Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels

We studied the effects of mutations of positively charged amino acid residues in the pore of X. tropicalis TMEM16A calcium-activated chloride channels: K613E, K628E, K630E; R646E and R761E. The activation and deactivation kinetics were not affected, and only K613E showed a lower current density. K628E and R761E affect anion selectivity without affecting Na⁺ permeation, whereas K613E, R646E and the double mutant K613E + R646E affect anion selectivity and permeability to Na⁺. Furthermore, altered blockade by the chloride channel blockers anthracene-9-carboxylic acid (A-9-C), 4, 4''-Diisothiocyano-2,2''-stilbenedisulfonic acid (DIDS) and T16inh-A01 was observed. These results suggest the existence of 2 binding sites for anions within the pore at electrical distances of 0.3 and 0.5. These sites are also relevant for anion permeation and blockade.     

The Development of a Phytoremediation Technique for the Detoxification of Soils Contaminated with Phenolic Compounds Using Horseradish Peroxidase (Armoracia rusticana): Preliminary Results

The proposed phytoremediation technique is based on the successful exploitation and optimization of oxidative coupling, mediated by horseradish peroxidase. Susceptibility to degradation of a selection of phenolic compounds, in solution, by horseradish peroxidase appears to be structurally related and was found to be of the order 2,4-dichlorophenol (2,4-DCP) > 4-chlorophenol (4-CP) > 2-chlorophenol (2-CP). Only 1.89% of 2,4-DCP, at an initial concentration of 5 mM, remained unchanged at the end of the experiment. Reaction rates between purified horseradish peroxidase and 2,4-DCP were found to be extremely rapid with 74% of the substrate removed from solution during the first 30 s. Inhibition of the reaction by the heavy metals Cd, Zn, Ni, and Pb at concentrations of 100 mg/l is of concern because these metals are often present in contaminated soils. H₂O₂ has a dominant role in optimizing peroxidase activity in crude horseradish extracts. Fluctuations in temperature and pH, normally experienced in soils, did not appear to have a detrimental impact on peroxidase activity. However, the functioning of the enzyme is seriously affected at a pH ≤ 3. All reactions in this study were carried out in solution.     

HIV-miR-H1 evolvability during HIV pathogenesis

The discovery of microRNAs (miRNAs) in viruses has generated considerable attention into their functional relevance in processes such as cell death, viral proliferation, and oncogenesis. Two early studies found no detectable miRNAs expressed within HIV; however, several studies have verified the existence and function of three HIV miRNAs, most notably HIV-miR-TAR, thus making the earlier results controversial. Although miRNAs are highly conserved within most species, HIV is known to have a high mutation rate, which could contribute to the opposing experimental findings and raises questions about whether all HIV miRNAs are robust enough to maintain their integrity, especially in viral regions prone to insertions and deletions. In addition, could the evolvability of HIV miRNAs contribute to the diversity in HIV disease pathogenesis? To address this question, we examined mutations in 1293 sequences in a suspect HIV miRNA, called miR-H1, derived from a large variety of tissues from seven patients. We found considerable diversity within the structures, including a patient-specific deletion and the potential for the development of new miRNAs as a result of deletions. We also note a potential disease association between a less stable miR-H1 and the development of AIDS-related lymphoma (ARL).     

Analysis of the introgression of Solanum bulbocastanum DNA into potato breeding lines

Somatic hybrids have been obtained between potato and Solanum bulbocastanum PI 245310, a Mexican diploid (2n=2x=24) species. Through restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) analyses it was found that the somatic hybrids contain each chromosome of the diploid parent and that the synteny of RFLP markers noted with tomato, potato and S. brevidens is largely maintained in S. bulbocastanum. RFLP analyses of BC1 progeny of two different hybrids indicated that a substantial number of markers were either lost or were heterozygous, in marked contrast with results previously noted with S. brevidens. A RAPD map for all 12 chromosomes of S. bulbocastanum was prepared and marker transmission was followed in three BC2 populations. Results with chromosomes 3, 8 and 10 from these populations are compared.     

Cryomicroscopy of liposome sytems as simple models to study cellular freezing response

Liposomes represent an attractive model system to study the freeze/thaw-induced alterations in biological membranes primarily because liposomes may be custom-manufactured for a particular investigation.This paper has reviewed briefly the degree of flexibility possible in terms of manufacturing liposomes with desired membrane composition and intraliposomal markers.Earlier research using liposomes as a model to study the freezing response of biological cells demonstrated their usefulness in this respect. Combining these earlier results with new results obtained in our laboratory, many analogies between the responses of biological cells and liposomes have been demonstrated. These analogies are summarized in Table 3.It is significant to note that in virtually every category thus far examined the relevant cryobiological behavior of biological cells can be mimicked qualitatively (and in some cases quantitatively) by a pure lipid membrane system. This is not to say that protein components of the membrane are insignificant. They are likely to be quite important in some cases. The liposome model system offers some interesting possibilities of examining the relative importance of lipids and proteins in model systems.Cryomicroscopic observation of liposome systems represents a promising approach to an improved understanding of membrane-related phenomena which occur during freezing and thawing. Especially interesting are the observations of membrane instabilities (“popping”) which appear to be induced osmotically.Real-time observation of well-defined membrane bilayer systems subjected to computer-controlled freeze/thaw protocols should lead to valuable insights into the nature of membrane freezing injury in the future.     

Fragments of plastid DNA in the nuclear genome of tomato: prevalence, chromosomal location, and possible mechanism of integration

Summary We have undertaken a systematic search for plastid DNA sequences integrated in the tomato nuclear genome, using heterologous probes taken from intervals of a plastid DNA region spanning 58 kb. A total of two short integrates (202 and 141 nucleotides) were isolated and mapped to chromosomes 9 and 5, respectively. The nucelotide sequence of the integrates and that of the flanking regions were determined. The integration sites contain direct repeat elements similar in position (but not in length or sequence) to the direct repeats previously observed with another plastid integrate in the tomato nuclear genome. Based on these results, a model for the process of movement and integration of plastid sequences into the nuclear genome is discussed.