Synergistic effects of corticosterone and kainic acid on neurite outgrowth in axotomized dorsal root ganglion.

Corticosterone is the main adrenal glucocorticoids induced by stress in rats. Therapeutic use of high concentration of synthetic glucocorticoids in clinical treatment of spinal cord injury suggests that pharmacological action of glucocorticoids might be beneficial for nerve repair. In this article we cultured axotomized rat dorsal root ganglion neurons to investigate the effects of corticosterone and a glutamate receptor agonist kainic acid on neurite outgrowth. Our results revealed a synergistic effect of corticosterone and kainic acid in promoting neurite outgrowth when applied as early as one and two days in vitro, but not effective at three and four days in vitro. In addition, applied corticosterone and kainic acid were neurotoxic at three and four days in vitro but not at one and two days in vitro. The minimal concentrations of corticosterone and kainic acid to be effective were 10 microM and 1 mM, respectively. The neurotrophic effect of corticosterone and kainic acid was attenuated by the receptor tyrosine kinase A (TrkA) inhibitor AG-879. Western blot analysis and immunocytochemical studies revealed an increase of expressions of both TrkA and growth-associated protein GAP-43 in dorsal root ganglion neurons with combined treatment of corticosterone and kainic acid. Immunocytochemistry showed that corticosterone+kainic acid increase nerve growth factor immunoreactivity in dorsal root ganglion neurites and enhance GAP-43 immunointensity in dorsal root ganglion neurons. These results suggest that the neurotrophic effect of glucocorticoids on axonal regeneration might require facilitation of excitatory stimulation at an early stage of nerve injury, and nerve growth factor may mediate a growth signaling to accomplish the effect.

Pharmacological basis of 'Yin-nourishing' and 'Yang-invigorating' actions of Cordyceps, a Chinese tonifying herb.

Cordyceps sinensis (Berk.) Sacc. (Cordyceps), a popular Chinese tonifying herb, was revered for being both 'Yin-nourishing' and 'Yang-invigorating' in Chinese medicine. In order to establish the pharmacological basis for the 'Yin-nourishing' and 'Yang-invigorating' action of Cordyceps, the effects of wild and cultured Cordyceps on concanavalin A (Con A)-stimulated splenocytes, an in vitro bioassay for 'Yin-nourishment', and myocardial ATP generation capacity, an ex vivo bioassay for 'Yang-invigoration', were investigated in mice. The results indicated that methanolic extracts of wild and cultured Cordyceps enhanced both the Con A-stimulated splenocyte proliferation in vitro and myocardial mitochondrial ATP generation ex vivo in mice, with no significant difference in potency of action between the two types of Cordyceps. While the immuno-potentiating effect was associated with the increase in interleukin II production, the stimulation of myocardial ATP generation was paralleled by an enhancement in mitochondrial electron transport. When compared with typical 'Yin' and 'Yang' tonifying Chinese herbs, Cordyceps was found to possess both 'Yin-nourishing' and 'Yang-invigorating' activities, with a lower potency in both modes of action. The pharmacological characterization of Cordyceps by means of contemporary bioassays is consistent with the time-honored clinical observation from Chinese herbalists.

Expression of c-met and WT-1.

Protooncogenes and tumor-suppressor genes are two types of genes associated with cancer development.

Cloning of a pig glucose transporter 4 cDNA fragment: use in developing a sensitive ribonuclease protection assay for quantifying low-abundance glucose transporter 4 mRNA in porcine adipose tissue.

A 246-bp fragment of porcine glucose transporter 4 (GLUT4) cDNA was cloned by polymerase chain reaction (PCR) from porcine adipose tissue RNA. Nucleotide sequences 1-138 and 139-246 of the GLUT4 cDNA share 78% sequence identity with exon 4a and 91% sequence identity with exon 4b of the human GLUT4 gene, respectively. The GLUT4 cDNA fragment was subcloned into pGEM-4Z vector to synthesize a highly specific riboprobe that hybridized only to human GLUT4 cDNA but not to human glucose transporter 1 (GLUT1) cDNA. Northern blot analysis of total RNA revealed the presence of a single transcript of 2.8 kb in porcine adipose tissue. Cloning a fragment of the GLUT4 cDNA enabled us to develop a ribonuclease protection assay for detecting porcine GLUT4 mRNA. The ribonuclease (RNase) protection assay is highly reproducible and retains a sensitivity level to as little as 2 pg of GLUT4 mRNA. The standard curve was linear between 2 and 128 pg of sense-strand GLUT4 RNA (r = .994). The ability to detect small quantities of GLUT4 mRNA is important when the abundance of GLUT4 mRNA is low and the quantity of tissue is limiting (e.g., when RNA is extracted from cultured adipose tissue). When porcine adipose tissue explants were cultured in the presence of insulin (10 ng/mL), GLUT4 mRNA abundance was increased. Development of a sensitive assay to quantify GLUT4 mRNA in porcine adipose tissue will enable us to conduct studies to increase our understanding of the molecular mechanisms by which porcine somatotropin (pST) regulates GLUT4 gene expression.

