Obligatory role of Src kinase in the signaling mechanism for TRPC3 cation channels

Members of the canonical transient receptor potential (TRPC) subfamily of cation channels are candidates for capacitative and non-capacitative Ca2+ entry channels. When ectopically expressed in cell lines, TRPC3 can be activated by phospholipase C-mediated generation of diacylglycerol or by addition of synthetic diacylglycerols, independently of Ca2+ store depletion. Apart from this mode of regulation, little is known about other receptor-dependent signaling events that modulate TRPC3 activity. In the present study the role of tyrosine kinases in receptor- and diacylglycerol-dependent activation of TRPC3 was investigated. In HEK293 cells stably expressing TRPC3, pharmacological inhibition of tyrosine kinases, and specifically of Src kinases, abolished activation of TRPC3 by muscarinic receptor stimulation and by diacylglycerol. Channel regulation was lost following expression of a dominant-negative mutant of Src, or when TRPC3 was expressed in an Src-deficient cell line. In both instances, wild-type Src restored TRPC3 regulation. We conclude that Src plays an obligatory role in the mechanism for receptor and diacylglycerol activation of TRPC3.     

Discovering an epidemic before it has reached a certain level of prevalence

In this communication we calculate the probability of discovering a simple epidemic in a large population before the epidemic has reached a given level of prevalence, by regularly taking a small random sample from the population for microbiological screening. Apart from the general formula which has to be evaluated numerically, we derive various simple approximation formulae which shed light on the properties of various monitoring regimes. These formulae are, moreover, rather robust against deviations from the model specifications. The results are applied to the evaluation of the efficiency of an infection-monitoring program in an animal breeding centre.     

Meta-analysis of how well measures of bone mineral density predict occurrence of osteoporotic fractures.

OBJECTIVE--To determine the ability of measurements of bone density in women to predict later fractures. DESIGN-- Meta-analysis of prospective cohort studies published between 1985 and end of 1994 with a baseline measurement of bone density in women and subsequent follow up for fractures. For comparative purposes, we also reviewed case control studies of hip fractures published between 1990 and 1994. SUBJECTS--Eleven separate study populations with about 90,000 person years of observation time and over 2000 fractures. MAIN OUTCOME MEASURES--Relative risk of fracture for a decrease in bone mineral density of one standard deviation below age adjusted mean. RESULTS--All measuring sites had similar predictive abilities (relative risk 1.5 (95% confidence interval 1.4 to 1.6)) for decrease in bone mineral density except for measurement at spine for predicting vertebral fractures (relative risk 2.3 (1.9 to 2.8)) and measurement at hip for hip fractures (2.6 (2.0 to 3.5)). These results are in accordance with results of case-control studies. Predictive ability of decrease in bone mass was roughly similar to (or, for hip or spine measurements, better than) that of a 1 SD increase in blood pressure for stroke and better than a 1 SD increase in serum cholesterol concentration for cardiovascular disease. CONCLUSIONS--Measurements of bone mineral density can predict fracture risk but cannot identify individuals who will have a fracture. We do not recommend a programme of screening menopausal women for osteoporosis by measuring bone density.     

Social influences and cardiovascular risk factors as determinants of plasma fibrinogen concentration in a general population sample of middle aged men.

OBJECTIVE--To analyse the relation between fibrinogen concentration and social class and other social factors found to be related to mortality. The results regarding cardiovascular disease are unpublished, as yet. DESIGN--Cross sectional population study. SETTING--City of Gothenburg, Sweden. SUBJECTS--639 Men from a population sample of 1016 men aged 50 in 1983. They were all employed and had no history of myocardial infarction or stroke. Fibrinogen values were available for all of them. MAIN OUTCOME MEASURE--Fibrinogen concentration in relation to socioeconomic state according to occupation, and other social influences determined as number of people in the household and scores of social activities and activities in and outside the house. RESULTS--Men with low scores for activities at home had a mean plasma fibrinogen concentration of 3.34 g/l (95% confidence interval 3.21 to 3.47), whereas those with an intermediate score had a mean concentration of 3.16 (3.00 to 3.32) g/l and those with a high score 3.02 (2.95 to 3.10) g/l. Similar inverse relations were noted for the two other activity scores and for occupational class (class 1 being unskilled and semiskilled workers and class 5 professionals and executives) and the number of people in the household. Smoking exerted a strong influence on fibrinogen concentration, the relations between fibrinogen concentration and social factors being evident only in non-smokers. The mean difference in fibrinogen value between the non-smokers with the lowest activity scores at home and those with the highest scores was 0.36 (0.19 to 0.54) g/l, and similar differences were seen for the two other activity scores. Multiple regression analyses showed smoking, body mass index, the sum of all activities (inverse relation), and diabetes to be independently associated with fibrinogen value, whereas occupational class (p = 0.81) and the number of people in the household (p = 0.09) were not. CONCLUSIONS--Psychosocial influences seem to influence the coagulation system in the body in a way that is associated with cardiovascular disease and premature death.     

