Sulfated HNK-1 epitope in developing and mature kidney: a new marker for thin ascending loop of Henle and tubular injury in acute tubular necrosis.

The HNK-1 carbohydrate epitope is a 3-sulfo-glucuronyl residue attached to lactosamine structures on glycoproteins, proteoglycans, or glycolipids mostly expressed in the nervous system. Here, using monoclonal antibodies against the sulfated HNK-1 carbohydrate epitope, we first examined its distribution in developing and adult kidneys, then its expression in kidneys with tubular necrosis and renal neoplasms. This HNK-1 epitope was expressed in the human, rabbit, and rat, but not mouse kidney. It was detected within a subset of epithelial cells in the renal vesicle and in comma- and S-shaped bodies during early stages of nephrogenesis. In ureteral bud derivatives, the epitope was present transiently in the area where the collecting duct fused with the nephron. In the adult kidney, expression of the HNK-1 epitope became mainly restricted to the thin ascending loop of Henle where this epitope was carried by heparan- and chondro-proteoglycan. In pathological conditions, HNK-1 epitope expression increased dramatically in proximal epithelial tubule cells in kidneys with acute tubular necrosis. In tumors, the HNK-1 epitope was expressed in the epithelial component of nephroblastomas and in a subgroup of papillary renal cell carcinomas. These data suggest that molecules carrying the sulfated HNK-1 carbohydrate epitope may play an important role in critical stages of renal development and in the physiology of thin ascending loop of Henle.     

Laser-scanning lithography (LSL) for the soft lithographic patterning of cell-adhesive self-assembled monolayers

We report the development of laser-scanning lithography (LSL), which employs a laser-scanning confocal microscope to pattern photoresists that can be utilized, for example, in the fabrication of masters for use in soft lithography. This convenient technique provides even exposure across the entire view field and facilitates accurate alignment of successive photoresist exposures. Features on the scale of 3 microm have been achieved to date with a 10x objective (NA 0.45). Virtual masks, instructions for laser irradiation, were drawn using the Region of Interest (ROI) function of a Zeiss LSM 510 microscope. These regions were then exposed to a 458 nm argon laser for 32 micros (0.9 mW/microm(2)). Differential interference contrast (DIC) imaging was utilized with a non-destructive 514 nm argon laser as an immediate quality check of each exposure, to align successive exposures, and to reduce chromatic aberration between imaging and exposure. Developed masters were replica-molded with poly(dimethylsiloxane) (PDMS); these masters were then utilized for microcontact printing of cell-adhesive self-assembled monolayers (SAMs) to demonstrate the utility of this process. Initial studies confirmed that human dermal fibroblast adhesion and spreading were limited to cell-adhesive SAM areas. LSL is a rapid, flexible, and readily available technique that will accelerate master design and preparation; moreover, it can be applied to additional forms of photolithography and photopolymerization for studies in cell biology, biomaterials design and evaluation, materials science, and surface chemistry.     

DnaJA4 is a SREBP-regulated chaperone involved in the cholesterol biosynthesis pathway

Using subtractive hybridization technique in 3T3-L1 adipocytes overexpressing constitutively active SREBP2, we have identified a DnaJ/Hsp40 chaperone, DnaJA4, as a new SREBP-responsive gene. SREBP2 regulation was demonstrated by changes in DnaJA4 mRNA under conditions of altered sterol status that were strictly parallel to that of well-characterized SREBP targets (LDL receptor and HMG-CoA reductase). The role of SREBP2 was further established using adenoviral overexpression of a dominant negative SREBP2, which abolished cholesterol-regulated changes in DnaJA4 expression. To determine the functional significance of this regulation, DnaJA4 was overexpressed in COS cells, which induced a specific increase in the synthesis of cholesterol from acetate. We also observed that DnaJA4 overexpression increased the activity and the protein content of HMG-CoA reductase, the rate limiting enzyme in this pathway. At the molecular level, DnaJA4 overexpression did not alter HMG-CoA reductase stability or mRNA levels, suggesting a co-translational effect of the chaperone. In the DnaJ/Hsp40 family, DnaJA4 uniquely exhibited SREBP-regulated expression, and also responded to heat shock. Through its responsiveness to SREBP, and its stimulatory effect on cholesterol synthesis, the DnaJA4 chaperone can be viewed as a new player in cholesterol synthesis. These data suggest a link between molecular chaperones, heat stress and cholesterol synthesis.     