A 'Yang-Invigorating' Chinese herbal formula protects against gentamicin-induced nephrotoxicity in rats.

The effects of VI-28 (a Yang-invigorating Chinese herbal formula) treatment on the renal mitochondrial antioxidant system and susceptibility to gentamicin-induced nephrotoxicity were investigated in rats. VI-28 treatment (80 or 240 mg/kg/day x 12) enhanced the renal mitochondrial antioxidant system, as indicated by dose-dependent increases in the level/activities of reduced glutathione, Mn-superoxide dismutase, Se-glutathione peroxidase and glutathione S-transferases. VI-28 treatment protected against nephrotoxicity induced by gentamicin administration (100 mg/kg/day x 8) and the nephroprotection was associated with an enhancement in the renal mitochondrial antioxidant system. In conclusion, VI-28 treatment enhanced the renal mitochondrial antioxidant system, thereby protecting against gentamicin nephrotoxicity.

A characteristic pharmacological action of 'Yang-invigorating' Chinese tonifying herbs: enhancement of myocardial ATP-generation capacity.

In order to investigate the pharmacological basis of 'Yang-invigorating' action, the effect of oral treatment with the methanolic extract of 'Yang-invigorating' herbs on ATP-generation capacity was examined, using heart homogenates prepared from herb-pretreated mice. Tonifying (i.e., health-promoting) herbs of other functional categories were also included for comparison. The results indicated that 'Yang-invigorating' Chinese tonifying herbs could invariably enhance myocardial ATP-generation capacity, with the extent of stimulation varying among the herbs. In contrast, 'Yin-nourishing' herbs either did not stimulate or even decreased myocardial ATP-generation capacity. While 'Qi-invigorating' herbs produced variable effects on myocardial ATP-generation capacity, most of the 'blood-enriching' herbs did not cause any significant changes. The results obtained from studies using myocardial mitochondrial fractions isolated from herb-pretreated mice suggest that 'Yang-invigorating' herbs might speed up ATP generation by increasing mitochondrial electron transport. The ensemble of results has provided evidence for the first time to support the pharmacological basis of 'Yang invigoration' in Chinese medicine.

Dang-Gui Buxue Tang produces a more potent cardioprotective effect than its component herb extracts and enhances glutathione status in rat heart mitochondria and erythrocytes.

The effects of pretreatment with Dang-Gui Buxue Tang (DBT, a decoction of Astragali and Angelica roots) and its component herb extracts on myocardial ischaemia-reperfusion (IR) injury were examined in rats ex vivo. DBT and its component herb extracts could protect against myocardial IR injury in a dose-dependent manner. The more potent cardioprotection afforded by DBT pretreatment than that of a mixture of Astragali and Angelica root extracts was associated with a much higher extraction yield of active ingredients from Angelica root in the herbal decoction. The high level of active ingredients might increase their bioavailability after oral administration. DBT pretreatment could enhance myocardial mitochondrial as well as red blood cell (RBC) glutathione status, thereby increasing their resistance to oxidative stress-induced injury in rats. The measurement of RBC glutathione status may serve as a useful index for the antioxidant effect produced by DBT treatment in human subjects.

Expression of divalent metal transporter 1 (DMT1) isoforms in first trimester human placenta and embryonic tissues.