Inhibitory effects of the HDAC inhibitor valproic acid on prostate cancer growth are enhanced by simultaneous application of the mTOR inhibitor RAD001

AimsTo analyze the combined impact of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 on prostate cancer cell growth.Main methodsPC-3, DU-145 and LNCaP cells were treated with RAD001, VPA or with an RAD001–VPA combination for 3 or 5 days. Tumor cell growth, cell cycle progression and cell cycle regulating proteins were then investigated by MTT assay, flow cytometry and Western blotting, respectively. Effects of drug treatment on cell signaling pathways were determined.Key findingsSeparate application of RAD001 or VPA distinctly reduced tumor cell growth and impaired cell cycle progression. Significant additive effects were evoked when both drugs were used in concert. Particularly, the cell cycle regulating proteins cdk1, cdk2, cdk4 and cyclin B were reduced, whereas p21 and p27 were enhanced by the RAD001–VPA combination. Signaling analysis revealed deactivation of EGFr, ERK1/2 and p70S6k. Phosphorylation of Akt was diminished in DU-145 but elevated in PC-3 and LNCaP cells.SignificanceThe RAD001–VPA combination exerted profound antitumor properties on a panel of prostate cancer cell lines. Therefore, simultaneous blockage of HDAC and mTOR related pathways should be considered when designing novel therapeutic strategies for treating prostate carcinoma.     

Analysis of cDNA Clones Encoding the Entire Ferredoxin I Precursor Polypeptide from Spinach

We report the isolation and characterization of a recombinant cDNA phage from a lambda gt11 expression library made from polyadenylated spinach RNA that encodes the entire precursor polypeptide of ferredoxin I. The deduced sequence predicts a molecular mass of 10.5 kDa (97 amino acid residues) for the mature protein and a transit peptide of 50 residues (5.2 kDa). In vitro synthesized ferredoxin precursor was used for import experiments with isolated unbroken spinach chloroplasts. The polypeptide was correctly directed to the organelle stroma and processed to the size of the mature protein. Northern analysis indicates a mRNA size of ca. 850 nucleotides which is close to the size of the cDNA insert (ca. 700 bp).     

The xenoestrogen bisphenol A in the Hershberger assay: androgen receptor regulation and morphometrical reactions indicate no major effects

We evaluated androgen-like effects of bisphenol A (BPA) using orchiectomized Wistar rats. Animals were treated p.o. either with vehicle or with 3, 50, 200, 500 mg/kgbw/day BPA (n=13) for 7 days. One group was treated s.c. with 1mg/kgbw/day testosterone propionate (TP). Flutamide (FL) (3mg/kgbw/day, p.o.) was used to antagonize androgen effects of the suprapharmacological dose (500 mg/kgbw/day) of BPA. Androgen-like effects of BPA on prostates and seminal vesicles were assessed by the Hershberger assay, densitometric analysis of androgen receptor (AR) immunoreactivity, cell proliferation-index and a morphometric analysis. Absolute weights of prostates and seminal vesicles were not increased by BPA, whereas the relative weights were increased at higher doses of BPA, most likely due to a decrease in body weight. Staining intensity for AR immunoreactivity was increased at low but not at higher doses of BPA in comparison to the orchiectomized rats. BPA at all doses tested did not cause an increase of the cell proliferation-index. Epithelial height and glandular luminal area were increased by low doses of BPA, whereas higher doses caused a decrease of these parameters. The data provide evidence that BPA does not exert major androgenic effects.     

Expression level of the canonical transient receptor potential 3 (TRPC3) channel determines its mechanism of activation

Studies on the mechanism of activation of canonical transient receptor potential (TRPC) channels have often yielded conflicting results. In the current study, we have investigated the influence of expression level on the mode of regulation of TRPC3 channels. At relatively low levels of expression in DT40 chicken B-lymphocytes, TRPC3 was activated by the depletion of Ca2+ stores. Expression was increased by either transfecting with a 10-fold greater concentration of plasmid or transfecting with TRPC3 under control of a more efficient avian beta-actin promoter. At higher levels of expression, TRPC3 was no longer store-operated but could be activated through receptor-coupled phospholipase C. Under these expression conditions, TRPC3 was efficiently activated in DT40 cells lacking inositol 1,4,5-trisphosphate receptors. The Ca2+ store-operated channels formed upon expression of TRPC3 at limited levels were blocked by gadolinium; the receptor-activated channels formed upon expression of higher levels of TRPC3 were insensitive to gadolinium. These findings indicate that a single ion channel protein can form or contribute to the formation of channels regulated in two very distinct ways, i.e. either by phospholipase C-derived messengers or Ca2+ store-depletion. The mechanism of regulation of the channels depends on their level of expression.