Lithium-mediated phosphorylation of glycogen synthase kinase-3β involves PI3 kinase-dependent activation of protein kinase C-α

Lithium, a known mood-stabilizer frequently used in treatment of bipolar disorders, is an effective glycogen synthase kinase-3beta (GSK-3beta) inhibitor. This led to the idea that GSK-3beta is an in vivo target directly inhibited by lithium. As lithium is a weak in vitro inhibitor of GSK-3beta (IC50=2 mM), however, we speculated that it inhibits GSK-3beta via an indirect, yet unknown, mechanism. The present studies show that lithium increased the phosphorylation of a key inhibitory site of GSK-3beta, serine-9 (Ser-9), in HEK293 cells and in PC12 cells. This phosphorylation was significantly reduced by protein kinase C (PKC) inhibitors GF109203X and Ro31-8425, as well as GO6976, an effective inhibitor toward conventional PKC isoforms (cPKC). Consistent with these results, lithium increased PKC-alpha activity approximately twofold in both cell lines. Because PI3 kinase is a potential upstream regulator of cPKC, its inhibition by wortmannin or LY294002 also abolished the lithium-induced serine phosphorylation of GSK-3beta in HEK293 and PC12 cells. Moreover, lithium did not activate PKB, and in addition, its activity was not dependent on the presence of medium inositol nor did it affect the autophosphorylation activity of GSK-3beta. Finally, intracerebroventricular injection of lithium increased GSK-3beta Ser-9 phosphorylation and enhanced PKC-alpha activity 1.8-fold in mouse hippocampus, confirming this lithium response in vivo. Our studies propose a new mechanism by which lithium indirectly inhibits GSK-3beta via phosphatidylinositol 3 kinase- dependent activation of PKC-alpha.     

Effect of D-003, a mixture of very high molecular weight aliphatic acids, on prednisolone-induced osteoporosis in Sprague-Dawley rats

BACKGROUND: Drugs inhibiting cholesterol biosynthesis may affect bone metabolism through inhibition of the mevalonate pathway resulting in the inhibition of protein prenylation required for osteoclast activity. D-003 is a mixture of high molecular weight aliphatic primary acids purified from sugar-cane (Saccharum officinarum) wax, with cholesterol-lowering effects demonstrated in experimental and clinical studies. D-003 inhibits cholesterol biosynthesis through indirect regulation of HMG-CoA reductase activity. A previous study demonstrated that D-003 prevented bone loss and bone resorption on ovariectomy-induced osteoporosis in rats. Corticosteroid-induced osteoporosis is the result of changes affecting calcium homeostasis, but the hallmark of corticosteroid-induced bone loss is the direct effects on bone cells, such as inhibition of osteoblastogenesis, promotion of apoptosis of osteoblasts and osteocytes, and decrease in bone formation. OBJECTIVE: To determine whether D-003 could prevent the bone loss induced with prednisolone in Sprague-Dawley rats. METHODS: Rats were randomly distributed in five groups (ten rats per group): a sham-operated control and four groups orally treated with prednisolone 6 mg/kg for 80 days; a positive control orally treated with vehicle; and three groups orally treated with D-003 at 5, 25 and 200 mg/kg, respectively. Rats were killed, bones removed and histological variables of bone resorption and formation studied for histomorphometry. RESULTS: Compared with the sham group, prednisolone significantly (p < 0.01) reduced trabecular bone volume (TBV), while D-003 significantly (p < 0.001) and dose-dependently prevented the prednisolone-induced reduction of TBV. Treatment with prednisolone lowered (p < 0.001) trabecular thickness (TbTh) and number (TbN), while increasing (p < 0.001) the gap between trabeculae. D-003 (5, 25 and 200 mg/kg/day) significantly (p < 0.001) and dose-dependently prevented the reduction of TbTh and TbN and the increase of trabecular gap induced with prednisolone. Treatment with prednisolone increased both the surface and number of osteoclasts compared with sham (p < 0.001). D-003 (5-200 mg/day), however, prevented this effect (p < 0.001 for all comparisons). D-003 also prevented (p < 0.001) the reduction of osteoblast surface (ObS/BS) induced by prednisolone. Osteonecrotic areas were observed in all positive controls, but in none of the sham animals. Positive controls showed hypertrophy of bone marrow adipocytes and lipid-laden pluripotential stromal cells in bones. A significant and dose-dependent reduction of the frequency of animals showing prednisolone-induced osteo-necrosis was observed across the doses of D-003 (5, 25 and 200 mg/kg) investigated here. CONCLUSIONS: D-003 (5, 25 and 200 mg/kg) prevented trabecular bone loss and femoral neck osteonecrosis induced with prednisolone in Sprague Dawley rats, also increasing osteoblast surface and reducing bone resorption parameters. These results suggest that D-003 could be useful for managing corticosteroid-induced osteoporosis.     

