Activation of NKCC1 by hyperosmotic stress in human tracheal epithelial cells involves PKC-delta and ERK

Hyperosmotic stress activates Na+-K+-2Cl- cotransport (NKCC1) in secretory epithelia of the airways. NKCC1 activation was studied as uptake of 36Cl or 86Rb in human tracheal epithelial cells (HTEC). Application of hypertonic sucrose or NaCl increased bumetanide-sensitive ion uptake but did not affect Na+/H+ and Cl-/OH-(HCO3-) exchange carriers. Hyperosmolarity decreased intracellular volume (Vi) after 10 min from 7.8 to 5.4 microl/mg protein and increased intracellular Cl- (Cl-i) from 353 to 532 nmol/mg protein. Treatment with an alpha-adrenergic agent rapidly increased Cl-i and Vi in a bumetanide-sensitive manner, indicating uptake of ions by NKCC1 followed by osmotically obligated water. These results indicate that HTEC act as osmometers but lose intracellular water slowly. Hyperosmotic stress also increased the activity of PKC-delta and of the extracellular signal-regulated kinase ERK subgroup of the MAPK family. Activity of stress-activated protein kinase JNK was not affected by hyperosmolarity. PD-98059, an inhibitor of the ERK cascade, reduced ERK activity and bumetanide-sensitive 36Cl uptake. PKC inhibitors blocked activation of ERK indicating that PKC may be a downstream activator of ERK. The results indicate that hyperosmotic stress activates NKCC1 and this activation is regulated by PKC-delta and ERK.     

Contractile function of rat myocardium is less susceptible to hypoxia/reoxygenation after acute infarction

The FHIT (fragile histidine triad) gene located at chromosome 3p14.2 has been proposed as a candidate tumor suppressor gene in human cancers. Fhit protein with the diadenosine 5',5'-P¹,P³-triphosphate (Ap₃A) hydrolase activity is the protein product of FHIT gene. The way in which Fhit exerts its tumor suppressor activity and the relationship of the Ap₃A hydrolase activity to tumor suppression are not known. As a step toward understanding of the Fhit function in the cell we have explored its intracellular localization and distribution in the rat tissues. Data obtained from immunoblot analysis showed that Fhit protein was most abundant in spleen and brain. Moderate amount of Fhit was detected in kidney and liver, whereas the level of Fhit protein in heart, skeletal muscle and kidney glomeruli was undetectable. RT-PCR performed on RNA isolated from these tissues showed no product, whereas the level of Fhit mRNA in spleen, brain, kidney, liver and lung correlated with the Fhit protein level. The immunoblot analysis performed on subcellular fractions of various rat tissues obtained by differential and density-gradient centrifugation showed that Fhit protein was localized exclusively in nucleus and at the plasma membrane. Presented data showing nuclear and plasma membrane localization of Fhit may support the hypothesis concerning Fhit as a signaling molecule.     

Substrate specificity of lipase B from Candida antarctica in the synthesis of arylaliphatic glycolipids

Arylaliphatic glycolipids are known for their pharmaceutical and medicinal properties. We found that a great variety of arylaliphatic esters can be synthesized from non-activated substrates like glucose or the natural occurring drug salicin using lipase B from Candida antarctica (CAL-B). However, esters based on aromatic carboxylic acids or unsaturated arylaliphatic acids, like cinnamic acid and its derivatives, which are known to display anticancer activity, could not be obtained. In this work, we performed computer-aided molecular modeling based on data of our work published recently and syntheses of new glycolipids to understand why some substances are accepted by CAL-B while some are not. For this purpose, we investigated the accessibility of the lipase binding site for the arylaliphatic acyl donors as well as the steric interactions between the aglycons of glucosides and the residues of the alcohol binding pocket in order to elucidate potentials and limitations of CAL-B for the synthesis of aromatic glycolipids.     

Estimations of Bacterial Growth Rates in Natural Waters

Specific growth rates as low as 0.005 hr⁻¹ (generation times of 20 to 200 hr) of aquatic bacteria in natural waters have been calculated from significant differences between dilution rates and washout rates in a chemostat. The measured growth rates were affected by the treatment of the water samples (type of sterilization) and by competition with the natural microflora for the unknown growth-limiting substrate.     

A site-directed mutagenesis study to identify amino acid residues involved in the catalytic function of the restriction endonuclease EcoRV.

We have used site-directed mutagenesis of the EcoRV restriction endonuclease to change amino acid side chains that have been shown crystallographically to be in close proximity to the scissile phosphodiester bond of the DNA substrate. DNA cleavage assays of the resulting mutant proteins indicate that the largest effects on nucleolytic activity result from substitution of Asp74, Asp90, and Lys92. We suggest on the basis of structural information, mutagenesis data, and analogies with other nucleases that Asp74 and Asp90 might be involved in Mg2+ binding and/or catalysis and that Lys92 probably stabilizes the pentacovalent phosphorus in the transition state. These amino acids are part of a sequence motif, Pro-Asp...Asp/Glu-X-Lys, which is also present in EcoRI. In both enzymes, it is located in a structurally similar context near the scissile phosphodiester bond. A preliminary mutational analysis with EcoRI indicates that this sequence motif is of similar functional importance for EcoRI and EcoRV. On the basis of these results, a proposal is made for the mechanism of DNA cleavage by EcoRV and EcoRI.     

Accumulation of Hydroxycoumarins During Post-harvest Deterioration of Tuberous Roots of Cassava (Manihot esculenta Crantz)

The use of the root crop Cassava (Manihot esculenta Crantz)is constrained by its rapid deterioration after harvest. Chemicaland spectroscopic examination revealed the accumulation of fourhydroxycoumarins (esculin, esculetin, scopolin and scopoletin),compounds derived from the phenylpropanoid pathway, during thetime course of post-harvest deterioration. Fluorescence-microscopyrevealed their localization in the apoplast of the parenchyma.Scopoletin and scopolin showed the most dramatic increases inconcentration, peaking by day 2 after harvesting. A smallersecondary peak of scopoletin tended to be more pronounced incultivars showing lower susceptibility to deterioration. Evidencefor the metabolism of scopoletin to an insoluble coloured productby means of a peroxidase is presented. This product may be thecause of the discolouration of the vascular tissue during storage.Copyright 2000 Annals of Botany Company Cassava, hydroxycoumarins, Manihot esculenta, peroxidases, post-harvest physiological deterioration, wound response