FIRST EVIDENCE OF PALYTOXIN ANALOGUES FROM AN OSTREOPSIS MASCARENENSIS (DINOPHYCEAE) BENTHIC BLOOM IN SOUTHWESTERN INDIAN OCEAN1

Benthic dinoflagellates of the genus Ostreopsis Schmidt are common in tropical and subtropical water, and some species produce toxins potentially involved in human intoxication events. A benthic bloom of Ostreopsis mascarenensis Quod was observed near Rodrigues Island during a survey of benthic dinoflagellates in the southwestern Indian Ocean. The morphology of O. mascarenensis was studied by LM and SEM. Preliminary screening of a crude extract of an O. mascarenensis bloom revealed neurotoxicity in mice similar to that induced by palytoxin. After partition of the crude extract, the highest toxicity was retained in the butanol‐soluble fraction, which retained hemolytic activity suggestive of palytoxin analogues. Two new toxins, mascarenotoxin‐A and ‐B, were resolved from this fraction by HPLC coupled to a diode array detector. The closed mass spectrum profile and fragmentation pattern obtained by advanced nano–electrospray ionization quadrupole time‐of‐flight mass spectrometry between purified toxins and a reference palytoxin confirmed the mascarenotoxins as palytoxin analogues. These results were confirmed by tandem mass spectrometry with the identification of specific fragment ion m/z 327. An on‐line liquid chromatography protocol coupled to tandem mass spectrometry was developed for detection of these palytoxin analogues. The present study describes the first purification, chemical, and toxicological characterization of new palytoxin analogues isolated from a benthic bloom of O. mascarenensis. These results suggest that O. mascarenensis, which is largely distributed in the southwestern Indian Ocean, could be a source of palytoxin poisoning in this tropical area.     

A plate assay for simultaneous screening of polysaccharide- and protein-degrading micro-organisms

AIMS: To develop a plate assay for simultaneous screening of polysaccharide-degrading and protein-degrading micro-organisms. METHODS AND RESULTS: A plate assay, based on the visible solubilization of small substrate particles and the formation of haloes on Petri dishes, containing a mixture of diversely coloured insoluble polysaccharides and dye-labelled collagen as chromogenic substrates, was developed. This method was successfully applied for isolating the diverse polysaccharide- and/or protein-degrading bacteria from soil and sludge samples. Selected strains were identified using 16S rDNA partial sequencing; most of them belong to the genera Bacillus, Cellulomonas and Cellulosimicrobium. CONCLUSIONS: This novel approach provides unique and valuable information for direct primary screening when the target of selection is micro-organisms exhibiting protein-degrading activity, polysaccharide-degrading activity or a specific combination of them. SIGNIFICANCE AND IMPACT OF THE STUDY: This plate assay is convenient and easy to perform, rapid, and more adaptable for screening of a large number of samples, compared with other existing methods in the literature.     

Preconception cystic fibrosis carrier screening: costs and consequences

The objective of the study was to determine net costs of four potential preconception cystic fibrosis (CF) carrier screening strategies, and to identify the most important cost-related parameters. An estimate was made of the costs of screening carried out during general practitioner (GP) consultations, and screening carried out during group educational sessions. Two test strategies were evaluated: single-entry two-step (SETS) and double- entry two-step (DETS). Several sensitivity analyses were performed. Under the baseline assumptions, SETS screening during GP consultations was the least expensive (net costs US dollar 1,111,354 per year). The sensitivity analysis showed that the costs depend greatly on the estimated lifetime costs of care for a CF patient and the strategy used to inform the target population. Adaptations could be made to the screening program, resulting in economic savings while the primary goal of the screening--to enable participants to make an autonomous informed choice--is guaranteed.     

Neonatal androgenization of hypogonadal (hpg) male mice does not abolish estradiol-induced FSH production and spermatogenesis

Background  

Testicular development is arrested in the hypogonadal (hpg) mouse due to a congenital deficiency in hypothalamic gonadotropin-releasing hormone (GnRH) synthesis. Chronic treatment of male hpg mice with estradiol induces FSH synthesis and secretion, and causes testicular maturation and qualitatively normal spermatogenesis. As estradiol negative feedback normally inhibits FSH production in the male, this study tested whether this paradoxical response to estradiol in the male hpg mouse might be due to inadequate masculinisation or incomplete defeminization in the neonatal period. Previous studies have demonstrated that treatment of hpg mice with testosterone propionate in the immediate neonatal period is necessary to allow full reproductive behaviors to be expressed following suitable endocrine stimulation at adult ages.     