BACKGROUND: Divalent metal transporter 1 (DMT1) is a transmembrane glycoprotein which mediates the proton-coupled transport of a variety of divalent metal ions. Two isoforms, which differ by the presence (DMT1-IRE) or absence (DMT1-nonIRE) of an iron-responsive element (IRE) in their 3' untranslated region, are implicated in apical iron transport and endosomal iron transport respectively. Although the expression pattern of DMT1 isoforms is tissue specific in adult, data regarding its expression in embryonic tissues are lacking. METHODS: Semiquantitative RT-PCR and immunohistochemistry were used to study the mRNA and protein expression of both DMT1 isoforms in embryonic tissues between 8 and 14 weeks gestational age. RESULTS: DMT1-IRE and DMT1-nonIRE expressions were ubiquitous in embryonic tissues examined. In the lung, statistically significant correlations were found between the levels of DMT1 isoform expression and gestational age. In the placenta, DMT1-IRE was the predominantly expressed isoform. Both isoform proteins were localized in embryonic epithelial cellular membrane. CONCLUSION: Both DMT1 isoforms are ubiquitously expressed in embryonic tissues in the first trimester. Predominant DMT1-IRE isoform expression in placenta suggests an iron-regulatory mechanism reminiscent of that in the adult duodenum. Epithelial distributions of both DMT1 isoforms are associated with the absorptive or excretory functions of the expressed tissues.

An in-vitro study of ginsenoside Rb1-induced teratogenicity using a whole rat embryo culture model.

BACKGROUND: Ginseng is a commonly used herbal medicine worldwide. However, there is limited information regarding its effects on the developing embryo. METHODS: The effect of ginsenoside on the developing embryo during the critical period of organogenesis was investigated using a whole rat embryo culture model. Embryos were exposed to various concentrations of ginsenoside Rb(1) and scored for growth and differentiation at the end of the culture period. RESULTS: Median total morphological scores in embryos exposed to 30 micro g/ml of ginsenoside Rb(1) was significantly lower (P < 0.05) than that in control embryos (35 versus 45). Morphological scores for flexion, forelimb and hindlimb were also significantly reduced. The median total morphological scores further decreased to 28 when the concentration of ginsenoside Rb(1) was increased to 50 micro g/ml. At this concentration, the embryonic crown-rump length and somite number were also significantly reduced compared with control embryos (2.8 versus 3.0 mm and 16.0 versus 21.0, respectively). CONCLUSIONS: Our study has demonstrated that ginsenoside exerts direct teratogenic effects on rat embryos. Until more is known about the effects of ginsenoside in women of reproductive age, we suggest its use should be treated with caution.

A study of diclofenac-induced teratogenicity during organogenesis using a whole rat embryo culture model.

BACKGROUND: Diclofenac is a non-steroidal anti-inflammatory drug, commonly used by reproductive age women for the treatment of a variety of conditions. However, there is limited information regarding the teratogenic effects of this drug. METHODS: The effect of diclofenac on the developing embryo during the critical period of organogenesis was investigated by using a whole rat embryo culture model. Embryos were exposed to various concentrations of diclofenac and scored for growth and differentiation at the end of the culture period. RESULTS: Total developmental score and score for caudal neural tube, flexion and hindlimb were significantly lower in embryos exposed to high concentrations of diclofenac (7.5 and 15.0 microg/ml), but no difference in these parameters was observed when embryos were exposed to low concentration of diclofenac (1.5, 2.5 and 5.0 microg/ml). No significant differences in yolk sac diameter, crown-rump length and number of somites was found between embryos in the experimental and the control group. CONCLUSIONS: Our study has demonstrated that diclofenac exerts direct teratogenic effects on rat embryos. Until more is known about the effects of diclofenac (especially in moderate to high doses) in women of reproductive age, we suggest its use should be treated with caution.

Detecting abnormal regional cerebral blood flow in patients with primary Sjögren's syndrome by technetium-99m ethyl cysteinate dimer single photon emission computed tomography of the brain--a preliminary report.

Technetium-99m ethyl cysteinate dimer (Tc-99m ECD) brain single photon emission computed tomography (SPECT) was used to detect abnormal regional cerebral blood flow (rCBF) in 32 female patients with primary Sjögren's syndrome (PSS) showing definite neuropsychiatric symptoms/signs and normal brain magnetic resonance imaging (MRI) findings. It demonstrated hypoperfusion brain lesions in 18 (56.3%) of the patients, most frequently in the parietal lobes, and appears to be a sensitive tool for this clinical application.

Objective evidence of abnormal regional cerebral blood flow in patients with systemic lupus erythematosus on Tc-99m ECD brain SPECT.