Policosanol prevents bone loss in ovariectomized rats

Osteoporosis is characterized by reduced bone mass, abnormal bone architecture and increased fracture risk. Ovariectomy impairs bone mass and metabolism in rats and ovariectomized rats are considered as a suitable model of postmenopausal osteoporosis. Mevalonate is required for producing lipoids that are important in osteoclast activity and thus drugs affecting mevalonate production can prevent bone loss in rodents. Policosanol is a cholesterol-lowering drug isolated from sugar cane wax that inhibits cholesterol biosynthesis through an indirect regulation of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase activity. The purpose of this study was to determine whether policosanol could prevent bone loss in the bones of ovariectomized rats by comparing its effects with those induced by estradiol. Sprague Dawley female rats were randomly distributed in four groups: a sham-operated group treated with Tween/H2O vehicle and three groups of ovariectomized rats treated with 17beta-estradiol (30 microg/kg/day) or policosanol (50 and 200 mg/kg/day), respectively, for 3 months. At treatment completion the rats were sacrificed, their bones removed and variables of bone resorption and formation were investigated by histomorphometry. Ovariectomy increased trabecular separation but diminished the number and thickness of trabecules. Estradiol and policosanol prevented these effects compared with ovariectomized controls. Both treatments also prevented an increase in the number of osteoclasts and their surface area induced by ovariectomy. Estradiol, but not policosanol, significantly prevented an increase of osteoblast surface area compared with ovariectomized controls. In conclusion, policosanol prevented bone loss and decreased bone resorption in ovariectomized rats, suggesting that it should be potentially useful in preventing bone loss in postmenopausal women.     

C-reactive protein as a marker for active coronary artery disease in patients with chest pain in the emergency room

BACKGROUND: Markers of inflammation, such as C-reactive protein (CRP), were found to be related to risk for cardiovascular disease (CVD) events in patients with angina pectoris. In addition, recent studies have shown that, in the case of atherosclerosis, increased CRP concentration reflects the inflammatory condition of the vascular wall. HYPOTHESIS: The study was undertaken to determine whether CRP levels in individuals with chest pain attending the emergency room (ER) may be used as a marker of active CVD. METHODS: Serum CRP level was measured in 226 of 326 consecutive patients (128 men, 98 women; mean age 61.3 +/- 5.9 years; range 19-87 years) referred to the ER with chest pain. The decision whether to admit orrelease the subjects was determined without taking the CRP level into account. Follow-up was then performed for 1 year. RESULTS: Eighty-four patients were admitted to the hospital. Of these, 9 with acute coronary syndrome (ACS) had very high levels of CRP (25-40 mg/l), 35 had had an acute coronary event within the preceding 3 months, with levels of CRP 14-20 mg/l. Only eight patients with nonsignificant CVD had elevated CRP levels. Twenty-eight subjects who were released from the ER had elevated CRP levels (7-14 mg/l); 8 of these, in addition to 4 subjects with normal CRP levels, had a late coronary event. CONCLUSION: This study indicates that in patients referred to the ER with chest pain and no other indication for hospitalization, a normal level of CRP suggests safe release. Most hospitalized patients with normal CRP will not have acute coronary syndrome. Patients who will develop early coronary events have very high CRP levels. High serum CRP level, after excluding other inflammatory sources, was proven to be a sensitive diagnostic and prognostic marker for significant coronary disease.     

Breast engorgement and galactorrhea during magnesium sulfate treatment of preterm labor

Breast engorgement and galactorrhea occasionally occur during tocolysis with ritodrine. We are not aware of breast engorgement and galactorrhea associated with other tocolytics. We report the first case of breast engorgement and galactorrhea during tocolysis with intravenous magnesium sulfate in a generally healthy 24-year-old woman admitted for tocolysis at 30 weeks' gestation. Breast engorgement and galactorrhea gradually subsided after magnesium sulfate was discontinued. The rapid disappearance of both the galactorrhea and the breast engorgement after discontinuation of magnesium sulfate treatment suggests a cause-effect relationship, the mechanism of which remains unclear.     

