Seasonal effect on tissue culture response and plant regeneration frequency from non-bombarded and bombarded immature scutella of barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) harvested from controlled environment

Scutella of immature embryos from two barley cultivars were used for cell culture and transformation. Explants were supplied by continuous growth of donor plants in a 2-week schedule under defined conditions at first plants were grown for 6–7 weeks in a growth chamber, followed by 10–13 weeks in a greenhouse with stringent control of temperature and light round the year. Strong seasonal variation in plant regeneration frequency was observed for both genotypes in non-bombarded (control) as well as bombarded and subsequent selected explants. Scutella from immature embryos of cv. Salome showed increased frequencies of plant regeneration from January to March, reaching highest values in March/April and followed by a continuous and strong decrease from May to December. This tendency was observed in all 3 years studied, although absolute numbers of plant regeneration varied between the years. The same seasonal effect was evident for plant regeneration from immature scutella of cv. Golden Promise. Frequency of embryogenic callus formation was also found to be influenced by season but this effect was not so pronounced as for plant regeneration.     

The Combined Effects of Pre- and Post-Insemination Sexual Selection on Extreme Variation in Male Body Size

Orb-weaving spiders of the genus Nephila are notable for their sexual size dimorphism, with dwarf males and giant females. However, less well known is the extreme size polymorphism of males that is characteristic of some species. For example, adult male body size in N. edulis varies by an order of magnitude. Previous experiments reveal that male mating behaviour covaries with body size, suggesting the size variation is maintained by opposing pre- and post-insemination sexual selection pressures. Here, we test this idea by allowing males of different sizes to compete directly and simultaneously for access to females. Using the sterile-male technique for paternity assessment, we show that two competing males drawn from the extremes of size variations, split paternity the same way as two males of the same size drawn from the intermediate sizes. The paternity of a large male dropped from 50% to 30% on average if he competed against two instead of one small male. The large male increased his mating frequency when there were more rivals but required a much lower total duration of copulation to achieve the same paternity share. These data are consistent with the idea that opposing pre- and post-insemination selection pressures at least partly explain the variation in male body size. Co-ordinating editor: L.D. Hurst     

Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1

Deleted in malignant brain tumours 1 (DMBT1) codes for a approximately 340kDa glycoprotein with highly repetitive scavenger receptor cysteine-rich (SRCR) domains. DMBT1 was implicated in cancer, defence against viral and bacterial infections, and differentiation of epithelial cells. Recombinant expression and purification of DMBT1 is an essential step for systematic standardized functional research and towards the evaluation of its therapeutical potential. So far, DMBT1 is obtained from natural sources such as bronchioalveolar lavage or saliva, resulting in time consuming sample collection, low yields, and protein preparations which may substantially vary due to differential processing and genetic polymorphism, all of which impedes functional research on DMBT1. Cloning of DMBT1 cDNAs is hampered because of the size and the 13 highly homologous SRCR exons. In this study, we report on the setup of a vector system that facilitates cloning of DMBT1 variants. We demonstrate applicability of the vector system by expression of the largest DMBT1 variant in a tetracycline-inducible mammalian expression system using the Chinese hamster ovary cell line. Yields up to 30 mg rDMBT1 per litre of cell culture supernatant could be achieved with an optimized production procedure. By harnessing the specific bacteria-binding property of DMBT1 we established an affinity purification procedure which allows the isolation of more than 3 mg rDMBT1 with a purity of about 95%. Although the glycosylation moieties of rDMBT1 are different from DMBT1(SAG) isolated from saliva, we demonstrate that rDMBT1 is functionally active in aggregating Gram-positive and Gram-negative bacteria and binding to C1q and lactoferrin, which represent two known endogenous DMBT1 ligands.     

Heterozygous deficiency of manganese superoxide dismutase results in severe lipid peroxidation and spontaneous apoptosis in murine myocardium in vivo

To circumvent the early lethality of manganese superoxide dismutase (SOD2)-deficient mice, we have used a skin-specific strategy with introduction of loxP sites flanking exon 3 of the SOD2 gene. To our surprise, when breeding a female keratin 14 Cre transgenic mouse to a SOD2 "floxed" male mouse, due to keratin 14 promoter-driven Cre expression in the oocytes, all offspring were heterozygous for SOD2. In sharp contrast to initial publications on SOD2(+/-) mice, the herein reported mice on a mixed genetic background (C57BL/6 x 129/Ola) in their heterozygous state (SOD(+/-)) revealed distinct ultrastructural damage of the myocard, with swelling and disruption of mitochondria and accumulation of lipid droplets, increased nitrotyrosine formation, and lipid peroxidation as well as activation of apoptosis signaling pathways in the heart in vivo. Strikingly, and so far unreported, we found a substantial decrease in the activity of the cytosolic copper, zinc superoxide dismutase (SOD1) in the heart tissue of SOD2(+/-) mice, suggesting that the breakdown of mitochondrial membranes in the heart of SOD2(+/-) mice results in the enhanced release of superoxide anion radicals or derivatives thereof with subsequent inactivation of cytosolic SOD1. This model may be particularly suited to long-term studies on age-related heart failure as well as other age-related diseases and the polygenic base of tissue-specific responses to oxidative injury.     