Cross-Sectional and Longitudinal Associations of Different Sedentary Behaviors with Cognitive Performance in Older Adults

Background

The deleterious health effects of sedentary behaviors, independent of physical activity, are increasingly being recognized. However, associations with cognitive performance are not known.

Purpose

To estimate the associations between different sedentary behaviors and cognitive performance in healthy older adults.

Methods

Computer use, time spent watching television (TV), time spent reading and habitual physical activity levels were self-reported twice (in 2001 and 2007) by participants in the SUpplémentation en Vitamines et MinérauX (SU.VI.MAX and SU.VI.MAX2) study. Cognitive performance was assessed at follow-up (in 2007–2009) via a battery of 6 neuropsychological tests used to derive verbal memory and executive functioning scores. Analyses (ANCOVA) were performed among 1425 men and 1154 women aged 65.6±4.5 at the time of the neuropsychological evaluation. We estimated mean differences with 95% confidence intervals (95%CI) in cognitive performance across categories of each type of sedentary behavior.

Results

In multivariable cross-sectional models, compared to non-users, participants using the computer for >1 h/day displayed better verbal memory (mean difference = 1.86; 95%CI: 0.95, 2.77) and executive functioning (mean difference = 2.15; 95%CI: 1.22, 3.08). A negative association was also observed between TV viewing and executive functioning. Additionally, participants who increased their computer use by more than 30 min between 2001 and 2007 showed better performance on both verbal memory (mean difference = 1.41; 95%CI: 0.55, 2.27) and executive functioning (mean difference = 1.41; 95%CI: 0.53, 2.28) compared to those who decreased their computer use during that period.

Conclusion

Specific sedentary behaviors are differentially associated with cognitive performance. In contrast to TV viewing, regular computer use may help maintain cognitive function during the aging process.

Clinical Trial Registration

clinicaltrial.gov (number {"type":"clinical-trial","attrs":{"text":"NCT00272428","term_id":"NCT00272428"}}NCT00272428).     

Acetaldehyde-induced mutational pattern in the tumour suppressor gene TP53 analysed by use of a functional assay, the FASAY (functional analysis of separated alleles in yeast)

Chronic alcohol consumption is a major risk factor for upper aero-digestive tract cancers, including cancer of the esophagus. Whereas alcohol as such is not thought to be directly carcinogenic, acetaldehyde, its first metabolite, has been proven genotoxic and mutagenic in the HPRT gene. As mutations in the tumour suppressor gene TP53 are the most common genetic alterations involved in human cancers, especially esophageal tumours, the aim of this work was to establish the mutational pattern induced by acetaldehyde in vitro on the TP53 gene, and to compare this pattern with that found in human alcohol-related tumours. For this purpose, we used a functional assay in yeast, the FASAY (functional analysis of separated alleles in yeast), after in vitro exposure of human normal fibroblasts AG1521 to acetaldehyde. We noted 35 mutations, of which 32 were single-nucleotide substitutions including 2 nonsense and 30 missense mutations. The pattern showed that the main mutations were G>A transitions (n=23, of which 14 in CpG sites), followed by G>T transversions (n=4), A>G transitions (n=2) and A>T transversions (n=2). Other mutations were one-base insertion and two deletions, leading to frameshifts. Eleven mutations (31%) were located in TP53 hot-spots in codons 245, 248, 249 and 273. Finally, we compared this pattern with that found for esophageal cancers in humans. These results support the notion that acetaldehyde plays a role in TP53 mutations in esophageal cancers. The key feature of this approach is that mutagenesis is directly studied in a key gene in human carcinogenesis, allowing direct comparison of mutational patterns with those in human tumours.     

The N-terminal domain of mammalian Lysyl-tRNA synthetase is a functional tRNA-binding domain.