Bacteremia due to beta-hemolytic Streptococcus group G: increasing incidence and clinical characteristics of patients

PURPOSE: To describe the epidemiology and clinical characteristics of patients diagnosed with Streptococcus group G bacteremia from 1990 to 1999 at a community teaching hospital in Israel. SUBJECTS AND METHODS: We calculated the annual rate of bacteremia with Streptococcus group G, expressed as a percentage of positive blood cultures (after excluding contaminants) and per 1000 admissions. Medical records of patients with Streptococcus group G were reviewed. RESULTS: During the 10-year study period, there was a total of 7415 positive blood cultures, 327 (4.4%) of which were beta-hemolytic Streptococcus species, of which 49 (15%) were group G. The rate of Streptococcus group G bacteremia per 1000 admissions increased from zero (0/18,783) in 1990 to 0.41 (13/31,440) in 1999 (P = 0.001), surpassing Streptococcus group A in frequency. Of the 47 patients with Streptococcus group G, 40 medical records were available for review: 25 patients (63%) were older than 75 years and 32 (80%) were men. The probable source of Streptococcus group G bacteremia was a skin or soft tissue infection in 37 patients (93%). Six of the 40 patients died. CONCLUSION: Community-acquired group G streptococcal bacteremia occurred with increasing frequency from 1990 to 1999 at our hospital. Most patients were elderly men, and the portal of entry was usually the skin or soft tissue. Our findings suggest a change in the epidemiology of bacteremia due to beta-hemolytic streptococci.     

Two types of intrinsic muscarinic responses in Xenopus oocytes

Fully grown Xenopus laevis oocytes display marked morphological asymmetry. The giant cell is divided into animal (pigmented) and vegetal hemispheres. We have developed methodology aimed at easy determination of hemispheric responses to the application of acetylcholine (ACh) and determination of the distribution of muscarinic receptors. Oocytes of common donors exhibit muscarinic responses that are similar when either the animal or the vegetal hemisphere of the cell is exposed to ACh. Oocytes of variant donors, however, exhibit markedly larger muscarinic responses when the animal hemisphere is exposed to ACh (ratio animal/vegetal, 5.8). The differences in hemispheric responsiveness correlate well with the hemispheric distribution of muscarinic receptors. While oocytes of common donors exhibit a modest excess of receptor number at the animal hemisphere (ratio animal/vegetal hemispheres, 1.4), oocytes of variant donors exhibit a large excess of receptors on the animal hemisphere (ratio animal/vegetal, 5.6). Upon further examination, we have found that the distribution of muscarinic receptors is non-homogeneous in either hemisphere in oocytes of both common and variant donors. The asymmetric distribution of receptors may be related to increased efficiency of signal transduction coupling in oocytes of variant donors.     

Messenger RNA Expression of Transporter and Ion Channel Genes in Undifferentiated and Differentiated Caco-2 Cells Compared to Human Intestines

Purpose. The purpose of this work was to study the influence of cell differentiation on the mRNA expression of transporters and channels in Caco-2 cells and to assess Caco-2 cells as a model for carrier-mediated drug transport in the intestines. Method. Gene mRNA expression was measured using a custom-designed microarray chip with 750 deoxyoligonucleotide probes (70mers). Each oligomer was printed four times on poly-lysine-coated glass slides. Expression profiles were expressed as ratio values between fluorescence intensities of Cy3 and Cy5 dye-labeled cDNA derived from poly(A) + RNA samples of Caco-2 cells and total RNA of human intestines. Results. Significant differences in the mRNA expression profile of transporters and channels were observed upon differentiation of Caco-2 cells from 5 days to 2 weeks in culture, including changes for MAT8, S-protein, and Nramp2. Comparing Caco-2 cells of different passage number revealed few changes in mRNAs except for GLUT3, which was down-regulated 2.4-fold within 13 passage numbers. Caco-2 cells had a similar expression profile when either cultured in flasks or on filters but differed more strongly from human small and large intestine, regardless of the differentiation state of Caco-2 cells. Expression of several genes highly transcribed in small or large intestines differed fourfold or more in Caco-2 cells. Conclusions. Although Caco-2 cells have proven a suitable model for studying carrier-mediated transport in human intestines, the expression of specific transporter and ion channel genes may differ substantially.