Trans-Atlantic genetic uniformity in the rare snowbed sedge <Emphasis Type="Italic">Carex rufina</Emphasis>

The red-listed, amphi-Atlantic sedge Carex rufina is highly specialized to certain alpine snowbeds, and threatened by current changes in snow cover duration and moisture conditions. Here we address its range-wide genetic diversity, history, and conservation using amplified fragment length polymorphisms (AFLPs). Despite extensive primer testing, we detected very low overall diversity (4.1% polymorphic markers). Only a single AFLP phenotype was found throughout Norway and across the Atlantic to Iceland and Greenland, while another was found in Canada, suggesting glacial survival in one East and one West Atlantic refugium. East Atlantic C. rufina has probably been heavily bottlenecked in a small refugium, possibly situated within the maximum limits of the ice sheets. Its lack of diversity is likely maintained through local clonal growth causing longevity of genotypes. Habitat availability appears as the main limiting factor for C. rufina, and its currently occupied habitats need to be preserved to ensure its long-time survival.     

Lack of Evidence for the Role of Human Adenovirus‐36 in Obesity in a European Cohort

Adenovirus infection has been shown to increase adiposity in chickens, mice, and nonhuman primates. Adenovirus type 36 (Ad‐36) DNA was detected in adipose tissues in these animal trials. In the United States, Ad‐36 significantly correlates with obesity as illustrated by an Ad‐36 seroprevalence of 30% in obese individuals and 11% in nonobese individuals. We investigated the possibility of a similar correlation of Ad‐36 in Dutch and Belgian persons. In total, 509 serum samples were analyzed for Ad‐36 antibodies using a serum neutralization assay. In addition, PCR was used to detect adenoviral DNA in visceral adipose tissue of 31 severely obese surgical patients. Our results indicated an overall Ad‐36 seroprevalence of 5.5% increasing with age. BMI of Ad‐36 seropositive humans was not significantly different from seronegative humans. No adenoviral DNA could be found using PCR on visceral adipose tissue. In conclusion, this first Ad‐36 study in the Netherlands and in Belgium indicates that Ad‐36 does not play a role as a direct cause of BMI increase and obesity in humans in Western Europe.     

Circulating endothelial progenitor cells in kidney transplant patients

Background

Kidney transplantation (RTx) leads to amelioration of endothelial function in patients with advanced renal failure. Endothelial progenitor cells (EPCs) may play a key role in this repair process. The aim of this study was to determine the impact of RTx and immunosuppressive therapy on the number of circulating EPCs.

Methods

We analyzed 52 RTx patients (58±13 years; 33 males, mean ± SD) and 16 age- and gender-matched subjects with normal kidney function (57±17; 10 males). RTx patients received a calcineurin inhibitor (CNI)-based (65%) or a CNI-free therapy (35%) and steroids. EPC number was determined by double positive staining for CD133/VEGFR2 and CD34/VEGFR2 by flow cytometry. Stromal cell-derived factor 1 alpha (SDF-1) levels were assessed by ELISA. Experimentally, to dissociate the impact of RTx from the impact of immunosuppressants, we used the 5/6 nephrectomy model. The animals were treated with a CNI-based or a CNI-free therapy, and EPCs (Sca+cKit+) and CD26+ cells were determined by flow cytometry.

Results

Compared to controls, circulating number of CD34+/VEGFR2+ and CD133+/VEGFR2+ EPCs increased in RTx patients. There were no correlations between EPC levels and statin, erythropoietin or use of renin angiotensin system blockers in our study. Indeed, multivariate analysis showed that SDF-1 – a cytokine responsible for EPC mobilization – is independently associated with the EPC number. 5/6 rats presented decreased EPC counts in comparison to control animals. Immunosuppressive therapy was able to restore normal EPC values in 5/6 rats. These effects on EPC number were associated with reduced number of CD26+ cells, which might be related to consequent accumulation of SDF-1.

Conclusions

We conclude that kidney transplantation and its associated use of immunosuppressive drugs increases the number of circulating EPCs via the manipulation of the CD26/SDF-1 axis. Increased EPC count may be associated to endothelial repair and function in these patients.     

Effect of poly(ADP-ribosyl)ation inhibitors on the genotoxic effects of the boron neutron capture reaction

The boron neutron capture (BNC) reaction results from the interaction of 10B with low-energy thermal neutrons and gives rise to highly damaging lithium and alpha-particles. In this work the genotoxicity caused by the BNC reaction in V79 Chinese hamster cells was evaluated in the presence of poly(ADP-ribosyl)ation inhibitors. Poly(ADP-ribose) polymerase-1 (PARP-1), the most important member of the PARP enzyme family, is considered to be a constitutive factor of the DNA damage surveillance network present in eukaryotic cells, acting through a DNA break sensor function. Inhibition of poly(ADP-ribosyl)ation was achieved with the classical compound 3-aminobenzamide (3-AB), and with two novel and very potent inhibitors, 5-aminoisoquinolinone (5-AIQ) and PJ-34. Dose-response increases in the frequencies of aberrant cells excluding gaps (%ACEG) and chromosomal aberrations excluding gaps per cell (CAEG/cell) were observed for increasing exposures to the BNC reaction. The presence of 3-AB did not increase the %ACEG or CAEG/cell, nor did it change the pattern of the induced chromosomal aberrations. Results with 5-AIQ and PJ-34 were in agreement with the results obtained with 3-AB. We further studied the combined effect of a PARP inhibitor and a DNA-dependent protein kinase (DNA-PK) inhibitors (3-AB and wortmannin, respectively) on the genotoxicity of the BNC reaction, by use of the cytokinesis-block micronucleus assay. DNA-PK is also activated by DNA breaks and binds DNA ends, playing a role of utmost importance in the repair of double-strand breaks. Our results show that the inhibition of poly(ADP-ribosyl)ation does not particularly modify the genotoxicity of the BNC reaction, and that PARP inhibition together with a concomitant inhibition of DNA-PK revealed barely the same sensitizing effect as DNA-PK inhibition per se.     

