Genetic variation and structure of fisher (Martes pennanti) populations across North America

Fishers are mid-sized forest carnivores indigenous to North America that experienced sharp population declines from the early 1800s through to the mid-1900s. To evaluate levels of genetic variation within and subdivision among northern fisher populations 459 individuals were genotyped using 13 microsatellite loci. Genetic diversity was found to be slightly lower in re-introduced populations than in adjacent indigenous populations. Furthermore, fisher populations revealed much more genetic structuring than two closely related mustelids. Further investigation is needed to determine if fishers are more philopatric than martens and wolverines or if barriers to dispersal explain the levels of structure identified in this study.     

Efficient expression of exogenous genes in primary vascular cells using IRES-based retroviral vectors

Analysis of gene function in primary vascular cells has been particularly limited by low transfection efficiencies. Using internal ribosomal entry site (IRES)-based retroviral vectors, we demonstrate efficient infection (range of 45%-95%) of primary human endothelial and smooth muscle cells with genes varying in size from 1.3 to 4.5 kb. Because IRES vectors are designed to allow the expression of two genes from a single mRNA, we can show excellent correlation between the expression of a reporter gene and an inserted gene of interest. Reporter gene expression allows rapid (24-48 h) and unambiguous identification of transduced cells. Additionally, reporter gene expression can be used to isolate subpopulations of cells that express distinct levels of cistron 1 genes by flow cytometry, and sorted cells maintain relative levels of gene expression over multiple passages in culture. Two examples of the usefulness of these vectors to characterize gene function in primary vascular cells include (i) the inhibition of endothelial cell inflammatory responses in a polyclonal population by the expression of a dominant negative inhibitor of nuclear factor-kappaB and (ii) monitoring the in vitro evolution of smooth muscle cells provided with a selective growth advantage by transduction with telomerase. Potential applications of retroviral expression strategies in vascular biology are also discussed.     

The phylogeny and a new classification of the gingers (Zingiberaceae): evidence from molecular data

The pantropical Zingiberaceae is the largest family in the order Zingiberales with 53 genera and over 1200 species. Classifications of the family first proposed in 1889 and refined by others since that time recognize four tribes (Globbeae, Hedychieae, Alpinieae, and Zingibereae) based on morphological features, such as number of locules and placentation in the ovary, development of staminodia, modifications of the fertile anther, and rhizome-shoot-leaf orientation. New phylogenetic analyses based on DNA sequences of the nuclear internal transcribed spacer (ITS) and plastid matK regions suggest that at least some of these morphological traits are homoplasious and three of the tribes are paraphyletic. The African genus Siphonochilus and Bornean genus Tamijia are basal clades. The former Alpinieae and Hedychieae for the most part are monophyletic taxa with the Globbeae and Zingibereae included within the latter. The results of these phylogenetic investigations are used to propose a new classification of the Zingiberaceae that recognizes four subfamilies and four tribes: Siphonochiloideae (Siphonochileae), Tamijioideae (Tamijieae), Alpinioideae (Alpinieae, Riedelieae), and Zingiberoideae (Zingibereae, Globbeae). Morphological features congruent with this classification and the taxonomic status of various monotypic genera are discussed.     

Protein kinase C epsilon-dependent regulation of cystic fibrosis transmembrane regulator involves binding to a receptor for activated C kinase (RACK1) and RACK1 binding to Na+/H+ exchange regulatory factor

Protein kinase C (PKC) regulation of cystic fibrosis transmembrane regulator (CFTR) chloride function has been demonstrated in several cell lines, including Calu-3 cells that express native, wild-type CFTR. We demonstrated previously that PKC epsilon was required for cAMP-dependent CFTR function. The goal of this study was to determine whether PKC epsilon interacts directly with CFTR. Using overlay assay, immunoprecipitation, pulldown and binding assays, we show that PKC epsilon does not bind to CFTR, but does bind to a receptor for activated C kinase (RACK1), a 37-kDa scaffold protein, and that RACK1 binds to Na(+)/H(+) exchange regulatory factor (NHERF1), a binding partner of CFTR. In vitro binding assays demonstrate dose-dependent binding of PKC epsilon to RACK1 which is inhibited by an 8-amino acid peptide based on the sequence of the sixth Trp-Asp repeat in RACK1 or by an 8-amino acid sequence in the V1 region of PKC epsilon, epsilon V1-2. A 4-amino acid sequence INAL (70-73) expressed in CFTR shares 50% homology to the RACK1 inhibitory peptide, but it does not bind PKC epsilon. NHERF1 and RACK1 bind in a dose-dependent manner. Immunofluorescence and confocal microscopy of RACK1 and CFTR revealed colocalization of the proteins to the apical and lateral regions of Calu-3 cells. The results indicate the RACK1 binds PKC epsilon and NHERF1, thus serving as a scaffold protein to anchor the enzyme in proximity to CFTR.     

