Repetin (Rptn), a New Member of the “Fused Gene” Subgroup within the S100 Gene Family Encoding a Murine Epidermal Differentiation Protein

We report the cloning and characterization of a murine epidermal differentiation gene, repetin (Rptn), exhibiting striking similarity to the genes of the intermediate filament-associated proteins profilaggrin and trichohyalin. The repetin gene consists of three exons and two introns. The first exon is short and untranslated. The deduced amino acid sequence distributed between exons II and III contains 1130 amino acids with a calculated molecular mass of 130 kDa and pIof 7.7. The amino terminus exhibits significant homology to the S100 proteins containing two calcium-binding motifs of the EF-hand type. The remainder coding sequence contains a central segment consisting of 49 tandem repeats of a 12-amino-acid sequence rich in glutamines. By fluorescencein situhybridization the repetin gene was localized to chromosome band 3 F1-2. Expression of repetin mRNA is detectable in the stratified internal epithelia of forestomach and tongue and to a lesser degree in normal skin epidermis, where it is restricted to the differentiated suprabasal cell layers. Based on its chromosomal localization, its genomic organization, and its stage-specific expression during late epidermal differentiation, as well as on the structural features of the encoded protein, we conclude that the repetin gene represents a novel member of the “fused gene” subgroup of the S100 gene family encoding multifunctional epidermal matrix proteins.     

Control of skeletal muscle fibres and adipose cells size in the flesh of rainbow trout

The distributions of the diameters of skeletal muscle fibres and adipocytes were studied in rainbow trout. The cellularity of perivisceral adipose tissues and subcutaneous ventral and dorsal adipose tissues were characterized more specifically. In these tissues, a population of small adipocytes was distinguishable from larger adipocytes. The same was observed in white muscle. The effects of extrinsic factors (dietary lipid in two different thermal conditions) and intrinsic factors (strains in two different saline conditions, growth hormone) on the long-term response of the cellularity of both muscle and adipose tissues were studied. The effects of thermal environment were tested on fish fed the same ration and the effects of saline environment on fish fed ad libitum. The mean size of white muscle fibres was relatively unaffected by the different treatments tested: genetic origin and dietary lipid in different environmental conditions. There were significant differences in growth rate due to genetic origin and saline environment. The possible involvement of hyperplasia in response to these different factors is discussed. Growth hormone supplementation enhanced the percentage of small diameter fibres indicating a role of this hormone in the control of muscle hyperplastic growth. The mean size of adipose cells was affected only slightly by the different treatments tested. An increase in adipose cell size with aging and lipid content was observed. The percentage of small adipocytes also increased with aging. Thus, it is proposed that the development of adipose tissues, and thus fat retention, both result from the recruitment of new adipocytes and from the increase in size of existing adipocytes. The hyperplastic process contributed significantly to the differences in fat retention due to different treatments tested (strains, thermal and saline environments). When partially substituting fish oils for corn oils in the diet, a large increase in the ventral adipose cell size was seen indicating a potential negative effect of n-6 fatty acids on cell proliferation. Growth hormone treatment, on the contrary, induced a decrease in the size of perivisceral adipocytes. Thus, diet and hormonal status affect adipose cells size through two different metabolic pathways: lipogenesis and lipolysis respectively.     

ChlaDub1 of Chlamydia trachomatis suppresses NF-kappaB activation and inhibits IkappaBalpha ubiquitination and degradation

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes various human diseases, including blindness caused by ocular infection and sexually transmitted diseases resulting from urogenital infection. After infecting host cells, Chlamydiae avoid alarming the host's immune system. Among the immune evasion mechanisms, Chlamydiae can inhibit NF-κB activation, a crucial pathway for host inflammatory responses. In this study, we show that Chla Dub1, a deubiquitinating and deNeddylating protease from C. trachomatis , is expressed in infected cells. In transfection experiments, Chla Dub1 suppresses NF-κB activation induced by several pro-inflammatory stimuli and binds the NF-κB inhibitory subunit IκBα, impairing its ubiquitination and degradation. Thus, we provide further insight into the mechanism by which C. trachomatis may evade the host inflammatory response by demonstrating that Chla Dub1, a protease produced by this microorganism, is capable of inhibiting IκBα degradation and blocking NF-κB activation.     

