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The size of human cervical intraepithelial neoplasia (CIN) biopsies is usually very small and standard methods do not allow an adequate number of keratinocytes to be isolated for culturing purposes. In this study, a new approach to establish keratinocyte cultures from small CIN a tissue fragments was developed. Neoplastic specimens and corresponding normal tissues, which were used as controls, were digested with collagenase. Tissue‐derived fibroblasts and keratinocytes were co‐cultured in calcium and serum medium. Single keratinocyte colonies from primary cultures were expanded using a culture medium optimized in our laboratory. Primary keratinocyte colonies, as well as expanded colonies, were tested for epithelial and cervical markers such as 5, 14, 17, and 19 keratins, and p63 by immunofluorescence. Our results indicate that a variable number of primary keratinocyte colonies could be detected in neoplastic cultures, depending on the grade of cervical lesions from which the colonies originated. Single colonies, when cultured with our new medium, grew at a high rate with uniform size and morphology for some passages. Epithelial and p63 markers were expressed in keratinocyte colonies, as well as in expanded colonies. In conclusion, our study reports a rapid and easy culturing system which enables keratinocyte colonies from minute cervical tumor tissues to be obtained. Moreover, using the new culture medium, keratinocyte colonies can be expanded at a high proliferative rate. J. Cell. Physiol. 227: 3787–3795, 2012. © 2012 Wiley Periodicals, Inc.  相似文献   
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Mating plugs have been proposed as a mechanism that has evolved to avoid sperm competition. Their structure and composition vary across taxa and are related to the effectiveness of its function. This effectiveness could be related to different evolutionary interests of the sexes. Urophonius brachycentrus and Urophonius achalensis (Scorpiones, Bothriuridae) are highly suitable species to study mating plugs because both are monandrous species with specific morphological and physiological responses in the female's genitalia. Here, we analyze (a) the morphology and fine structure of the mating plugs of both species, (b) the site of production in males and the formation process of the mating plug, and (c) the changes that it undergoes over time in the female's reproductive tract. In both species, a complex mating plug obliterates the female's genital aperture and fills the genital atrium. We observed considerable interspecific variation in the mating plug morphology. A mating hemi-plug was found surrounding the capsular lobes of the hemispermatophore, which could have a mixed composition (involving portions of the hemispermatophore and glandular products). The glandular portion was transferred in a semi-solid state filling the female's genital atrium and then hardening. Changes that the plug undergoes in the female's genitalia (darkening and increase of the “distal” area of the plug) indicate a participation of the female to the formation of this type of plug. Our study provides new insights into the plugging phenomenon in scorpions, and we discussed the adaptive significance as a post-copulatory mechanism to avoid sperm competition.  相似文献   
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Changes in the position of the minimum of the parabolic capacitance-voltage curve allow the measurement of the amount of ganglioside present in artificial bilayers made with phosphatidylcholine-ganglioside mixtures and asymmetrically shielded with Ca2+. The screening effect of the ionic solution must be considered. With ganglioside/phospholipid molar ratios of up to 15%, all glycolipids can be found at the membrane surfaces.  相似文献   
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The aim of this study was to determine the effect of both acute exercise and maintained training during a period of competition (3 mo, at the start of the season) on iron metabolism in sportsmen on a professional volleyball team. Twelve sportsmen volunteered for this study. The exercise test was performed on a mechanically braked Monark cycle ergometer and consisted of a triangular progressive test. Three blood samples were obtained in each test: at rest, just after exercise, and after recovery. The following hematological parameters were determined: red blood count (RBC), hemoglobin (Hb) and hematocrit (Hto), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), total proteins (TP), serum iron (Fe) and total iron-binding capacity (TIBC), ferritin (FER), transferrin (TRF), haptoglobin (HPT), and serum cortisol (COR) concentrations. We have found changes in hematological and biochemical variables related to Fe metabolism during the study. The changes observed could be the result of hemoconcentration processes after exercise and, at least in part, to physical stress and muscular damage. We conclude that athletes, after a period of adaptation, with a good plan of work/recovery series, undergo a biological redistribution on hematological and biochemical parameters concerning Fe metabolism during the training and competition period. Also, daily Fe supplementation could restore and mask the true repercussions of maintained training observed in other sports.  相似文献   
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Bacillus sp. GRE1 isolated from an Ethiopian hyperthermal spring produced raw-starch digesting, Ca2+-independent thermostable α-amylase. Enzyme production in shake flask experiments using optimum nutrient supplements and environmental conditions was 2,360 U l−1. Gel filtration chromatography yielded a purification factor of 33.6-fold and a recovery of 46.5%. The apparent molecular weight of the enzyme was 55 kDa as determined by SDS-PAGE. Presence or absence of Ca2+ produced similar temperature optima of 65–70°C. The optimum pH was in the range of 5.5–6.0. The enzyme maintained 50% of its original activity after 45 min of incubation at 80°C and was stable at a pH range of 5.0–9.0. The V max and K m values for soluble starch were 42 mg reducing sugar min−1 and 4.98 mg starch ml−1, respectively. Strong inhibitors of enzyme activity included Cu2+, Zn2+ and Fe2+. The enzyme coding gene and the deduced protein translation revealed a characteristic but markedly atypical homology to Bacillus species α-amylase sequences. The enzyme hydrolyzed wheat, corn and tapioca starch granules efficiently below their gelatinization temperatures. Rather than the higher oligosaccharides normally produced by Bacillus α-amylases operating at high temperatures, maltose was the major hydrolysis product with the present enzyme.  相似文献   
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Bacterial lipoproteins are attractive vaccine candidates because they represent a major class of cell surface-exposed proteins in many bacteria and are considered as potential pathogen-associated molecular patterns sensed by Toll-like receptors with built-in adjuvanticity. Although Gram-negative lipoproteins have been extensively characterized, little is known about Gram-positive lipoproteins. We isolated from Streptococcus pyogenes a large amount of lipoproteins organized in vesicles. These vesicles were obtained by weakening the bacterial cell wall with a sublethal concentration of penicillin. Lipid and proteomic analysis of the vesicles revealed that they were enriched in phosphatidylglycerol and almost exclusively composed of lipoproteins. In association with lipoproteins, a few hypothetical proteins, penicillin-binding proteins, and several members of the ExPortal, a membrane microdomain responsible for the maturation of secreted proteins, were identified. The typical lipidic moiety was apparently not necessary for lipoprotein insertion in the vesicle bilayer because they were also recovered from the isogenic diacylglyceryl transferase deletion mutant. The vesicles were not able to activate specific Toll-like receptor 2, indicating that lipoproteins organized in these vesicular structures do not act as pathogen-associated molecular patterns. In light of these findings, we propose to name these new structures Lipoprotein-rich Membrane Vesicles.Bacterial lipoproteins (Lpps)1 are a subset of membrane proteins that are covalently modified with a lipidic moiety at their N-terminal cysteine residue. It is commonly reported that Lpps of Gram-positive bacteria are processed by two key enzymes; the prolipoprotein diacylglyceryl transferase (Lgt) and the lipoprotein signal peptidase (Lsp). The Lgt enzyme recognizes a so-called lipobox motif in the C-terminal region of the signal peptide of a premature lipoprotein and transfers a diacylglyceryl moiety to the cysteine residue of the lipobox (1), (2). Subsequently, the Lsp enzyme cleaves the signal peptide resulting in a mature Lpp (3), (4). Nevertheless, recent reports have suggested that N-acylation occurs in bacteria that lack the Gram-negative homologous apolipoprotein N-acyltransferase (Lnt) gene responsible for this modification (5, 6), and that Lpp N-terminal