An inhibitor of O-glycosylation induces apoptosis in NIH3T3 cells and developing mouse embryonic mandibular tissues

The family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases) is responsible for initiating mucin-type O-linked glycosylation in higher eukaryotes. To begin to examine the biological role of O-linked glycosylation, mammalian cells were treated with a small molecule inhibitor (designated 1-68A, Ref. 15) of ppGaNTase activity. NIH3T3 cells exposed to the inhibitor were shown to undergo a significant reduction in cell surface O-glycosylation as detected by staining with jacalin and peanut agglutinin lectins after 30 min of treatment; no reduction in staining using antibodies to O-linked N-acetylglucosamine or the lectin concanavalin A was detected. Apoptosis was also observed in treated cells after 45 min of exposure, ostensibly following the O-glycosylation reduction. Overexpression of several different ppGaNTase isoforms restored cell surface O-glycosylation and rescued inhibitor-induced apoptosis. Additionally, mouse embryonic mandibular organ cultures exposed to 1-68A developed abnormally, presumably because of epithelial and mesenchymal apoptosis that followed a reduction in jacalin and peanut agglutinin staining. Our studies suggest that mucin-type O-linked glycosylation may be required for normal development and that ppGaNTases may play a role in the regulation of apoptosis.     

Smad7-induced beta-catenin degradation alters epidermal appendage development

To assess whether Smad signaling affects skin development, we generated transgenic mice in which a Smad antagonist, Smad7, was induced in keratinocytes, including epidermal stem cells. Smad7 transgene induction perturbed hair follicle morphogenesis and differentiation, but accelerated sebaceous gland morphogenesis. Further analysis revealed that independent of its role in anti-Smad signaling, Smad7 bound beta-catenin and induced beta-catenin degradation by recruiting an E3 ligase, Smurf2, to the Smad7/beta-catenin complex. Consequently, Wnt/beta-catenin signaling was suppressed in Smad7 transgenic hair follicles. Coexpression of the Smurf2 and Smad7 transgenes exacerbated Smad7-induced abnormalities in hair follicles and sebaceous glands. Conversely, when endogenous Smad7 was knocked down, keratinocytes exhibited increased beta-catenin protein and enhanced Wnt signaling. Our data reveal a mechanism for Smad7 in antagonizing Wnt/beta-catenin signaling, thereby shifting the skin differentiation program from forming hair follicles to sebaceous glands.     

Regulatory factors and cell populations involved in skeletal muscle regeneration

Skeletal muscle regeneration is a complex process, which is not yet completely understood. Satellite cells, the skeletal muscle stem cells, become activated after trauma, proliferate, and migrate to the site of injury. Depending on the severity of the myotrauma, activated satellite cells form new multinucleated myofibers or fuse to damaged myofibers. The specific microenvironment of the satellite cells, the niche, controls their behavior. The niche contains several components that maintain satellite cells quiescence until they are activated. In addition, a great diversity of stimulatory and inhibitory growth factors such as IGF‐1 and TGF‐β1 regulate their activity. Donor‐derived satellite cells are able to improve muscle regeneration, but their migration through the muscle tissue and across endothelial layers is limited. Less than 1% of their progeny, the myoblasts, survive the first days upon intra‐muscular injection. However, a range of other multipotent muscle‐ and non‐muscle‐derived stem cells are involved in skeletal muscle regeneration. These stem cells can occupy the satellite cell niche and show great potential for the treatment of skeletal muscle injuries and diseases. The aim of this review is to discuss the niche factors, growth factors, and other stem cells, which are involved in skeletal muscle regeneration. Knowledge about the factors regulating satellite cell activity and skeletal muscle regeneration can be used to improve the treatment of muscle injuries and diseases. J. Cell. Physiol. 224:7–16, 2010 © 2010 Wiley‐Liss, Inc.     