Lysyl-tRNA synthetase from higher eukaryotes possesses a lysine-rich N-terminal polypeptide extension appended to a classical prokaryotic-like LysRS domain. Band shift analysis showed that this extra domain provides LysRS with nonspecific tRNA binding properties. A N-terminally truncated derivative of LysRS, LysRS-DeltaN, displayed a 100-fold lower apparent affinity for tRNA(3)Lys and a 3-fold increase in K(m) for tRNA(3)Lys in the aminoacylation reaction, as compared with the native enzyme. The isolated N-domain of LysRS also displayed weak affinity for tRNA, suggesting that the catalytic and N-domains of LysRS act synergistically to provide a high affinity binding site for tRNA. A more detailed analysis revealed that LysRS binds and specifically aminoacylates an RNA minihelix mimicking the amino acid acceptor stem-loop structure of tRNA(3)Lys, whereas LysRS-DeltaN did not. As a consequence, merging an additional RNA-binding domain into a bacterial-like LysRS increases the catalytic efficiency of the enzyme, especially at the low concentration of deacylated tRNA prevailing in vivo. Our results provide new insights into tRNA(Lys) channeling in eukaryotic cells and shed new light on the possible requirement of native LysRS for triggering tRNA(3)Lys packaging into human immunodeficiency virus, type 1 viral particles.     

The Arabidopsis BLAP75/Rmi1 Homologue Plays Crucial Roles in Meiotic Double-Strand Break Repair

In human cells and in Saccharomyces cerevisiae, BLAP75/Rmi1 acts together with BLM/Sgs1 and TopoIIIα/Top3 to maintain genome stability by limiting crossover (CO) formation in favour of NCO events, probably through the dissolution of double Holliday junction intermediates (dHJ). So far, very limited data is available on the involvement of these complexes in meiotic DNA repair. In this paper, we present the first meiotic study of a member of the BLAP75 family through characterisation of the Arabidopsis thaliana homologue. In A. thaliana blap75 mutants, meiotic recombination is initiated, and recombination progresses until the formation of bivalent-like structures, even in the absence of ZMM proteins. However, chromosome fragmentation can be detected as soon as metaphase I and is drastic at anaphase I, while no second meiotic division is observed. Using genetic and imunolocalisation studies, we showed that these defects reflect a role of A. thaliana BLAP75 in meiotic double-strand break (DSB) repair—that it acts after the invasion step mediated by RAD51 and associated proteins and that it is necessary to repair meiotic DSBs onto sister chromatids as well as onto the homologous chromosome. In conclusion, our results show for the first time that BLAP75/Rmi1 is a key protein of the meiotic homologous recombination machinery. In A. thaliana, we found that this protein is dispensable for homologous chromosome recognition and synapsis but necessary for the repair of meiotic DSBs. Furthermore, in the absence of BLAP75, bivalent formation can happen even in the absence of ZMM proteins, showing that in blap75 mutants, recombination intermediates exist that are stable enough to form bivalent structures, even when ZMM are absent.     

Sexual reproduction of Daphnia in a deep temperate reservoir: the phenology and genetics of male formation

Long-term persistence of a Daphnia population is allowed by the production of dormant stages, produced mostly through sexual reproduction. We investigated the spatio-temporal dynamics of male production in a spring population of Daphnia galeata in the reservoir, and compared the genetic structure of three groups within this population: male-, female-producing females, and adult males. With a fine resolution sampling design and the use of highly variable microsatellite markers we revealed that: (1) the spring period of male offspring production was delimited in time with minimum interannual variation to about 3 weeks, and in space to the upper 5-m water layer; (2) there were no remarkable changes in the clonal composition of male-producing females within the period of male production; (3) overall certain clones exhibited a higher tendency to produce male offspring and therefore the clonal structure of male-producing lineages was significantly different from that of female-producing lineages; and (4) the clonal structure of male-producing females was not significantly different from that of adult males occurring later in the reservoir. This suggests that males were not subjected to any significant selective forces till maturity and the male-producing females confer a long-term fitness advantage over female-producing females.