Phase-transition-driven synaptic exocytosis: a hypothesis and its physiological and evolutionary implications

It is proposed that the plasma membrane in the active zones of synaptic terminals contains self-assembling cooperative domains whose Ca2+-induced solidification may be the driving force of the fast neurotransmitter release in the central synapses. This hypothesis and a qualitative model of the phase-transition-driven exocytosis provide formulation of a unitary approach to a number of general problems in the physiology of animals. It allows answering the following questions, among others: (i) What is the physical reason for the existence of a narrow optimum range of body temperatures in warm-blooded species? (ii) What is the physical reason for the inevitable necessity of regular sleep in animals? (iii) Does there indeed exist any general mechanism of general anesthesia?     

Telomeric DNA localization on dinoflagellate chromosomes: structural and evolutionary implications

Dinoflagellates are eukaryotic microalgae with distinct chromosomes throughout the cell cycle which lack histones and nucleosomes. The molecular organization of these chromosomes is still poorly understood. We have analysed the presence of telomeres in two evolutionarily distant and heterogeneous dinoflagellate species (Prorocentrum micans and Amphidinium carterae) by FISH with a probe containing the Arabidopsis consensus telomeric sequence. Telomere structures were identified at the chromosome ends of both species during interphase and mitosis and were frequently associated with the nuclear envelope. These results identify for the first time telomere structures in dinoflagellate chromosomes, which are formed in the absence of histones. The presence of telomeres supports the linear nature of dinoflagellate chromosomes.     

VDAC inhibition by tubulin and its physiological implications

Regulation of mitochondrial outer membrane (MOM) permeability has dual importance: in normal metabolite and energy exchange between mitochondria and cytoplasm, and thus in control of respiration, and in apoptosis by release of apoptogenic factors into the cytosol. However, the mechanism of this regulation involving the voltage-dependent anion channel (VDAC), the major channel of MOM, remains controversial. For example, one of the long-standing puzzles was that in permeabilized cells, adenine nucleotide translocase is less accessible to cytosolic ADP than in isolated mitochondria. Still another puzzle was that, according to channel-reconstitution experiments, voltage regulation of VDAC is limited to potentials exceeding 30mV, which are believed to be much too high for MOM. We have solved these puzzles and uncovered multiple new functional links by identifying a missing player in the regulation of VDAC and, hence, MOM permeability - the cytoskeletal protein tubulin. We have shown that, depending on VDAC phosphorylation state and applied voltage, nanomolar to micromolar concentrations of dimeric tubulin induce functionally important reversible blockage of VDAC reconstituted into planar phospholipid membranes. The voltage sensitivity of the blockage equilibrium is truly remarkable. It is described by an effective "gating charge" of more than ten elementary charges, thus making the blockage reaction as responsive to the applied voltage as the most voltage-sensitive channels of electrophysiology are. Analysis of the tubulin-blocked state demonstrated that although this state is still able to conduct small ions, it is impermeable to ATP and other multi-charged anions because of the reduced aperture and inversed selectivity. The findings, obtained in a channel reconstitution assay, were supported by experiments with isolated mitochondria and human hepatoma cells. Taken together, these results suggest a previously unknown mechanism of regulation of mitochondrial energetics, governed by VDAC interaction with tubulin at the mitochondria-cytosol interface. Immediate physiological implications include new insights into serine/threonine kinase signaling pathways, Ca(2+) homeostasis, and cytoskeleton/microtubule activity in health and disease, especially in the case of the highly dynamic microtubule network which is characteristic of cancerogenesis and cell proliferation. In the present review, we speculate how these findings may help to identify new mechanisms of mitochondria-associated action of chemotherapeutic microtubule-targeting drugs, and also to understand why and how cancer cells preferentially use inefficient glycolysis rather than oxidative phosphorylation (Warburg effect). This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

Yeasts in floral nectar: a quantitative survey

Background and Aims

One peculiarity of floral nectar that remains relatively unexplored from an ecological perspective is its role as a natural habitat for micro-organisms. This study assesses the frequency of occurrence and abundance of yeast cells in floral nectar of insect-pollinated plants from three contrasting plant communities on two continents. Possible correlations between interspecific differences in yeast incidence and pollinator composition are also explored.