Implications of mitochondrial DNA polyphyly in two ecologically undifferentiated but morphologically distinct migratory birds, the masked and white-browed woodswallows Artamus spp. of inland Australia

The white‐browed woodswallow Artamus superciliosus and masked woodswallow A. personatus (Passeriformes: Artamidae) are members of Australia's diverse arid‐ and semi‐arid zone avifauna. Widely sympatric and among Australia's relatively few obligate long‐distance temperate‐tropical migrants, the two are well differentiated morphologically but not ecologically and vocally. They are pair breeders unlike other Artamus species, which are at least facultative cooperative breeders. For these reasons they are an excellent case in which to use molecular data in integrative study of their evolution from ecological and biogeographical perspectives. We used mitochondrial DNA (mtDNA) to test whether they are each other's closest relatives, whether they evolved migration independently, whether they have molecular signatures of population expansions like some other Australian arid zone birds, and to estimate the timing of any inferred population expansions. Their mtDNAs are monophyletic with respect to other species of Artamus but polyphyletic with respect to each other. The two species appear not to have evolved migration independently of each other but their morphological and mtDNA evolution have been strongly decoupled. Some level of hybridization and introgression cannot be dismissed outright as being involved in their mtDNA polyphyly but incomplete sorting of their most recent common ancestor's mtDNA is a simpler explanation consistent with their ecology. Bayesian phylogenetic inference and analyses of diversity within the two species (n=77) with conventional diversity statistics, statistical parsimony, and tests for population expansion vs stability (Tajima's D, Fu's Fs and Ramos‐Onsin and Rozas's R₂) all favour recent population increases. However, a non‐starlike network suggests expansion(s) relatively early in the Pleistocene. Repeated population bottlenecks corresponding with multiple peaks of Pleistocene aridity could explain our findings, which add a new dimension to accruing data on the effects of Pleistocene aridity on the Australian biota.     

Carbohydrate microarrays reveal sulphation as a modulator of siglec binding

Siglecs are receptors on cells of the immune, haemopoietic, and nervous systems that recognize sialyl-glycans with differing preferences for sialic acid linkage and oligosaccharide backbone sequence. We investigate here siglec binding using microarrays of Lewis(x) (Le(x))- and 3'-sialyl-Le(x)-related probes with different sulphation patterns. These include sulphation at position 3 of the terminal galactose of Le(x), position 6 of the galactose of Le(x) and sialyl-Le(x), position 6 of N-acetylglucosamine of Le(x) and sialyl-Le(x), or both positions of sialyl-Le(x). Recombinant soluble forms of five siglecs have been investigated: human Siglec-7, -8, -9, and murine Siglec-F and CD22 (Siglec-2). Each siglec has a different binding pattern. Unlike two C-type lectins of leukocytes, L-selectin and Langerin, which also bind to sulphated analogues of sialyl-Le(x), the siglecs do not give detectable binding signals with sulphated analogues that are lacking sialic acid. The sulphate groups modulate, however, positively or negatively the siglec binding intensities to the sialyl-Le(x) sequence.     

Two short, daily activity bouts vs. one long bout: are health and fitness improvements similar over twelve and twenty-four weeks?

This study sought to determine whether a 12-week intermittent (INT; 2 x 15 min.d(-1)) exercise program yielded similar improvements in cardiovascular health and fitness, compared with a traditional 12-week, 30-minute continuous (CON; 1 x 30 min.d(-1)) exercise program. A second purpose was to determine the effects of switching exercise programs and continuing training for an additional 12 weeks. Twenty women and 17 men, (age 48.8 +/- 9.0 years) were divided randomly into 2 groups: INT (n = 20) and CON (n = 17). Aerobic exercise was performed 4 d.wk(-1) for 12 weeks. Subjects then crossed over to the opposite training program for an additional 12 weeks of training. Subjects exercised incrementally for weeks 1-4 and training was conducted at 70-80% heart rate reserve for weeks 5-24. Both groups showed comparable exercise adherence, completing 96.6 +/- 12.2% (CON) and 96.3% +/- 17.7% (INT) of the prescribed exercise time. The INT walked at a lower percentage of Vo(2)max, maximum heart rate, systolic blood pressure, and diastolic blood pressure (p < 0.05). Maximal oxygen consumption increased by 4.5% in CON and by 8.7% in INT. Following the second 12 weeks, Vo(2)max increased by 3.6 and 7.7% in CON and INT, respectively. Treadmill test time increased by 41 seconds in CON (p < 0.05) and 71 seconds in INT (p < 0.05) after 12 weeks of training. High-density lipoproteins significantly increased in the INT group following the first 12 weeks of training. This study suggests that an INT exercise program, which is incremental in nature, provides comparable, and in some cases greater, health and fitness benefits than those expected following traditional CON exercise training.