Spectrin loss during in vitro red cell lysis

Spectrin was extracted from washed erythrocyte ghosts in 1 mM EDTA buffer (pH 8.0) and purified to homogeneity by gel filtration. Anti-human spectrin was raised in rabbits. Specificity of the antibody was demonstrated by immunodiffusion, immunoelectrophoresis and immunofluorescent techniques. Membrane-free hemolysate prepared by lysing red cells in 5 mM phosphate buffer (pH 8.0) for variable intervals (5--60 min) at 4 degrees C was found to contain spectrin identifiable by immunodiffusion, immunoelectrophoresis, immunofluorescence and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Spectrin was demonstrable in ultracentrifuged membrane-free hemolysate and, in progressively decreasing amounts, in membrane washes. Membrane-free hemolysate contained more spectrin when erythrocytes were lysed for 60 min than for 5 min. The data indicate that a significant amount of spectrin is detached from the membrane following sysis in hypotonic buffer for different time intervals. Spectrin lost in this manner might be part of spectrin attached to the lipid bilayer.     

Cellular and genetic analysis of wound healing in Drosophila larvae

To establish a genetic system to study postembryonic wound healing, we characterized epidermal wound healing in Drosophila larvae. Following puncture wounding, larvae begin to bleed but within an hour a plug forms in the wound gap. Over the next couple of hours the outer part of the plug melanizes to form a scab, and epidermal cells surrounding the plug orient toward it and then fuse to form a syncytium. Subsequently, more-peripheral cells orient toward and fuse with the central syncytium. During this time, the Jun N-terminal kinase (JNK) pathway is activated in a gradient emanating out from the wound, and the epidermal cells spread along or through the wound plug to reestablish a continuous epithelium and its basal lamina and apical cuticle lining. Inactivation of the JNK pathway inhibits epidermal spreading and reepithelialization but does not affect scab formation or other wound healing responses. Conversely, mutations that block scab formation, and a scabless wounding procedure, provide evidence that the scab stabilizes the wound site but is not required to initiate other wound responses. However, in the absence of a scab, the JNK pathway is hyperinduced, reepithelialization initiates but is not always completed, and a chronic wound ensues. The results demonstrate that the cellular responses of wound healing are under separate genetic control, and that the responses are coordinated by multiple signals emanating from the wound site, including a negative feedback signal between scab formation and the JNK pathway. Cell biological and molecular parallels to vertebrate wound healing lead us to speculate that wound healing is an ancient response that has diversified during evolution.     

Biocides formulation of essential oils having antimicrobial activity

Essential oils of fennel, peppermint, caraway, eucalyptus, geranium and lemon were tested for their antimicrobial activities against some plant pathogenic micro-organisms (Fusarium oxysporum, Alternaria alternate, Penicilium italicum Penicilium digitatum and Botyritus cinerea). Essential oils of fennel, peppermint, caraway were selected as an active ingredient for the formulation of biocides due to their efficiency in controlling the tested micro-organisms. Successful emulsifiable concentrates (biocides) were prepared from these oils using different emulsifiers (Emulgator B.L.M. Tween20 and Tween80) and different fixed oils (sesame, olive, cotton and soybean oils). Physico-chemical properties of the formulated biocide (spontaneous emulsification, emulsion stability test, cold stability and heat stability tests as well as viscosity, surface tension and pH) were measured. The prepared biocides were ready to be tested for application in a future work as a safe pesticide against different pathogens.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

The relationship between anogenital distance, fatherhood, and fertility in adult men

Background

Anogenital distance (AGD), a sexually dimorphic measure of genital development, is a marker for endocrine disruption in animal studies and may be shorter in infant males with genital anomalies. Given the correlation between anogenital distance and genital development, we sought to determine if anogenital distance varied in fertile compared to infertile adult men.

Methods

A cross sectional study of consecutive men being evaluated for infertility and men with proven fertility was recruited from an andrology clinic. Anogenital distance (the distance from the posterior aspect of the scrotum to the anal verge) and penile length (PL) were measured using digital calipers. ANOVA and linear regression were used to determine correlations between AGD, fatherhood status, and semen analysis parameters (sperm density, motility, and total motile sperm count).

Findings

A total of 117 infertile men (mean age: 35.3±17.4) and 56 fertile men (mean age: 44.8±9.7) were recruited. The infertile men possessed significantly shorter mean AGD and PL compared to the fertile controls (AGD: 31.8 vs 44.6 mm, PL: 107.1 vs 119.5 mm, p<0.01). The difference in AGD persisted even after accounting for ethnic and anthropomorphic differences. In addition to fatherhood, on both unadjusted and adjusted linear regression, AGD was significantly correlated with sperm density and total motile sperm count. After adjusting for demographic and reproductive variables, for each 1 cm increase in a man''s AGD, the sperm density increases by 4.3 million sperm per mL (95% CI 0.53, 8.09, p = 0.03) and the total motile sperm count increases by 6.0 million sperm (95% CI 1.34, 10.58, p = 0.01). On adjusted analyses, no correlation was seen between penile length and semen parameters.

Conclusion

A longer anogenital distance is associated with fatherhood and may predict normal male reproductive potential. Thus, AGD may provide a novel metric to assess reproductive potential in men.     

Micelle‐enhanced spectrofluorimetric method for determination of sitagliptin and identification of potential alkaline degradation products using LC‐MS

A novel, quick, simple and highly sensitive spectrofluorimetric method was developed and validated for the determination of sitagliptin (SG) in its pharmaceutical formulations. The proposed method is based on investigation of the fluorescence spectral behavior of sitagliptin in an SDS micellar system. In an aqueous solution of phosphate buffer pH 4.0, the fluorescence intensity of SG in the presence of SDS was greatly enhanced, by 200%, i.e. twofold enhancement. The fluorescence intensity of SG was measured at 300 nm after excitation at 270 nm. The method showed good linearity in the range 0.03–10.0 µg/mL with a good correlation coefficient (r = 0.9998). The limits of detection and quantitation values were 5.31 and 16.1 ng/mL, respectively. The proposed method was successfully applied to the analysis of SG in its single and co‐formulated commercial tablets; the results were in good agreement with those obtained using a reference method. Application of the proposed method was extended to stability studies of SG after exposure to different forced degradation conditions according to the ICH guidelines, such as acidic, alkaline, thermal, photo‐ and oxidative stress. The chemical structure of certain potential degradation products (DPs) were investigated using LC‐MS. Copyright © 2013 John Wiley & Sons, Ltd.     

Neural networks in intestinal immunoregulation

Key physiological functions of the intestine are governed by nerves and neurotransmitters. This complex control relies on two neuronal systems: an extrinsic innervation supplied by the two branches of the autonomic nervous system and an intrinsic innervation provided by the enteric nervous system. As a result of constant exposure to commensal and pathogenic microflora, the intestine developed a tightly regulated immune system. In this review, we cover the current knowledge on the interactions between the gut innervation and the intestinal immune system. The relations between extrinsic and intrinsic neuronal inputs are highlighted with regards to the intestinal immune response. Moreover, we discuss the latest findings on mechanisms underlying inflammatory neural reflexes and examine their relevance in the context of the intestinal inflammation. Finally, we discuss some of the recent data on the identification of the gut microbiota as an emerging player influencing the brain function.