Engrailed homeoprotein secretion is a regulated process

Chicken Engrailed 2 homeoprotein is transported between cells in culture. This intercellular transfer is based on unconventional secretion and internalisation mechanisms: Engrailed 2 has access to vesicles but lacks a signal sequence for secretion and is internalised by a non-endocytic process. We show that phosphorylation of a serine-rich domain within Engrailed 2 by the protein kinase CK2 specifically inhibits Engrailed 2 secretion. The availability of the serine-rich domain to CK2 is highly increased when it is displaced from its normal position to the C terminus of Engrailed 2, leading to a constitutive blockage of Engrailed 2 intercellular transfer. This indicates that intercellular transfer of Engrailed 2 is a highly regulated process.     

Adipose tissue selective insulin receptor knockout protects against obesity and obesity-related glucose intolerance

Insulin signaling in adipose tissue plays an important role in lipid storage and regulation of glucose homeostasis. Using the Cre-loxP system, we created mice with fat-specific disruption of the insulin receptor gene (FIRKO mice). These mice have low fat mass, loss of the normal relationship between plasma leptin and body weight, and are protected against age-related and hypothalamic lesion-induced obesity, and obesity-related glucose intolerance. FIRKO mice also exhibit polarization of adipocytes into populations of large and small cells, which differ in expression of fatty acid synthase, C/EBP alpha, and SREBP-1. Thus, insulin signaling in adipocytes is critical for development of obesity and its associated metabolic abnormalities, and abrogation of insulin signaling in fat unmasks a heterogeneity in adipocyte response in terms of gene expression and triglyceride storage.     

Oocyte Ca2+ spike acquisition during in vitro development of early preantral follicles: influence of age and hormonal supplementation

Calcium signalling is involved in important events in oocytes, such as meiotic competence acquisition. We have previously demonstrated the positive influence of animal age and gonadotropin stimulation in vivo regarding the ability of oocytes recovered from preantral follicles to exhibit calcium spikes. In the present work we determined whether preantral follicle development in vitro also allows oocytes to acquire calcium signalling activity. We also aimed to verify the influence of animal age, FSH + LH and/or insulin on oocyte calcium spike acquisition during preantral follicle culture. Early preantral follicles were isolated from 12-day-old and 1- to 3-month-old F1 hybrid mice and cultured individually for either 2 or 6 days. At the end of the culture period the oocytes were processed for calcium imaging by confocal microscopy. We show that oocytes recovered from cultured preantral follicles exhibit variable calcium spike activity rates, depending on animal age, culture duration and hormonal supplementation. Oocytes recovered from adult animals continue to exhibit calcium spikes, and those recovered from juveniles acquire that activity after culture. Insulin and gonadotropins in combination account for an early and maintained inhibitory effect on calcium signalling acquisition by oocytes. Insulin alone also leads to an early inhibitory effect, which, however, disappears with longer culture periods. Contrary to the complex in vivo situation, the acquisition of calcium signalling by oocytes in a controlled in vitro environment does not seem to be dependent on gonadotropins alone.     

Human CDC25B and CDC25C differ by their ability to restore a functional checkpoint response after gene replacement in fission yeast

In fission yeast, inactivation of the Cdc25 phosphatase by checkpoint kinases participates in the signaling cascade that temporarily stops cell cycle progression after DNA damage. In human, CDC25B and C are also known to be targeted by a similar checkpoint machinery. We have examined by homologous recombination, whether CDC25B and CDC25C were able to substitute for the function of fission yeast Cdc25. We demonstrate that (i) CDC25B and C efficiently replace Cdc25 for vegetative growth, (ii) CDC25C is able to restore a functional checkpoint in response to ionizing radiation in both a Chk1- and Cds1-dependent manner, (iii) CDC25B and C are equally efficient in the response to UV irradiation, CDC25B being only dependent on Chk1, while CDC25C depends on both Chk1 and Cds1, and (iv) CDC25C is able to restore a functional DNA replication checkpoint induced by hydroxyurea in a Cds1-dependent manner. The consequences of these findings on our current view of the checkpoint cascade are discussed.     

Glucocorticoids, 11 beta-hydroxysteroid dehydrogenase type 1, and visceral obesity

Glucocorticoids are implicated as a pathophysiological mediator of obesity and its accompanying metabolic and cardiovascular complications. Obese patients exhibit normal circulating cortisol levels, related to increased glucocorticoid production and degradation. However, it has been demonstrated that local production of active cortisol from inactive cortisone driven by 11 beta-hydroxysteroid dehydrogenase type 1 is exaggerated in adipose tissue of obese subjects. Such local hypercortisolism may be responsible for increased adipocyte differentiation and enhanced secretion of free fatty acids and other substances involved in the metabolic and cardiovascular complications observed in obesity.     

