Transmodulation between phospholipase D and c-Src enhances cell proliferation

Phospholipase D (PLD) has been implicated in the signal transduction pathways initiated by several mitogenic protein tyrosine kinases. We demonstrate for the first time that most notably PLD2 and to a lesser extent the PLD1 isoform are tyrosine phosphorylated by c-Src tyrosine kinase via direct association. Moreover, epidermal growth factor induced tyrosine phosphorylation of PLD2 and its interaction with c-Src in A431 cells. Interaction between these proteins is via the pleckstrin homology domain of PLD2 and the catalytic domain of c-Src. Coexpression of PLD1 or PLD2 with c-Src synergistically enhances cellular proliferation compared with expression of either molecule. While PLD activity as a lipid-hydrolyzing enzyme is not affected by c-Src, wild-type PLDs but not catalytically inactive PLD mutants significantly increase c-Src kinase activity, up-regulating c-Src-mediated paxillin phosphorylation and extracellular signal-regulated kinase activity. These results demonstrate the critical role of PLD catalytic activity in the stimulation of Src signaling. In conclusion, we provide the first evidence that c-Src acts as a kinase of PLD and PLD acts as an activator of c-Src. This transmodulation between c-Src and PLD may contribute to the promotion of cellular proliferation via amplification of mitogenic signaling pathways.     

Conformational preferences in diastereomeric(5S)-methyl-3-(o-aryl)-2,4-oxazolidinediones

The thermally interconvertible diastereomers of the (5S)-methyl-3-(o-aryl)-2,4-oxazolidinediones were synthesized and their conformers studied by (1)H NMR and HPLC. The barriers to rotation about the N-C(aryl) bond were found to be very much solvent dependent. For the o-fluoro oxazolidinedione, difference in barriers to rotation in deuterated methanol and deuterated chloroform amounted to 34 kJ/mol. ortho-Bromo substitution increased the barrier to rotation up to 100 kJ/mol in ethanol, which enabled the analytical separation of the diastereomers and observation of the thermodynamic enrichment of the S-P conformer by HPLC. In CDCl(3) by (1)H NMR, on the other hand, a barrier of only 89 kJ/mol was determined. The S-M and S-P conformers of the diastereomers of o-methyl, alpha-naphthyl and o-iodo derivatives have been assigned by NOESY experiments and the kinetic and thermodynamic constants for the interconversion between the S-M and S-P conformers were determined.     

Model of ionic transport for bovine ciliary epithelium: effects of acetazolamide and HCO

The possible existence of transepithelial bicarbonate transport across the isolated bovine ciliary body was investigated by employing a chamber that allows for the measurement of unidirectional, radiolabeled fluxes of CO2 + HCO. No net flux of HCO was detected. However, acetazolamide (0.1 mM) reduced the simultaneously measured short-circuit current (I(sc)). In other experiments in which (36)Cl- was used, a net Cl- flux of 1.12 microeq. h(-1). cm(-2) (30 microA/cm(2)) in the blood-to-aqueous direction was detected. Acetazolamide, as well as removal of HCO from the aqueous bathing solution, inhibited the net Cl- flux and I(sc). Because such removal should increase HCO diffusion toward the aqueous compartment and increase the I(sc), this paradoxical effect could result from cell acidification and partial closure of Cl- channels. The acetazolamide effect on Cl- fluxes can be explained by a reduction of cellular H+ and HCO (generated from metabolic CO2 production), which exchange with Na+ and Cl- via Na+/H+ and Cl-/HCO exchangers, contributing to the net Cl- transport. The fact that the net Cl- flux is about three times larger than the I(sc) is explained with a vectorial model in which there is a secretion of Na+ and K+ into the aqueous humor that partially subtracts from the net Cl- flux. These transport characteristics of the bovine ciliary epithelium suggest how acetazolamide reduces intraocular pressure in the absence of HCO transport as a driving force for fluid secretion.     

The protective effect of lisinopril on membrane-bound enzymes in myocardial preservation

