Evidence for a role of NA+/H+ exchange in platelets activated with calcium-ionophore A 23187

We have investigated the release of protons from human platelets and platelet aggregation induced by the calcium ionophore, A 23187. Addition of the ionophore to suspensions of washed platelets resulted in fast liberation of H+. In the presence of 0.2 mM amiloride, a potent inhibitor of Na+/H+ countertransport, the amount of protons liberated was decreased by 50% and was further reduced to about 10% by 1 mM amiloride. Similar inhibition of H+-release was observed after decreasing Na+ in the incubation medium. Both results suggest that increasing internal Ca2+ by the ionophore induces Na+/H+ exchange in human platelets. Platelet aggregation could be induced by adding the ionophore to the platelet suspension. This aggregation was inhibited by amiloride, at least when induced by low ionophore concentrations. The results suggest that stimulation of Na+/H+ exchange, and the concomitant increase in intraplatelet pH, are important mechanisms in platelet activation.     

Gas mixing in the airways of dog lungs during high-frequency ventilation

Washout of insoluble inert test gases of different diffusivity (He and SF6 or He and Ar) from dog lungs was studied during high-frequency ventilation (HFV). Test gas equilibrium and subsequent washout were performed with HFV, succeeding measurements being performed at different stroke volumes (1.5-2.5 ml/kg body wt), oscillation frequencies (10-30 Hz), and with different lung volumes (32-74 ml X kg-1). Test gas concentrations were continuously measured by a mass spectrometer. The time course of washout could be described as the sum of two exponentials. There were no consistent differences in the time courses of washout between He and SF6 or between He and Ar. It is concluded that gas mixing in the airways during HFV is not significantly limited by diffusion, and this is suggested to apply during HFV to steady-state transport of respiratory gases (e.g., O2 and CO2) as well as to the transient state of inert gas washout.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

Monocarbonyl Analogs of Curcumin with Potential to Treat Colorectal Cancer

Curcumin has a plethora of biological properties, making this compound potentially effective in the treatment of several diseases, including cancer. However, curcumin clinical use is compromised by its poor pharmacokinetics, being crucial to find novel analogs with better pharmacokinetic and pharmacological properties. Here, we aimed to evaluate the stability, bioavailability and pharmacokinetic profiles of monocarbonyl analogs of curcumin. A small library of monocarbonyl analogs of curcumin 1a–q was synthesized. Lipophilicity and stability in physiological conditions were both assessed by HPLC-UV, while two different methods assessed the electrophilic character of each compound monitored by NMR and by UV-spectroscopy. The potential therapeutic effect of the analogs 1a–q was evaluated in human colon carcinoma cells and toxicity in immortalized hepatocytes. Our results showed that the curcumin analog 1e is a promising agent against colorectal cancer, with improved stability and efficacy/safety profile.     

Requirement of N-terminal amino acid residues of gp41 for human immunodeficiency virus type 1-mediated cell fusion.

An expression vector was designed to test the structural requirements of the gp41 N terminus for human immunodeficiency virus type 1-induced membrane fusion. Mutations in the region coding for the N terminus of gp41 were found to disrupt glycoprotein expression because of deleterious effects on the Rev-responsive element (RRE). Insertion of an additional RRE in the 3'-noncoding sequence of env made possible efficient glycoprotein expression, irrespective of the mutations introduced into the RRE in the natural location. This permitted the insertion of the unique restriction site SpeI within the N-terminal sequences of gp41, allowing convenient and efficient mutation of the gp41 N terminus by using double-stranded synthetic oligonucleotides. Mutants with deletions of 1 to 7 amino acids of the N terminus were constructed. Expression and cleavage of all mutants were confirmed by Western immunoblot analysis with anti-gp41 antibodies. The capability of mutants to induce membrane fusion was monitored following transfection of HeLa-T4+ cell lines with wild-type and mutant expression vectors by electroporation and microinjection. The efficiency of cell-fusing activity decreased drastically with deletion of 3 and 4 amino acids and was completely lost with deletion of 5 amino acids. Cotransfection of the parent and mutant expression vectors resulted in reduced cell-fusing activity. The extent of this dominant interference by mutant glycoprotein paralleled the decrease in cell-fusing activity of the mutants alone. This suggests the existence of a specific N-terminal structure required for fusing activity. However, there does not appear to be a stringent requirement for the precise length of the N terminus. This finding is supported by the length variation of this region among natural human immunodeficiency virus type 1 isolates and is in contrast to the apparent stringency in the length of analogous N-terminal structures of influenza A virus and paramyxovirus fusion glycoproteins.     

Sampling properties of DNA sequence data in phylogenetic analysis

We inferred phylogenetic trees from individual genes and random samples of nucleotides from the mitochondrial genomes of 10 vertebrates and compared the results to those obtained by analyzing the whole genomes. Individual genes are poor samples in that they infrequently lead to the whole-genome tree. A large number of nucleotide sites is needed to exactly determine the whole-genome tree. A relatively small number of sites, however, often results in a tree close to the whole-genome tree. We found that blocks of contiguous sites were less likely to lead to the whole-genome tree than samples composed of sites drawn individually from throughout the genome. Samples of contiguous sites are not representative of the entire genome, a condition that violates a basic assumption of the bootstrap method as it is applied in phylogenetic studies.      

Effect of the TP5 analogue of thymopoietin on the rejection of male skin by aged and thymectomized female mice

Although young adult C3H/HeJ (OH) females do not reject C3H male skin grafts, OH females older than 1 year commonly do so, as also do many thymectomized, young adult C3H females. Therapy With TP5, a synthetic pentapeptide analogue of thymopoietin which has biological properties of the parent molecule, substantially reduced the capacity of aged OH females and of thymectomized, young OH females to reject OH male skin.     

Lack of in vivo transcription of Acetabularia mediterranea 1175 bp ctDNA fragment homologous to the Drosophila per locus.

The period (per) locus of Drosophila melanogaster has a fundamental role in the expression of biological rhythms. A DNA sequence homologous to a short region of the Drosophila per locus was detected in the chloroplast of Acetabularia mediterranea. A 1175 bp DNA fragment containing the sequence was used as a probe in 'Northern' hybridization experiments. It was found that this DNA was not transcribed or only marginally transcribed in A. mediterranea, at least at the developmental stage just prior to cap formation. It seems that the 1175 bp ctDNA fragment is not involved in the Acetabularia biological rhythm mechanism.