Structure and evolution of <Emphasis Type="Italic">Apetala3</Emphasis>, a sex-linked gene in <Emphasis Type="Italic">Silene latifolia</Emphasis>

Background  

The evolution of sex chromosomes is often accompanied by gene or chromosome rearrangements. Recently, the gene AP3 was characterized in the dioecious plant species Silene latifolia. It was suggested that this gene had been transferred from an autosome to the Y chromosome.     

A single amino acid change (Y318F) in the L-arabitol dehydrogenase (LadA) from Aspergillus niger results in a significant increase in affinity for D-sorbitol

Background  

L-arabitol dehydrogenase (LAD) and xylitol dehydrogenase (XDH) are involved in the degradation of L-arabinose and D-xylose, which are among the most abundant monosaccharides on earth. Previous data demonstrated that LAD and XDH not only differ in the activity on their biological substrate, but also that only XDH has significant activity on D-sorbitol and may therefore be more closely related to D-sorbitol dehydrogenases (SDH). In this study we aimed to identify residues involved in the difference in substrate specificity.     

An error in dystrophin mRNA processing in golden retriever muscular dystrophy, an animal homologue of Duchenne muscular dystrophy.

Golden retriever muscular dystrophy (GRMD) is a spontaneous, X-linked, progressively fatal disease of dogs and is also a homologue of Duchenne muscular dystrophy (DMD). Two-thirds of DMD patients carry detectable deletions in their dystrophin gene. The defect underlying the remaining one-third of DMD patients is undetermined. Analysis of the canine dystrophin gene in normal and GRMD dogs has failed to demonstrate any detectable loss of exons. Here, we have demonstrated a RNA processing error in GRMD that results from a single base change in the 3' consensus splice site of intron 6. The seventh exon is then skipped, which predicts a termination of the dystrophin reading frame within its N-terminal domain in exon 8. This is the first example of dystrophin deficiency caused by a splice-site mutation.     

A novel role for defensins in intestinal homeostasis: regulation of IL-1beta secretion

Impaired expression of alpha-defensin antimicrobial peptides and overproduction of the proinflammatory cytokine IL-1beta have been associated with inflammatory bowel disease. In this study, we examine the interactions between alpha-defensins and IL-1beta and the role of defensin deficiency in the pathogenesis of inflammatory bowel disease. It was found that matrix metalloproteinase-7-deficient (MMP-7(-/-)) mice, which produce procryptdins but not mature cryptdins (alpha-defensins) in the intestine, were more susceptible to dextran sulfate sodium-induced colitis. Furthermore, both baseline and dextran sulfate sodium-induced IL-1beta production in the intestine were significantly up-regulated in MMP-7(-/-) mice compared with that in control C57BL/6 mice. To elucidate the molecular mechanism for the increased IL-1beta production in defensin deficiency in vivo, we evaluated the effect of defensins on IL-1beta posttranslational processing and release. It was found that alpha-defensins, including mouse Paneth cell defensins cryptdin-3 and cryptdin-4, human neutrophil defensin HNP-1, and human Paneth cell defensin HD-5, blocked the release of IL-1beta from LPS-activated monocytes, whereas TNF-alpha expression and release were not affected. Unlike alpha-defensins, human beta-defensins and mouse procryptdins do not have any effect on IL-1beta processing and release. Thus, alpha-defensins may play an important role in intestinal homeostasis by controlling the production of IL-1beta.     

Cloning the quinic acid (aq) gene cluster from Neurospora crassa: identification of recombinant plasmids containing both qa-2+ and qa-3+

A 22.2-kb insert of Neurospora crassa DNA containing at least two of the genes from the inducible catabolic quinic acid pathway has been cloned into the cosmid vehicle pHC79 resulting in a recombinant plasmid, pMSK308. The qa-2+ locus (which encodes catabolic dehydroquinase) is functionally expressed in both Escherichia coli and qa-2 mutants of N. crassa transformed with pMSK308 plasmid DNA. Expression of the qa-3 gene (which encodes quinate dehydrogenase) is only detected upon reintroduction into N. crassa. Results were also obtained which suggested that the qa-4 gene, which maps between qa-2 and qa-3, may also be present on both pMSK308 and the previously described plasmid pVK88. Certain anomalies in the types of N. crassa transformants obtained with pMSK308 plasmid DNA were noted.     