Effects of light and nutrients on plankton stoichiometry and biomass in a P-limited lake

A field enclosure experiment was performed over 12 weeks in a P-limited lake to test the hypothesis that light:nutrient balance affects pelagic communities by altering the C:P stoichiometry of seston and by influencing exudation of labile DOC by algae. Three levels of light intensity (ambient, 50% of ambient, 25% of ambient) were cross-classified with three levels of nutrients in a factorial design (n=2). Dissolved nutrient concentrations, seston C concentration and C:P ratios in small (<1 m) and larger (1–85 m) size fractions were monitored, along with chlorophyll a concentration, abundance of bacteria and protozoa, and biomass and P-content of macrozooplankton. Algal exudation of recently-fixed C into the dissolved pool was also measured at the end of the experiment in selected enclosures. Treatments had no effect on seston C concentration but reduction of light intensity significantly decreased whole seston and large (1–85 m) seston C:P ratios. However, the magnitude of these effects was modest and not likely to be ecologically significant. There were no effects of nutrient addition or light×nutrient interaction on seston stoichiometry. Algae tended to release a higher percentage of fixed C as DOC in high light enclosures but this difference was not statistically significant. There were no effects of treatments on the abundance of bacteria or protozoa but nutrient enrichment led to a statistically significant but generally modest increase in macrozooplankton biomass. No effects on zooplankton community composition or P-content were observed. Comparison of effect sizes and treatment variances indicated a high probability of type II error and thus our confidence in failing to reject the null hypothesis in most of the above cases was low. Thus, our data provide support for only some aspects of the light:nutrient hypothesis but more appropriate tests of the hypothesis should involve stronger treatments and/or increased replication in order to be better able to evaluate its validity.     

Identification of frequent cytogenetic aberrations in hepatocellular carcinoma using gene-expression microarray data

Background  

Hepatocellular carcinoma (HCC) is a leading cause of death worldwide. Frequent cytogenetic abnormalities that occur in HCC suggest that tumor-modifying genes (oncogenes or tumor suppressors) may be driving selection for amplification or deletion of these particular genetic regions. In many cases, however, the gene(s) that drive the selection are unknown. Although techniques such as comparative genomic hybridization (CGH) have traditionally been used to identify cytogenetic aberrations, it might also be possible to identify them indirectly from gene-expression studies. A technique we have called comparative genomic microarray analysis (CGMA) predicts regions of cytogenetic change by searching for regional gene-expression biases. CGMA was applied to HCC gene-expression profiles to identify regions of frequent cytogenetic change and to identify genes whose expression is misregulated within these regions.     

Potent bivalent inhibition of human tryptase-beta by a synthetic inhibitor

Human tryptase-beta (HTbeta) is a unique serine protease exhibiting a frame-like tetramer structure with four active sites directed toward a central pore. Potent inhibition of HTbeta has been attained using CRA-2059. This compound has two phenylguanidinium head groups connected via a linker capable of spanning between two active sites. The properties of the CRA-2059:HTbeta interaction were defined in this study. Tight-binding reversible inhibition was observed with an inhibition constant (Ki) of 620 pM, an association rate constant of 7x10(7) M(-1) s(-1) and a relatively slow dissociation rate constant of 0.04 s(-1). Bivalent inhibition was demonstrated by displacement of p-aminobenzamidine from the primary specificity pocket with a stoichiometry, [CRA-2059]0/[HTbeta]0, of 0.5. The potency of the bivalent interaction was illustrated by CRA-2059 inhibition of HTbeta, 24% or 53% inhibited by pre-incubation with an irreversible inhibitor. Two interactions were observed consistent with mono- and bi-valent binding; the Ki value for bivalent inhibition was at least 10(4)-fold lower than that for monovalent inhibition. Comparison of the affinities of CRA-2059 and phenylguanidine for HTbeta finds an approximate doubling of the free energy change upon bivalent binding. This doubling suggests that the linker portion minimally hinders the binding of CRA-2059 to HTbeta. The potency of CRA-2059 is thus attributable to effective bivalent binding.     

A new mitochondrial fluorescent zinc sensor

A novel cationic fluorescent zinc (Zn2+) indicator (RhodZin-3) with nanomolar affinity for Zn2+ has been synthesized. RhodZin-3 exhibits large pH-independent fluorescence increases in the orange region of the visible wavelength spectrum with increasing zinc concentrations, and no sensitivity to physiologically relevant Ca2+ concentrations. Experiments in neuronal cell cultures show that RhodZin-3 effectively localizes into mitochondria and detects changes of intramitochondrial free Zn2+ ([Zn2+]m).