Typing of Genetic Markers Involved in Stress Response by Fluorescent cDNA-Amplified Fragment Length Polymorphism Technique

Identification of genetic markers involved in stress response to physical factors or chemical substances in organisms is a challenging task. Typing of upregulated gene expression due to selective antibacterial pressure is a promising approach in the search of molecular mechanisms responsible for development of resistance. cDNA-Fluorescent Amplified Fragment Length Polymorphism (cDNA-FAFLP) strategy was developed and applied in the search of antimycotic drug resistance marker(s) in medically important fungi as an alternative method to microarray analysis. We compared differential gene expression of two sensitive Candida albicans reference strains (ATCC 10231 and ATCC 60133) and two of their paired resistant to fluconazole and itraconazole mutants. Resistant mutants Candida albicans FLC-R, resistant to fluconazole (MIC > 128 μg/ml) and Candida albicans ICZ-R, resistant to itraconazole (MIC > 4 μg/ml) were obtained in subcultures with gradual increase of the antifungal in the culture medium. cDNA-AFLP profile in both itraconazole resistant mutants showed specific spectrophotometric peaks with 5–6-fold RNA overexpression product of 500 bp length compared to the sensitive strains. Fluconazole mutants do not reveal RNA level changes under tested by us typing conditions. These results indicate that the cDNA-FAFLP strategy is a relatively rapid, simple, and reliable method for simultaneous typing of both constitutive and induced differences in expression of host genes providing insight into the biological processes involved in response to drugs in bacteria and fungi. Moreover, this methodology could be tested for typing of the genome response of any organism to physical or chemical stress factors.     

Predicted impact of exotic vines on an endangered ecological community under future climate change

Potential interactions between climate change and exotic plant invasions may affect areas of high conservation value, such as land set aside for the protection of endangered species or ecological communities. We investigated this issue in eastern Australia using species distribution models for five exotic vines under climate regimes for 2020 and 2050. We examined how projected changes in the distribution of climatically suitable habitat may coincide with the remaining remnants of an endangered ecological community—littoral rainforests—in this region. The number of known infestations of each weed in tropical, subtropical and temperate areas was used to assess the likelihood of further expansion into areas projected to provide suitable habitat under future conditions. Littoral rainforest reserves were consistently predicted to provide bioclimatically suitable habitat for the five vines examined under both current and future climate scenarios. We explore the consequences and potential strategies for managing exotic plant invasions in these protected areas in the coming decades.     

Screening of Drug Metabolizing Enzymes for the Ginsenoside Compound K In Vitro: An Efficient Anti-Cancer Substance Originating from Panax Ginseng

Ginsenoside compound K (CK), a rare ginsenoside originating from Panax Ginseng, has been found to possess unique pharmacological activities specifically as anti-cancers. However, the role of cytochrome P450s (CYPs) in the metabolism of CK is unclear. In this study, we screened the CYPs for the metabolism of CK in vitro using human liver microsomes (HLMs) or human recombinant CYPs. The results showed that CK inhibited the enzyme activities of CYP2C9 and CYP3A4 in the HLMs. The K_m and V_max values of CK were 84.20±21.92 μM and 0.28±0.04 nmol/mg protein/min, respectively, for the HLMs; 34.63±10.48 μM and 0.45±0.05 nmol/nmol P450/min, respectively, for CYP2C9; and 27.03±5.04 μM and 0.68±0.04 nmol/nmol P450/min, respectively, for CYP3A4. The IC₅₀ values were 16.00 μM and 9.83 μM, and K_i values were 14.92 μM and 11.42μM for CYP2C9 and CYP3A4, respectively. Other human CYP isoforms, including CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on CK metabolism. The results suggested that CK was a substrate and also inhibitors for both CYP2C9 and CYP3A4. Patients using CK in combination with therapeutic drugs that are substrates of CYP2C9 and CYP3A4 for different reasons should be careful, although the inhibiting potency of CK is much poorer than that of enzyme-specific inhibitors.     

Temporal and spatial differences in the emission of calls by pipistrelle bats<Emphasis Type="Italic">Pipistrellus pipistrellus</Emphasis> and<Emphasis Type="Italic">P. pygmaeus</Emphasis>

The number of orientation, feeding and social calls emitted by pipistrelle batsPipistrellus pipistrellus Schreber, 1774 andP. pygmaeus Leach, 1825 was recorded throughout the night at eight different sites. Social calls were unaffected by weather variables, whereas orientation calls and feeding buzzes were both significantly affected by cloud and temperature conditions. The number of emissions of each call type was significantly different between sites, indicating that the bats used different sites for different activities. Significant positive correlations between all three combinations of call types occurred only during the middle of the night, corresponding with the nadir of flying insects. This suggests that bats were engaged in activities other than feeding at this time, such as territory protection or mate attraction.     