Dopamine transporter-dependent and -independent actions of trace amine beta-phenylethylamine

Beta-phenylethylamine (beta-PEA) is an endogenous amine that is found in trace amounts in the brain. It is believed that the locomotor-stimulating action of beta-PEA, much like amphetamine, depends on its ability to increase extracellular dopamine (DA) concentrations owing to reversal of the direction of dopamine transporter (DAT)-mediated DA transport. beta-PEA can also bind directly to the recently identified G protein-coupled receptors, but the physiological significance of this interaction is unclear. To assess the mechanism by which beta-PEA mediates its effects, we compared the neurochemical and behavioral effects of this amine in wild type (WT), heterozygous and 'null' DAT mutant mice. In microdialysis studies, beta-PEA, administered either systemically or locally via intrastriatal infusion, produced a pronounced outflow of striatal DA in WT mice whereas no increase was detected in mice lacking the DAT (DAT-KO mice). Similarly, in fast-scan voltammetry studies beta-PEA did not alter DA release and clearance rate in striatal slices from DAT-KO mice. In behavioral studies beta-PEA produced a robust but transient increase in locomotor activity in WT and heterozygous mice. In DAT-KO mice, whose locomotor activity and stereotypy are increased in a novel environment, beta-PEA (10-100 mg/kg) exerted a potent inhibitory action. At high doses, beta-PEA induced stereotypies in WT and heterozygous mice; some manifestations of stereotypy were also observed in the DAT-KO mice. These data demonstrate that the DAT is required for the striatal DA-releasing and hyperlocomotor actions of beta-PEA. The inhibitory action on hyperactivity and certain stereotypies induced by beta-PEA in DAT-KO mice indicate that targets other than the DAT are responsible for these effects.     

Effects of aromatic cationic molecules on bovine viral diarrhea virus and embryonic development

Bovine viral diarrhea virus (BVDV) has been shown to replicate in embryo culture systems and remain associated with bovine embryos developing in vitro. In this study, novel antiviral agents were evaluated for capability to inhibit replication of BVDV without affecting embryonic development. Serial concentrations of 2-[5(6)-{2-imidazolinyl}-2-benzimidazolyl]-5-(4-aminophenyl)furan (DB456) or 2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan (DB606) were prepared in IVC medium. Then, bovine uterine tubal epithelial cells (UTC) were placed in IVC media with varying concentrations of DB456 or DB606. Within 1h, a genotype I or II strain of BVDV was added to the cultures. Cultures were maintained for 7 days. Infectious virus was quantitated in IVC media collected on days 3 and 7 and in UTC lysates harvested on day 7. The effective antiviral concentrations of DB606 were much lower than effective antiviral concentrations of DB456. In subsequent experiments, IVF presumptive zygotes were cultured in IVC medium with or without DB456 or DB606 at multiple concentrations for 7 days to evaluate effect of the compound on conceptus development. On day 7, stage of embryonic development was observed, and blastocysts were harvested and stained using Hoechst 33342 to enumerate embryonic cells. While DB456 inhibited blastocyst development, DB606 at 20 times the effective antiviral concentration did not hinder blastocyst development or reduce the mean number of cells per blastocyst. These preliminary results indicated that bovine embryo cultures might be safely supplemented with effective concentrations of an antiviral agent.     

Inhibition of topoisomerases from Pneumocystis carinii by aromatic dicationic molecules.

Pentamidine and related derivatives inhibit an ATP-dependent topoisomerase activity from Pneumocystis carinii extracts. Since it would be extremely difficult to purify ample quantities of the organisms to allow characterization of the enzyme and carry out drug binding experiments, we have begun the cloning of the topoisomerase genes with a goal towards expression of each gene in a heterologous system. Following construction of genomic libraries in the vectors lambda DASH and lambda ZAP, oligonucleotides corresponding to conserved regions of both topoisomerases I and II were used in the polymerase chain reaction (PCR) of P. carinii DNA to generate probes. Candidate clones for both genes have been identified. Partial DNA sequence of the topoisomerase II gene has been determined.     

Instantaneous benthic response to different organic matter quality: In situ experiments in the Benguela Upwelling System

The benthic community in continental slope and deep-sea sediments of the Benguela Upwelling System was supplied with ¹³C-labelled organic matter (OM) of two different qualities using a benthic chamber lander. Freeze-dried cultures of Skeletonema costatum served as 'fresh' OM. 'Altered' OM of the same material had been additionally dialysed to remove low-molecular weight compounds. In order to investigate the benthic response pattern, mineralization of labelled OM, uptake by macrofauna and incorporation into bacteria were followed over 18-36 h. Total oxygen uptake was not affected beyond natural variation by the OM addition. Mineralization dominated the ¹³C-labelled phytodetritus processing, constituting 71-95% of the total processed OM. Bacterial incorporation of phytodetrital carbon exceeded macrofaunal uptake at all stations. Stations situated in a major centre of OM deposition showed phytodetritus processing rates on average twice as high as outside the depocentre. Phytodetritus processing was 1.5, 2.5 and 4.3 times higher for fresh than for altered OM at 605, 1019 and 1335 m water depth, respectively. Our observations clearly indicate the importance of OM quality on mineralization rates.