Technetium-99m ethyl cysteinate dimer (Tc-99m ECD) brain single photon emission computed tomography (SPECT) was used to detect abnormal regional cerebral blood flow (rCBF) in 78 SLE patients with neuropsychiatric manifestations. These patients were separated into two subgroups: group 1 including 48 cases with definite neuropsychiatric symptoms/signs and group 2 with 30 cases having no neuropsychiatric symptoms/signs. Tc-99m ECD brain SPECT demonstrated hypoperfusion brain lesions in 90% and 20% of patients in groups 1 and 2, respectively. In both groups, parietal lobe and cerebellum are the most and least common areas with hypoperfusion lesions, respectively. This study suggests that Tc-99m ECD brain SPECT may provide objective information for detection of hypoperfusion brain lesions in SLE patients.

In vivo antioxidant action of a lignan-enriched extract of Schisandra fruit and an anthraquinone-containing extract of Polygonum root in comparison with schisandrin B and emodin.

The in vivo antioxidant action of a lignan-enriched extract of the fruit of Schisandra chinensis (FS) and an anthraquinone-containing extract of the root of Polygonum multiflorum (PME) was compared with their respective active constituents schisandrin B (Sch B) and emodin by examining their effect on hepatic mitochondrial glutathione antioxidant status in control and carbon tetrachloride (CCl 4 )-intoxicated mice. FS and PME pretreatments produced a dose-dependent protection against CCl 4 hepatotoxicity, with the effect of FS being more potent. Pretreatment with Sch B, emodin or alpha-tocopherol (alpha-Toc) also protected against CCl 4 hepatotoxicity, with the effect of Sch B being more potent. The extent of hepatoprotection afforded by FS/Sch B and PME/emodin pretreatment against CCl 4 toxicity was found to correlate well with the degree of enhancement in hepatic mitochondrial glutathione antioxidant status, as evidenced by increases in reduced glutathione level and activities of glutathione reductase, glutathione peroxidase as well as glutathione S-transferases, in both control and CCl 4 -intoxicated mice. alpha-Toc, which did not enhance mitochondrial glutathione antioxidant status, seemed to be less potent in protecting against CCl 4 hepatotoxicity. The ensemble of results indicates that FS/PME produced a more potent in vivo antioxidant action than alpha-Toc by virtue of their ability to enhance hepatic mitochondrial glutathione antioxidant status and that the differential potency of FS and PME can be attributed to the difference in in vivo antioxidant potential between Sch B and emodin. Abbreviations. ALT:alanine aminotransferases CCl 4 :carbon tetrachloride FS:lignan-enriched extract of Schisandra fruit GRD:glutathione reductase GSH:reduced glutathione GSH-Px: Se-glutathione peroxidase GST:glutathione S-transferases mt:mitochondrial MDA:malondialdehyde PME:anthraquinone-containing fraction of Polygonum root Sch B:schisandrin B SDH:sorbitol dehydrogenase alpha-Toc:alpha-tocopherol

Levels of cord blood thyroid stimulating hormone after external cephalic version.

OBJECTIVE: To investigate the relationship between breech presentation, external cephalic version and levels of cord blood thyroid stimulating hormone. DESIGN: Case-control study. SETTING: University teaching hospital. POPULATION: The study group consisted of 289 consecutive singleton deliveries at term over a four-year period, all of whom had an attempt at external cephalic version performed near term for breech presentation. The control group included 23,001 singleton term deliveries during the same four-year period. METHODS: Between group differences were compared with the Mann-Whitney U test or chi2 test when appropriate. MAIN OUTCOME MEASURES: Levels of cord blood thyroid stimulating hormone and the incidence of false positive screening results for congenital hypothyroidism. RESULTS: Babies who were born vaginally after prior successful external cephalic version had significantly higher median levels of cord blood thyroid stimulating hormone (6.4 vs 6.0 mlU/L, P = 0.034) and the incidence of false positive screening for thyroid stimulating hormone (12.9% vs 7.2%, P = 0.016, OR 1.9) compared with babies with spontaneous cephalic presentation. In babies with a breech presentation born by elective caesarean section, previous attempts at external cephalic version had no effect on cord blood thyroid stimulating hormone levels. There was also no difference in the levels of cord blood thyroid stimulating hormone between cephalic and breech-presenting fetuses born by elective caesarean section. However, breech-presenting babies born by emergency caesarean section after onset of labour had higher median levels of cord thyroid stimulating hormone than those with cephalic presentation (5.1 vs 4.5 mIU/L, P= 0.008). CONCLUSION: Levels of cord blood thyroid stimulating hormone are elevated in babies born vaginally after a successful external cephalic version. This finding may represent a biological difference in fetal response to the stress of labour in breech-presenting fetuses, which is not correctable by a successful external cephalic version.