Oleanolic glycosides from Pometia ridleyi

Six triterpenoid saponins were isolated from the stem bark of Pometia ridleyi along with two known saponins, acutoside A and calenduloside C. Their structures were established using one- and two-dimensional NMR and mass spectrometry as 3-O-beta-D-apiofuranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucopyranosyl-, 3-O-beta-D-apiofuranosyl-(1-->3)-alpha-L-arabinopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucopyranosyl-, 3-O-beta-D-apiofuranosyl-(1-->3)-beta-D-galactopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucopyranosyl-, 3-O-alpha-L-arabinopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl-, 3-O-beta-D-galactopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl-, 3-O-beta-D-apiofuranosyl-(1-->3)-beta-D-galactopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl-oleanolic acid. The EtOH and EtOAc extracts of the stem bark showed no cytotoxic activity. At a concentration of 23 microg/ml, the saponin mixture showed haemolytic activity and caused 50% haemolysis of a 10% suspension of sheep erythrocytes.     

Evolution of ascariasis in humans and pigs: a multi-disciplinary approach

The nematode parasite Ascaris lumbricoides infects the digestive tracts of over 1.4 billion people worldwide, and its sister species, Ascaris suum, has infected a countless number of domesticated and feral pigs. It is generally thought that the putative ancestor to these worms infected either humans or pigs, but with the advent of domestication, they had ample opportunity to jump to a new host and subsequently specialize and evolve into a new species. While nuclear DNA makers decisively separate the two populations, mitochondrial sequences reveal that three major haplotypes are found in A. suum and in A. lumbricoides, indicating either occasional hybridization, causing introgression of gene trees, or retention of polymorphism dating back to the original ancestral species. This article provides an illustration of the combined contribution of parasitology, archaeoparasitology, genetics and paleogenetics to the history of ascariasis. We specifically investigate the molecular history of ascariasis in humans by sequencing DNA from the eggs of Ascaris found among ancient archeological remains. The findings of this paleogenetic survey will explain whether the three mitochondrial haplotypes result from recent hybridization and introgression, due to intensive human-pig interaction, or whether their co-occurrence predates pig husbandry, perhaps dating back to the common ancestor. We hope to show how human-pig interaction has shaped the recent evolutionary history of this disease, perhaps revealing the identify of the ancestral host.     

Proteomics application exercise of the Swiss Proteomics Society: report of the SPS'02 session

After the success of the mass spectrometry (MS) round table that was held at the first Swiss Proteomics Society congress (SPS'01) in Geneva, the SPS has organized a proteomics application exercise and allocated a full session at the SPS'02 congress. The main objective was to encourage the exchange of expertise in protein identification, with a focus on the use of mass spectrometry, and to create a bridge between the users' questions and the instrument providers' solutions. Two samples were sent to fifteen interested labs, including academic groups and MS hardware providers. Participants were asked to identify and partially characterize the samples. They consisted of a complex mixture of peptide/proteins (sample A) and an almost pure recombinant peptide carrying post-translational modifications (sample B). Sample A was an extract of snake venom from the species Bothrops jararaca. Sample B was a recombinant and modified peptide derived from the shrimp Penaeus vannamei penaeidin 3a. The eight labs that returned results reported the use of a wide range of MS instrumentation and techniques. They mentioned a variety of time and manpower allocations. The origin of sample A was generally identified together with a number of database protein entries. The difficulty of the sample identification lay in the incomplete knowledge of the Bothrops species genome sequence and is discussed. Sample B was generally and correctly identified as penaeidin. However, only one group reported the full primary structure. Interestingly, the approaches were again varied and are discussed in the text.     

Obligatory symbiotic Wolbachia endobacteria are absent from Loa loa

BACKGROUND: Many filarial nematodes harbour Wolbachia endobacteria. These endobacteria are transmitted vertically from one generation to the next. In several filarial species that have been studied to date they are obligatory symbionts of their hosts. Elimination of the endobacteria by antibiotics interrupts the embryogenesis and hence the production of microfilariae. The medical implication of this being that the use of doxycycline for the treatment of human onchocerciasis and bancroftian filariasis leads to elimination of the Wolbachia and hence sterilisation of the female worms. Wolbachia play a role in the immunopathology of patients and may contribute to side effects seen after antifilarial chemotherapy. In several studies Wolbachia were not observed in Loa loa. Since these results have been doubted, and because of the medical significance, several independent methods were applied to search for Wolbachia in L. loa. METHODS: Loa loa and Onchocerca volvulus were studied by electron microscopy, histology with silver staining, and immunohistology using antibodies against WSP, Wolbachia aspartate aminotransferase, and heat shock protein 60. The results achieved with L. loa and O. volvulus were compared. Searching for Wolbachia, genes were amplified by PCR coding for the bacterial 16S rDNA, the FTSZ cell division protein, and WSP. RESULTS: No Wolbachia endobacteria were discovered by immunohistology in 13 male and 14 female L. loa worms and in numerous L. loa microfilariae. In contrast, endobacteria were found in large numbers in O. volvulus and 14 other filaria species. No intracellular bacteria were seen in electron micrographs of oocytes and young morulae of L. loa in contrast to O. volvulus. In agreement with these results, Wolbachia DNA was not detected by PCR in three male and six female L. loa worms and in two microfilariae samples of L. loa. CONCLUSIONS: Loa loa do not harbour obligatory symbiotic Wolbachia endobacteria in essential numbers to enable their efficient vertical transmission or to play a role in production of microfilariae. Exclusively, the filariae cause the immunopathology of loiasis is patients and the adverse side effects after antifilarial chemotherapy. Doxycycline cannot be used to cure loiais but it probably does not represent a risk for L. loa patients when administered to patients with co-infections of onchocerciasis.