) rats can display features similar to those observed in menopausal women with Alzheimer's disease (AD), and oestrogen seems to play a key role. Preliminary studies on young coconut juice (YCJ) have reported the presence of oestrogen-like components in it. The aim of the study was to investigate the effects of YCJ on the AD pathological changes in the brains of ovx rats. Rat groups included sham-operated, ovx, ovx+oestradiol benzoate (EB) and ovx+YCJ. Brain sections (4 µm) were taken and were immunostained with ß-amyloid (Aß) 1-42, glial fibrillary acidic protein (GFAP) (an intermediate neurofilament of astrocytes) and Tau-1 antibodies. Aß 1-42, GFAP and Tau-1 are considered as reliable biomarkers of amyloidosis, astrogliosis and tauopathy (neurofibrillary tangles), respectively, which in turn are characteristic features associated with AD. The serum oestradiol (E2) level was measured using a chemiluminescent immunoassay technique. YCJ restored the serum E2 to levels significantly (P < 0·001) higher than that of the ovx group, and even that of the sham group. Aß deposition was significantly (P < 0·0001) reduced in the cerebral cortex of the YCJ group, as compared with the ovx group and with the sham and ovx+EB groups (P < 0·01). A similar trend was observed in relation to GFAP expression in the cerebral cortex and to Tau-1 expression in the hippocampus. This is a novel study demonstrating that YCJ could have positive future implications in the prevention and treatment of AD in menopausal women.

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.

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.

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.

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.

Immunohistochemical localization of parvalbumin calcium-binding protein in the heart tissues of various species.

Parvalbumin (PV), a cytoplasmic high-affinity Ca(2+)-binding protein, was recently identified in rat heart tissue and has been implicated in mediating relaxation in cardiac myocytes. The presence of PV in the heart of mouse, chicken, rabbit and pig was studied using immunohistochemistry. PV immunoreactivity (PV-ir) was identified in the heart of all four species. All cardiac myocytes of each species had an identical pattern of PV-ir in their cytoplasm. The highest intensity of PV-ir was observed in mouse and chicken cardiac myocytes. The intensity of PV-ir was lower in rabbit cardiac myocytes and lowest in pig cardiac myocytes compared to those of chicken and mouse. PV-ir was observed in the walls of all four cardiac chambers (left and right atria and left and right ventricles), with the left ventricle, in general, having the highest labeling intensity. The intensity of PV-ir may be correlated with the physical activity of the heart of each species. Some potential applications of these data for treatment of human diastolic heart dysfunctions are discussed.

Fos-like immunoreactivity in rat dorsal raphe nuclei induced by alkaloid extract of Mitragyna speciosa.

Mitragyna speciosa (MS) has been traditionally used for medicinal purposes especially in southern Thailand. Previously, an alkaloid extract of this plant was demonstrated to mediate antinociception, partly, through the descending serotonergic system. The present study investigated the stimulatory effect of the MS extract on the dorsal raphe nucleus and its antidepressant-like activity. The MS extract containing approximately 60% mitragynine as a major indole alkaloid was used to treat the animals. The stimulatory effect of the MS extract was determined by detecting the expression of the immediate early gene, cfos, in the dorsal raphe nucleus of male Wistar rats. The immunohistochemistry was used to detect Fos protein, the protein product of cfos gene. The present data show that a significant increase in Fos expression was observed following long-term administration of the MS extract (40 mg/kg) for 60 consecutive days. In addition, the antidepressant-like activity of the MS extract was determined by using the forced swimming test (FST) in male mice. The results show that a single injection (either 60 or 90 mg/kg doses) significantly decreased immobility time in the FST. These findings indicate that the MS extract has a stimulatory effect on the dorsal raphe nucleus and an antidepressant-like activity. Stimulation of this brain area has been known to cause antinociception. These findings suggest that the MS extract might produce antinociceptive and/or antidepressive actions partly through activation of the dorsal raphe nucleus. Moreover, the dorsal raphe nucleus may be one of site of MS action in the central nervous system.