A study of teratogenicity of hydrosalpinx fluid using a whole rat embryo culture model.

BACKGROUND: Hydrosalpinx fluid may be toxic to sperm and early embryo growth. Information concerning the effect of hydrosalpinx fluid on embryo development during organogenesis is lacking. METHODS: Rat embryos at gestational day 9.5 were cultured for 48 h with 80% rat serum and 20% of either hydrosalpinx fluid (study group) or lactated Ringer's solution (control group). Embryos were scored for growth and development at the end of the culture period. RESULTS: Hydrosalpinx fluid, collected from 10 patients, was tested for embryotoxicity individually. Median total morphological scores were significantly lower in embryos exposed to hydrosalpinx fluid from three of the 10 patients (43.0 versus 47.0, P = 0.01; 36.0 versus 45.0, P < 0.001; 36.0 versus 46.5, P = 0.003). This was accompanied by a significant reduction in median yolk sac diameter (4.0 versus 5.2 mm, P < 0.001 and 4.0 versus 5.0 mm, P < 0.001) and somite number (17.5 versus 22.5, P < 0.001 and 17.0 versus 21.5, P = 0.008) in the latter two patients. CONCLUSIONS: Hydrosalpinx fluid in some patients may contain toxin(s) that is potentially teratogenic.

Porcine somatotrophin differentially down-regulates expression of the GLUT4 and fatty acid synthase genes in pig adipose tissue.

The present study was conducted to determine whether porcine somatotropin (pST) differentially regulates expression of the GLUT4 and fatty acid synthase (FAS) genes in pig adipose tissue. Three different experiments were conducted in which pigs were treated daily with different doses of pST for different time periods (7 or 14 d and from 60 to 90 kg of body wt). In these experiments, pST significantly and consistently decreased FAS mRNA levels (80%, 66% and 85%, respectively); however, GLUT4 mRNA was not affected by pST in two of the three experiments, and in the one showing an effect (Experiment 2), the decrease was less than observed for FAS (44%). Because of these results, we conducted subsequent experiments to see if the effects of pST on glucose metabolism in cultured pig adipose tissue (48 h) differed when glucose concentrations were changed from 1 to 5 mmol/L. These studies revealed that the antagonistic effect of pST on insulin action was more potent when glucose transport was saturated (5 mmol/L) than when glucose concentration limited glucose entry into the cell (1 mmol/L). In summary, these results suggest that the effects of pST on glucose transport in pig adipocytes are secondary to changes elicited by the hormone on intracellular glucose use for lipogenesis. When considered in the context of the decrease previously observed in glucose transport in pig adipocytes, the findings reported herein suggest that pST acts to decrease GLUT4 protein activity and/or distribution between the plasma membrane and the intracellular pool with little alteration in GLUT4 gene expression or total cell GLUT4 protein.

A yang-promoting Chinese herbal suppository preparation enhances the antioxidant status of red cells in male human subjects.

In the 16-week pilot study, the effect of a Yang-promoting Chinese herbal suppository preparation (VI-28) on the red cell antioxidant status was examined in 31 healthy male subjects aged 41-66 years old. VI-28 treatment for 12 weeks (one suppository (0.3 g) daily for week 1-4; one every 2 days for week 5-8; one every 3 days for week 9-12) produced a time/dose-dependent alteration in red cell antioxidant status. The VI-28-induced change is characterized by a slight depletion in cellular reduced glutathione (GSH) level and a decrease in susceptibility to peroxide-induced lipid peroxidation as well as increases in catalase (CAT) and Cu-Zn-superoxide dismutase (SOD) activities. While a reversal trend of change was observed in cellular GSH level, the susceptibility to lipid peroxidation as well as the CAT activity after the cessation of treatment for 4 weeks, the SOD activity exhibited a protracted increase. The results indicate that VI-28 treatment enhances red cell antioxidant status in male subjects. The beneficial effect of VI-28 treatment on red cells may re fl ect a corresponding change in antioxidant status of peripheral tissues.