Methods

The study was conducted at three widely separated areas, two in the Iberian Peninsula (Spain) and one in the Yucatán Peninsula (Mexico). Floral nectar samples from 130 species (37–63 species per region) in 44 families were examined microscopically for the presence of yeast cells. For one of the Spanish sites, the relationship across species between incidence of yeasts in nectar and the proportion of flowers visited by each of five major pollinator categories was also investigated.

Key Results

Yeasts occurred regularly in the floral nectar of many species, where they sometimes reached extraordinary densities (up to 4 × 10⁵ cells mm⁻³). Depending on the region, between 32 and 44 % of all nectar samples contained yeasts. Yeast cell densities in the order of 10⁴ cells mm⁻³ were commonplace, and densities >10⁵ cells mm⁻³ were not rare. About one-fifth of species at each site had mean yeast cell densities >10⁴ cells mm⁻³. Across species, yeast frequency and abundance were directly correlated with the proportion of floral visits by bumble-bees, and inversely with the proportion of visits by solitary bees.

Conclusions

Incorporating nectar yeasts into the scenario of plant–pollinator interactions opens up a number of intriguing avenues for research. In addition, with yeasts being as ubiquitous and abundant in floral nectars as revealed by this study, and given their astounding metabolic versatility, studies focusing on nectar chemical features should carefully control for the presence of yeasts in nectar samples.Key words: Bumble-bees, floral nectar, microbial communities, pollination, yeasts

‘Il y a évidemment un intérêt très grand, dans une question encore peu connue, à recenser exactement et cela dans diverse régions, les organismes qu''on rencontre.’Schoellhorn (1919, p. 155)

     

DNA topology in a chromatin model system

The synthetic copolypeptide (Lys³³, Leu⁶⁷)₁₀₀-Orn₂₀, modeled on some general features of the histone sequences, has been found to supercoil the DNA double helix, wrapping it into a micelle, as a result of cohesive interactions between the polypeptide hydrophobic moieties. X-ray low-angle diffraction of complexes between the polypeptide and DNA is characterized by maxima at 50, 32, and 23 Å, reminiscent of the chromatin pattern. The existence of a nucleosome-like structure along the DNA is suggested by gel electrophoresis analysis of DNA fragments after micrococcal nuclease digestion, showing the presence of a fragment of about 100 basepairs (bp) long. Topological experiments on the complexes with supercoiled as well as relaxed circular DNA by two-dimentional gel electrophoresis show the presence of left-handed superhelical turns. The results are in agreement with an intrinsic propensity of B-DNA to writhe into left-handed supercoils.     

DNA topology in a chromatin model system

The synthetic copolypeptide (Lys33, Leu67)100-Orn20, modeled on some general features of the histone sequences, has been found to supercoil the DNA double helix, wrapping it into a micelle, as a result of cohesive interactions between the polypeptide hydrophobic moieties. X-ray low-angle diffraction of complexes between the polypeptide and DNA is characterized by maxima at 50, 32, and 23 A, reminiscent of the chromatin pattern. The existence of a nucleosome-like structure along the DNA is suggested by gel electrophoresis analysis of DNA fragments after micrococcal nuclease digestion, showing the presence of a fragment of about 100 basepairs (bp) long. Topological experiments on the complexes with supercoiled as well as relaxed circular DNA by two-dimensional gel electrophoresis show the presence of left-handed superhelical turns. The results are in agreement with an intrinsic propensity of B-DNA to writhe into left-handed supercoils.     

DNA topology: feeling the pulse of a topoisomerase

The action of individual type II DNA topoisomerases has been followed in real time by observing the elastic response of single DNA molecules to sequential strand passage events. Micromanipulation methods provide a complementary approach to biochemical studies for investigating the mechanism of DNA topoisomerases.     