Mast cell-specific Cre/loxP-mediated recombination in vivo

Mast cells are important effectors of type I allergy but also essential regulators of innate and adaptive immune responses. The aim of this study was to develop a Cre recombinase-expressing mouse line that allows mast cell-specific inactivation of genes in vivo. Following a BAC transgenic approach, Cre was expressed under the control of the mast cell protease (Mcpt) 5 promoter. Mcpt5-Cre transgenic mice were crossed to the ROSA26-EYFP Cre excision reporter strain. Efficient Cre-mediated recombination was observed in mast cells from the peritoneal cavity and the skin while only minimal reporter gene expression was detected outside the mast cell compartment. Our results show that the Mcpt5 promoter can drive Cre expression in a mast cell-specific fashion. We expect that our Mcpt5-Cre mice will be a useful tool for the investigation of mast cell biology. Julia Scholten and Karin Hartmann contributed equally to this work. Supported by grants from the German Research Counsil (Deutsche Forschungsgemeinschaft, RO 2133/2-2) to A.R. and K.H. and the Koeln Fortune Program/Faculty of Medicine, University of Cologne, to A.R. and K.H.. The authors have no conflict of interest     

Pivotal role for alpha1-antichymotrypsin in skin repair

α1-Antichymotrypsin (α1-ACT) is a specific inhibitor of leukocyte-derived chymotrypsin-like proteases with largely unknown functions in tissue repair. By examining human and murine skin wounds, we showed that following mechanical injury the physiological repair response is associated with an acute phase response of α1-ACT and the mouse homologue Spi-2, respectively. In both species, attenuated α1-ACT/Spi-2 activity and gene expression at the local wound site was associated with severe wound healing defects. Topical application of recombinant α1-ACT to wounds of diabetic mice rescued the impaired healing phenotype. LC-MS analysis of α1-ACT cleavage fragments identified a novel cleavage site within the reactive center loop and showed that neutrophil elastase was the predominant protease involved in unusual α1-ACT cleavage and inactivation in nonhealing human wounds. These results reveal critical functions for locally acting α1-ACT in the acute phase response following skin injury, provide mechanistic insight into its function during the repair response, and raise novel perspectives for its potential therapeutic value in inflammation-mediated tissue damage.     

Using exomarkers to assess mitochondrial reactive species in vivo

Background

The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging.

Scope of review

One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules.

Major conclusions and general significance

Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Assessment of therapeutic responses to gametocytocidal drugs in Plasmodium falciparum malaria

Background

Effective mating between laboratory-reared males and wild females is paramount to the success of vector control strategies aiming to decrease disease transmission via the release of sterile or genetically modified male mosquitoes. However mosquito colonization and laboratory maintenance have the potential to negatively affect male genotypic and phenotypic quality through inbreeding and selection, which in turn can decrease male mating competitiveness in the field. To date, very little is known about the impact of those evolutionary forces on the reproductive biology of mosquito colonies and how they ultimately affect male reproductive fitness.

Methods

Here several male reproductive physiological traits likely to be affected by inbreeding and selection following colonization and laboratory rearing were examined. Sperm length, and accessory gland and testes size were compared in male progeny from field-collected females and laboratory strains of Anopheles gambiae sensu stricto colonized from one to over 25 years ago. These traits were also compared in the parental and sequentially derived, genetically modified strains produced using a two-phase genetic transformation system. Finally, genetic crosses were performed between strains in order to distinguish the effects of inbreeding and selection on reproductive traits.

Results

Sperm length was found to steadily decrease with the age of mosquito colonies but was recovered in refreshed strains and crosses between inbred strains therefore incriminating inbreeding costs. In contrast, testes size progressively increased with colony age, whilst accessory gland size quickly decreased in males from colonies of all ages. The lack of heterosis in response to crossing and strain refreshing in the latter two reproductive traits suggests selection for insectary conditions.

Conclusions

These results show that inbreeding and selection differentially affect reproductive traits in laboratory strains overtime and that heterotic ‘supermales’ could be used to rescue some male reproductive characteristics. Further experiments are needed to establish the exact relationship between sperm length, accessory gland and testes size, and male reproductive success in the laboratory and field settings.