Up-regulation of parvalbumin expression in newborn and adult rat heart.

Parvalbumin (PV), a cytoplasmic calcium-binding protein, functions as a relaxing factor and has recently been detected in rat heart. Developmental changes in PV localization and expression were investigated in the heart of Wistar rats at different ages. Ten hearts from newborn, 3-month-old (young), 6-month-old (young adult), and 12-month-old (adult) rats were processed for immunohistochemistry and Western blot assay. PV was detected in hearts of all the age groups of the rats from newborn to 12-month-old by both immunohistochemistry and Western blotting. A variable distribution of PV immunoreactivity was present in newborn cardiac myocytes. In the 3-, 6-, and 12-month-old rat hearts, identical PV immunoreactivity was found in all cardiac myocytes and the intensity of PV immunoreactivity increased with increasing age. By using Western blotting, it was found that the expression of PV was low in the newborn rat heart and increased with increasing age. The presence of PV may correlate with the physiological age, and possibly serves to maintain proper relaxation of the cardiac myocytes to cope with an increasing workload of the heart during body growth.

Suppressive effects of dichloromethane fraction from the Areca catechu nut on naloxone-precipitated morphine withdrawal in mice.

In the present study, we investigated the effect of the dichloromethane fraction from Areca catechu nut on the severity of naloxone-precipitated morphine withdrawal in morphine-dependent mice. A single intraperitoneal injection of dichloromethane fraction at dose of 125 and 175 mg/kg significantly delayed the onset of withdrawal jumping behavior in a concentration-dependent manner compared to that of saline controls. The dichloromethane fractions also significantly decreased jumping numbers and faecal and urinary excretions during the withdrawal period.

Angiotensin II may mediate apoptosis via AT1-receptors in the rat cardiac conduction system.

INTRODUCTION: Apoptosis and angiotensin II (Ang II) have been suggested as possible causes of arrhythmias. In addition, Ang II via Ang II type I (AT(1)-) receptors, has been demonstrated to induce cardiomyocyte apoptosis. The transgenic m(Ren-2)27 (TG) rat carries the additional Ren-2 gene, the expression of which results in an increase in cardiac Ang II, thus potentially affecting the cell growth/death equilibrium. In this study we have investigated the effect of Ang II, via AT(1)-receptors, on mediating apoptosis in a cardiac conduction system (SA node and AV nodes). MATERIALS AND METHODS: Heart sections from male two-day, one-week and two-week TG and Sprague-Dawley (SD) rats were stained with Masson Trichrome to localise the SA and AV nodes. The sections containing SA or AV nodes were processed for quantitation of apoptotic nuclei and AT(1)-receptors. RESULTS: The number of apoptotic nuclei/mm(2) in the SA and AV nodes were found to decrease from two days to two weeks in both the TG and the SD rats, and the number of apoptotic nuclei/mm(2) in the TG groups was significantly higher than that of the SD groups for all ages (p<0.05). The number of AT(1)-receptors/mm(2) in the SA node were found to decrease with increasing age, whereas the number of AT(1)-receptors/mm(2) in the AV node was increased in both TG and SD rats and the number of AT(1)-receptors/mm(2) in the three TG groups was significantly more than that of the three SD groups (p<0.05). DISCUSSION AND CONCLUSION: As a consequence of the additional renin gene in the TG rats, which results in the alteration of the local renin-angiotensin system, the numbers of AT(1)-receptors/mm(2) and apoptotic nuclei/mm(2) are increased. The number of apoptotic nuclei/mm(2) and AT(1)-receptors/mm(2) in the SA node decrease with maturation, whereas, the number of AT(1)-receptors in the AV node increase. Thus, there may be a correlation between Ang II and apoptosis in the SA node, which does not appear to be present in the AV node.