Identification and functional analysis of NOL7 nuclear and nucleolar localization signals

Background  

NOL7 is a candidate tumor suppressor that localizes to a chromosomal region 6p23. This locus is frequently lost in a number of malignancies, and consistent loss of NOL7 through loss of heterozygosity and decreased mRNA and protein expression has been observed in tumors and cell lines. Reintroduction of NOL7 into cells resulted in significant suppression of in vivo tumor growth and modulation of the angiogenic phenotype. Further, NOL7 was observed to localize to the nucleus and nucleolus of cells. However, the mechanisms regulating its subcellular localization have not been elucidated.     

Separating the climatic signal from tree-ring width and maximum latewood density records

We propose a technique for separating the climatic signal which is contained in two tree-ring parameters widely used in dendroclimatology. The method is based on the removal of the relationship between tree-ring width (TRW) and maximum latewood density (MXD) observed for narrow tree rings from high latitudes. The new technique is tested on data from three larch stands located along the northern timberline in Eurasia. Correlations were calculated between the temperatures of pentads (five consecutive days), TRW chronologies and MXD chronologies calculated according to the standard and proposed methods. The analysis confirms the great importance of summer temperature for tree radial growth and tree-ring formation. TRW is positively correlated with the temperature of four to eight pentads (depending on the region) at the beginning of the growth season, but MXD as obtained by the standard technique is correlated with temperature over a much longer period. For maximum density series from which the relationship between MXD and TRW has been removed (MXD′), there is a clear correlation with temperatures in the second part of the growing season. These results are consistent with the known dynamics of tree-ring growth in high latitudes and mechanisms of tree-ring formation.     

α‐ketoglutarate delays age‐related fertility decline in mammals

The fecundity reduction with aging is referred as the reproductive aging which comes earlier than that of chronological aging. Since humans have postponed their childbearing age, to prolong the reproductive age becomes urgent agenda for reproductive biologists. In the current study, we examined the potential associations of α‐ketoglutarate (α‐KG) and reproductive aging in mammals including mice, swine, and humans. There is a clear tendency of reduced α‐KG level with aging in the follicle fluids of human. To explore the mechanisms, mice were selected as the convenient animal model. It is observed that a long term of α‐KG administration preserves the ovarian function, the quality and quantity of oocytes as well as the telomere maintaining system in mice. α‐KG suppresses ATP synthase and alterations of the energy metabolism trigger the nutritional sensors to down‐regulate mTOR pathway. These events not only benefit the general aging process but also maintain ovarian function and delay the reproductive decline. Considering the safety of the α‐KG as a naturally occurring molecule in energy metabolism, its utility in reproduction of large mammals including humans deserves further investigation.     

Origin of the cell nucleus,mitosis and sex: roles of intracellular coevolution

Background  

The transition from prokaryotes to eukaryotes was the most radical change in cell organisation since life began, with the largest ever burst of gene duplication and novelty. According to the coevolutionary theory of eukaryote origins, the fundamental innovations were the concerted origins of the endomembrane system and cytoskeleton, subsequently recruited to form the cell nucleus and coevolving mitotic apparatus, with numerous genetic eukaryotic novelties inevitable consequences of this compartmentation and novel DNA segregation mechanism. Physical and mutational mechanisms of origin of the nucleus are seldom considered beyond the long-standing assumption that it involved wrapping pre-existing endomembranes around chromatin. Discussions on the origin of sex typically overlook its association with protozoan entry into dormant walled cysts and the likely simultaneous coevolutionary, not sequential, origin of mitosis and meiosis.     

Cloning of genes that suppress an Escherichia coli K-12 alanine auxotroph when present in multicopy plasmids.

To facilitate molecular analyses of a previously uncharacterized gene involved in alanine synthesis, attempts were made to clone the wild-type allele of this gene, alaA, with a mini-Mu plasmid element used for in vivo cloning. Seventy-six independent Ala+ plasmids were isolated and characterized. Physiological, enzymological, and restriction endonuclease analyses indicated that three different genes, none of them alaA, were cloned. These genes were avtA+, which encodes the alanine-valine transaminase (transaminase C); tyrB+, which encodes the tyrosine-repressible transaminase (transaminase D); and a previously undescribed gene, called alaB, which encodes an alanine-glutamate transaminase.