Relationship between an increase of juvenile hormone titer in early instars and the induction of diapause in fully grown larvae of Sesamia nonagrioides

The larvae of Sesamia nonagrioides (Lepidoptera: Noctuidae) grown at 25 degrees C and long photoperiod (16:8h light:dark) pupate in the 5th or 6th (mostly) larval instar, while the larvae reared under a short photoperiod (12:12h) enter diapause during which they consume some food and undergo up to 12 (usually 3-4) stationary larval molts. Diapause programming includes an increase of juvenile hormone (JH) titer in the hemolymph from about 20 to 50 nM in the 4th and 5th instar larvae (titer in earlier instars was not measured). JH I, II, and III are present in approximate ratio 1-2:10:1. The JH titer drops to zero before pupation but remains around 20 nM during diapause. Perfect extra larval molts associated with a body weight increase can be induced in the non-diapausing larvae with a JH analogue (JHA). The weight rise is due to accumulation of reserves and not to a general body growth. The timing of extra molts is similar to the molting pattern of the diapausing larvae only when JHA is present since early larval instars. In the diapausing larvae, JHA application affects neither molting periodicity nor the body weight. It is concluded that (1) Increased JH titer in early larval instars is a part of diapause programming; (2) The extension of larval stage in the diapausing larvae, but not the timing pattern of extra molts, is due to continuously high JH titer; (3) The diapause program includes low food intake, maintenance of a certain body weight, and periodic larval molts.     

Fibroblast growth and H-7 protein kinase inhibitor response monitored in microimpedance sensor arrays

Functional genomic studies and drug candidate testing both require high throughput, parallel experimentation strategies to screen for variable cellular behaviors. In this article we describe the use of an impedance sensing electrode array that is capable of sensing cell "presence" as well as the extent of cell (focal) attachment to the substrate. The signals provided by mouse fibroblasts on a sensing structure containing four different sized electrodes are reported. In the absence of cells, each electrode's impedance was found to depend as expected on electrode size and frequency. The impedance increased by several-fold when fibroblasts attached and spread out over time. More notably, the sensors also detected the cellular response to the protein kinase C inhibitor, H-7. H-7 inhibits actomyosin contractility; thereafter, the loss of focal adhesion complexes occurs. The sensors, in turn, detected an impedance decrease after H-7 addition and an increase in impedance after H-7 removal.     

Potential application of low-stringency single specific primer-PCR in the identification of Leptospira in the serum of patients with suspected leptospirosis

In this study we tested the potential use of low-stringency single specific primer-PCR (LSSP-PCR) for genetically typing Leptospira directly from biological samples. Serum samples obtained from 29 patients with clinically suspected leptospirosis were amplified by specific PCR, using the previously selected G1 and G2 primers. The PCR products of approximately 300 bp were subsequently used as a template for LSSP-PCR analysis. We were able to produce genetic signatures from the leptospires present in the human samples, which permitted us to make a preliminary identification of the infective serovar by comparing the LSSP-PCR profiles obtained directly from serum samples with those from reference leptospires. Thus, LSSP-PCR has the potential to become a useful diagnostic tool for identifying leptospires in biological samples without the need for bacteria isolation and culture.     

Identification and molecular characterization of Mnk1b, a splice variant of human MAP kinase-interacting kinase Mnk1

In this paper, we report the identification and molecular characterization of a splice variant of human Mnk1 which has been named as Mnk1b. Human Mnk1b mRNA is homologous to human Mnk1 mRNA but lacking a region corresponding to exon 19, which causes a change in the reading frame generating a stop codon. The resulting protein lacks the last 89 amino acids at the C-terminal region that are replaced by 12 amino acids with an entirely new sequence. The C-terminal end in Mnk1 corresponds to the extracellular signal-regulated kinase (ERK1/2) binding site. Although Mnk1b lacks this domain and, consequently, is not phosphorylated by ERK1/2, it is able, however, to phosphorylate eIF4E in vitro and in vivo in a mitogen-activated protein kinases-independent manner. This result suggests that Mnk1b may play a key role in regulating protein translation in the absence of stimuli. Interestingly, a significant population of cells shows Mnk1b within the nucleus whereas Mnk1 is always detected in the cytoplasm. This fact may be explained because Mnk1b maintains the nuclear localization signal (NLS) but lacks the nuclear export sequence (NES).     

Extracellular calcium acts as a "third messenger" to regulate enzyme and alkaline secretion

It is generally assumed that the functional consequences of stimulation with Ca2+ -mobilizing agonists are derived exclusively from the second messenger action of intracellular Ca2+, acting on targets inside the cells. However, during Ca2+ signaling events, Ca2+ moves in and out of the cell, causing changes not only in intracellular Ca2+, but also in local extracellular Ca2+. The fact that numerous cell types possess an extracellular Ca2+ "sensor" raises the question of whether these dynamic changes in external [Ca2+] may serve some sort of messenger function. We found that in intact gastric mucosa, the changes in extracellular [Ca2+] secondary to carbachol-induced increases in intracellular [Ca2+] were sufficient and necessary to elicit alkaline secretion and pepsinogen secretion, independent of intracellular [Ca2+] changes. These findings suggest that extracellular Ca2+ can act as a "third messenger" via Ca2+ sensor(s) to regulate specific subsets of tissue function previously assumed to be under the direct control of intracellular Ca2+.