Side-chain hydroxylation of vitamin D3 and its physiological implications

Evidence is accumulating that, in vivo and in vitro, both 25-OH-D3 and 1,25-(OH)2D3 undergo side-chain modification leading to side-chain cleaved metabolites lacking the 24, 25, 26, and 27 carbons. The enzymes involved are D-dependent and are located in the kidney, bone, intestine, and perhaps other sites. We speculate that the extra-renal side-chain pathway may be primarily for target organ destruction of 1,25-(OH)2D3, whereas the renal pathway may be primarily for destruction of 25-OH-D3 formed in large amounts in hypervitaminosis D.     

Lay perspective on pharmacogenetics and its application to future drug treatment: a Danish quantitative survey

The purpose of the present survey is to describe the Danish perspective on pharmacogenetics and public willingness to adopt and utilize it as part of drug treatment. An Internet-based quantitative survey of a representative segment of the Danish population from ages 18 to 70 was conducted in March 2005. A total of 3,000 participated in the survey, with a response rate of 59%. Only 14.1% of respondents indicated they had heard of pharmacogenetics, while 79% said they had not. However, 81% indicated that they would choose pharmacogenetic drugs rather than ordinary drugs, while 6% would choose the drugs currently available. In addition, 89% indicated willingness to receive pharmacogenetic treatment in future. The population in general has a low level of knowledge about pharmacogenetics, and certain aspects are seen as disadvantageous: the lack of drug treatment for everyone, for example. Nonetheless, the attitude towards the use of pharmacogenetics is generally positive, and pharmacogenetic treatment is considered better than the treatment available at present.     

Direct measurement of DNA bending by type IIA topoisomerases: implications for non-equilibrium topology simplification

Type IIA topoisomerases modify DNA topology by passing one segment of duplex DNA (transfer or T–segment) through a transient double-strand break in a second segment of DNA (gate or G–segment) in an ATP-dependent reaction. Type IIA topoisomerases decatenate, unknot and relax supercoiled DNA to levels below equilibrium, resulting in global topology simplification. The mechanism underlying this non-equilibrium topology simplification remains speculative. The bend angle model postulates that non-equilibrium topology simplification scales with the bend angle imposed on the G–segment DNA by the binding of a type IIA topoisomerase. To test this bend angle model, we used atomic force microscopy and single-molecule Förster resonance energy transfer to measure the extent of bending imposed on DNA by three type IIA topoisomerases that span the range of topology simplification activity. We found that Escherichia coli topoisomerase IV, yeast topoisomerase II and human topoisomerase IIα each bend DNA to a similar degree. These data suggest that DNA bending is not the sole determinant of non-equilibrium topology simplification. Rather, they suggest a fundamental and conserved role for DNA bending in the enzymatic cycle of type IIA topoisomerases.     

Epigenetics DNA methylation in the core ataxin-2 gene promoter: novel physiological and pathological implications

Pathogenic CAG (cytosine-adenine-guanine) expansions beyond certain thresholds in the ataxin-2 (ATXN2) gene cause spinocerebellar ataxia type 2 (SCA2) and were shown to contribute to Parkinson disease, amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Regulation of ATXN2 gene expression and the function of the protein product are not known. SCA2 exhibits an inverse correlation between the size of the CAG repeat and the age at disease onset. However, a wide range of age at onset are typically observed, with CAG repeat number alone explaining only partly this variability. In this study, we explored the hypothesis that ATXN2 levels could be controlled by DNA methylation and that the derangement of this control may lead to escalation of disease severity and influencing the age at onset. We found that CpG methylation in human ATXN2 gene promoter is associated with pathogenic CAG expansions in SCA2 patients. Different levels of methylation in a SCA2 pedigree without an intergenerational CAG repeat instability caused the disease anticipation in a SCA2 family. DNA methylation also influenced the disease onset in SCA2 homozygotes and SCA3 patients. In conclusion, our study points to a novel regulatory mechanism of ATXN2 expression involving an epigenetic event resulting in differential disease course in SCA2 patients.