A number of studies have reported that oxidant stress reduces the activity of isolated Na(+)-K(+) ATPase and Ca(2+) ATPase which are known to affect the cell membrane integrity. The aim of the study is to determine whether the administration of lisinopril is able to protect the membrane-bound enzyme levels in isolated guinea pig hearts and also ascertain whether or not a relationship exists between oxygen free radicals and membrane bound Na(+)-K(+) ATPase and Ca(2+) ATPase. Forty guinea pig hearts were studied in an isolated Krebs-Henseleit solution-perfused Langendorff cardiac model. In all groups cardioplegic arrest was achieved by administering St. Thomas' Hospital cardioplegic solution (STHCS). Group 1 (control, n=10) received only STHCS. Group 2 (n=10) were arrested with lisinopril (l micromol l(-1)) added STHCS. Group 3 (n=10) were pretreated with oral lisinopril (0.2 mg kg(-1) twice a day) for 10 days and then arrested with STHCS. Group 4 were also pretreated with oral lisinopril (0.2 mg kg(-1) twice a day for 10 days), arrested with STHCS and reperfused with lisinopril added to Krebs-Henseleit solution (l micromol l(-1)). Hearts were subjected to normothermic global ischaemia for 90 min and then reperfused at 37 degrees C. Pretreatment and addition of lisinopril in the reperfusion buffer improved the levels of membrane-bound enzymes. When the treated groups were compared with control hearts, the best results were achieved in group 4. The Na(+)-K(+) and Ca(2+) ATPase levels increased from 466.38+/-5.99 to 560.12+/-18.02 and 884.69+/-9.13 to 1287.71+/-13.01 nmolPi mg(-1) protein h(-1) respectively (p<0.05). These results suggest that lisinopril protects the cell membrane integrity and lessens free radical-induced oxidant stress.     

Ion channels-related diseases

There are many diseases related to ion channels. Mutations in muscle voltage-gated sodium, potassium, calcium and chloride channels, and acetylcholine-gated channel may lead to such physiological disorders as hyper- and hypokalemic periodic paralysis, myotonias, long QT syndrome, Brugada syndrome, malignant hyperthermia and myasthenia. Neuronal disorders, e.g., epilepsy, episodic ataxia, familial hemiplegic migraine, Lambert-Eaton myasthenic syndrome, Alzheimer's disease, Parkinson's disease, schizophrenia, hyperekplexia may result from dysfunction of voltage-gated sodium, potassium and calcium channels, or acetylcholine- and glycine-gated channels. Some kidney disorders, e.g., Bartter's syndrome, policystic kidney disease and Dent's disease, secretion disorders, e.g., hyperinsulinemic hypoglycemia of infancy and cystic fibrosis, vision disorders, e.g., congenital stationary night blindness and total colour-blindness may also be linked to mutations in ion channels.     

Candida albicans and Saccharomyces cerevisiae cell wall mannans produce fever in rats: role of nitric oxide and cytokines

Mannan components of C. albicans (5 mg/kg, i.p.) and S. cerevisiae (2.5 mg/kg, i.p.) cell walls produced pyrogenic responses which were completely inhibited by indomethacin (5 mg/kg, s.c.) pretreatment in rats. A non-selective NOS inhibitor, L-NAME (10 mg/kg, s.c.), also inhibited the pyrogenic effectiveness of C. albicans mannan, whereas it was ineffective on the fever induced by S. cerevisiae mannan. A selective elevation in the serum TNF-alpha levels was observed at the initial phase of the fever due to S. cerevisiae mannan, whereas there was no significant change on the serum levels of TNF-alpha, IL-1beta and IFN-gamma during the latent period or at the initial phase of the fever induced by C. albicans mannan. Injections of N-linked and/or O-linked oligomannosides of the either mannan did not cause any significant change in the body temperature and serum cytokine levels. These data suggest that the mannan components of C. albicans and S. cerevisiae cell walls produce a prostaglandin-dependent fever in rats. The initial signal for fever seems to be different for each mannan. Data also indicate that integrity of the mannans is necessary for the pyrogenic response.     

Production of poly-γ-glutamic acid by fed-batch culture of Bacillus licheniformis

Fed-batch cultures of Bacillus licheniformis produced poly--glutamic acid (PGA), a water-soluble biodegradable polymer. PGA reached 35 g l^–1 with a productivity of 1 g l^–1 h^–1 by pulsed-feeding of citric acid (1.44 g h^–1) and l-glutamic acid (2.4 g h^–1) when citric acid was depleted from the culture medium.     

Rapid regeneration of whole plants in large crabgrass (Digitaria sanguinalis L.) using thin-cell-layer culture

A method was designed to produce rapidly (10–14 days) and directly (without intermediate callus) whole plants of Digitaria sanguinalis with a high yield without subculture. These plants developed from new structures, designated “pseudo-embryogenic structures”, initiated only 1 week after the culture of tranverse thin cell layers (tTCLs), i.e., thin stem sections, on a Murashige and Skoog medium containing 3% sucrose and a combination of a low concentration of 2,4-dichlorophenoxyacetic acid (1 μm) and a high concentration of 6-benzylaminopurine (10 μm). The fresh weight of plants regenerated per tTCL on gelrite was 6 times higher than with agar and 30 times higher than with agarose. Received: 25 August 1997 / Revision received: 18 February 1998 / Accepted: 10 March 1998