The human solute carrier gene SLC35B4 encodes a bifunctional nucleotide sugar transporter with specificity for UDP-xylose and UDP-N-acetylglucosamine

The transport of nucleotide sugars from the cytoplasm into the Golgi apparatus is mediated by specialized type III proteins, the nucleotide sugar transporters (NSTs). Transport assays carried out in vitro with Golgi vesicles from mammalian cells showed specific uptake for a total of eight nucleotide sugars. When this study was started, NSTs with transport activities for all but two nucleotide sugars (UDP-Xyl and UDP-Glc) had been cloned. Aiming at identifying these elusive NSTs, bioinformatic methods were used to display putative NST sequences in the human genome. Ten open reading frames were identified, cloned, and heterologously expressed in yeast. Transport capabilities for UDP-Glc and UDP-Xyl were determined with Golgi vesicles isolated from transformed cells. Although a potential UDP-Glc transporter could not be identified due to the high endogenous transport background, the measurement of UDP-Xyl transport was possible on a zero background. Vesicles from yeast cells expressing the human gene SLC35B4 showed specific uptake of UDP-Xyl, and subsequent testing of other nucleotide sugars revealed a second activity for UDP-GlcNAc. Expression of the epitope-tagged SLC35B4 in mammalian cells demonstrated strict Golgi localization. Because decarboxylation of UDP-GlcA is known to produce UDP-Xyl directly in the endoplasmic reticulum and Golgi lumen, our data demonstrate that two ways exist to deliver UDP-Xyl to the Golgi apparatus.     

The use of mini-organ cultures of human upper aerodigestive tract epithelia in ecogenotoxicology

The carcinogenic potential of xenobiotics and possible confounders are often difficult to differentiate in in vivo studies. In contrast, in vitro studies allow investigation of the impact of carcinogens on human target cells under standardized conditions. The aim of the present study is to demonstrate whether three-dimensional mini organ-cultures (MOCs) of human inferior nasal turbinate epithelia may represent a useful model to study genotoxic effects of xenobiotics in vitro. Culture of mini organs was performed by cutting 1mm3 pieces from fresh specimens of inferior nasal turbinates. After a period of 5-6 days the specimens were fully covered with epithelium. On days 7, 9, and 11 of culture, intact MOCs from 25 tissue donors were incubated with dimethyl sulfoxide (DMSO) as a negative control, or with mono(2-ethylhexyl) phthalate (MEHP), benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). On days 7 and 11, MOCs were analyzed by the alkaline Comet assay to detect DNA-single-strand breaks, alkali-labile sites and incomplete excision-repair sites. DNA migration after single exposure of non-cultivated fresh specimens was also analyzed. In order to detect regimen-specific effects, DNA fragmentation after single exposure of intact MOCs was compared with that of cells after separation of MOCs on day 7 of culture and consecutive exposure of individual cells. Significant DNA migration as a measure of DNA single-strand breaks, alkali-labile sites and incomplete excision repair sites, was found after electrophoresis due to single and triple exposure of MOCs to MEHP, BPDE and MNNG. Triple exposure of MOCs compared to single exposure revealed no difference after exposure to DMSO or MEHP, and an increased migration after exposure to BPDE and MNNG. When single exposure of isolated cells from fresh specimens was compared with that of intact MOCs, DMSO and MNNG had no significantly different effect, whereas exposure to MEHP or BPDE caused a reduced migration in cells from MOCs. When exposure of isolated cells harvested from MOCs was compared with exposure of intact MOCs, MEHP and BPDE caused a significantly lower DNA migration in intact MOCs. MOCs provide an in vitro model suitable for the assessment of genotoxic effects of environmental pollutants both after single or repetitive exposure. Due to the intact structure of the exposed mucosa this model may be a helpful tool in mimicking the in vivo situation in ecogenotoxicology studies.     

Strong genetic evidence of DCDC2 as a susceptibility gene for dyslexia

We searched for linkage disequilibrium (LD) in 137 triads with dyslexia, using markers that span the most-replicated dyslexia susceptibility region on 6p21-p22, and found association between the disease and markers within the VMP/DCDC2/KAAG1 locus. Detailed refinement of the LD region, involving sequencing and genotyping of additional markers, showed significant association within DCDC2 in single-marker and haplotype analyses. The association appeared to be strongest in severely affected patients. In a second step, the study was extended to include an independent sample of 239 triads with dyslexia, in which the association--in particular, with the severe phenotype of dyslexia--was confirmed. Our expression data showed that DCDC2, which contains a doublecortin homology domain that is possibly involved in cortical neuron migration, is expressed in the fetal and adult CNS, which--together with the hypothesized protein function--is in accordance with findings in dyslexic patients with abnormal neuronal migration and maturation.     

The mechanism of action of ramoplanin and enduracidin

The lipoglycodepsipeptide antibiotic ramoplanin is proposed to inhibit bacterial cell wall biosynthesis by binding to intermediates along the pathway to mature peptidoglycan, which interferes with further enzymatic processing. Two sequential enzymatic steps can be blocked by ramoplanin, but there is no definitive information about whether one step is inhibited preferentially. Here we use inhibition kinetics and binding assays to assess whether ramoplanin and the related compound enduracidin have an intrinsic preference for one step over the other. Both ramoplanin and enduracidin preferentially inhibit the transglycosylation step of peptidoglycan biosynthesis compared with the MurG step. The basis for stronger inhibition is a greater affinity for the transglycosylase substrate Lipid II over the MurG substrate Lipid I. These results provide compelling evidence that ramoplanin's and enduracidin's primary cellular target is the transglycosylation step of peptidoglycan biosynthesis.