could also be modified with an acetyl group in some Gram-positive (7).Lpps have been described as virulence factors because they play critical roles in membrane stabilization, nutrient uptake, antibiotic resistance, bacterial adhesion to host cells, protein maturation and secretion and many of them still have unknown function (8). Several studies have suggested that bacterial Lpps are pathogen-associated molecular patterns (PAMPs) sensed by the mammalian host through Toll-like receptor 2 (TLR2) heterodimerized with TLR1 or TLR6 to induce innate immunity activation and to control adaptive immunity (912). TLR2 plays a critical role in the host response to the Gram-positive bacteria Staphylococcus aureus (13) and Streptococcus agalactiae (14). Although TLR2 has been considered a receptor for various structurally unrelated PAMPs, recent studies have suggested that, via their lipid moiety, bacterial Lpps function as the major, if not the sole, ligand molecules responsible for TLR2 activation (15). Although Gram-negative Lpps have been widely studied, little information is available for Gram-positive Lpps (16) and the ways they are released into the bacterial extracellular compartment and reach the host immune system remain unclear.We focused our attention on Lpps release by Streptococcus pyogenes. This Gram-positive bacterium is an important human pathogen that causes a wide range of diseases from superficial and self-limiting infection, e.g. pharyngitis and impetigo, to more systemic or invasive diseases like necrotizing fasciitis and septicemia (17). Understanding the role of bacterial Lpps in mediating innate and acquired immunity can be instrumental for the therapy and prophylaxis of human S. pyogenes infections. In this study, we showed that in S. pyogenes Lpps are released into the growth medium within vesicle-like structures in minute amounts. Conditions weakening the bacterial cell wall, such as the addition of sublethal concentrations of penicillin to the bacterial growth medium enhanced this phenomenon and allowed the recovery of sufficient material to enable an in-depth characterization. Proteomic analysis of the vesicles revealed that they were almost exclusively constituted of Lpps. A total of 28 Lpps were identified, representing more than 72% of the Lpps predicted from the genome of the strain under investigation. In addition, multiple transmembrane domain proteins were not found in abundance associated to the vesicles, indicating that vesicles were not representative of the bacterial membrane. We defined these vesicles as Lipoprotein-rich Membrane Vesicles (LMVs).Common characteristics are shared between the LMVs and the ExPortal described for the first time by Rosch and Caparon (18). This asymmetric and distinct membrane microdomain has been reported to be enriched in anionic phospholipids and acts in promoting the biogenesis of secreted proteins by coordinating interactions between nascent unfolded secretory proteins and the accessory factors required for their maturation (1921). An association between ExPortal and peptidoglycan synthesis has also been reported (22). Similarly, LMVs are enriched in anionic phosphatidylglycerol, enzymes involved in protein maturation/secretion and cell wall biogenesis, suggesting that LMVs might derive from the ExPortal. Finally, we showed that LMVs do not induce TLR2 activation, indicating that the Lpps did not act as PAMPs when integrated into the LMVs.  相似文献   
8.
The echinoid fauna of the Gulf of Mexico collected during three research cruises (20-1260 m depth) was surveyed from samples were taken at 43 stations. A total of 190 individuals were identified (eight orders, 11 families, 15 genera and 18 species). Six species are new records for the Gulf of Mexico: Stylocidaris lineata, Phormosoma placenta placenta, Plesiodiadema antillarum, Plethotaenia spatangoides, Brissopsis atlantica and Hypselaster limicolus. This adds to the little information available on the echinoid fauna of Tamaulipas, Veracruz, Tabasco, Campeche and Yucatan states in Mexico.  相似文献   
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We have previously shown that dendrite morphology of cultured hippocampal neurones is controlled by Notch receptor activation or binding of nerve growth factor (NGF) to its low affinity receptor p75NTR, i.e. processes that up-regulate the expression of the Homologue of enhancer of split 1 and 5. Thus, the increased expression of these genes decreases the number of dendrites, whereas abrogation of Homologue of enhancer of split 1/5 activity stimulates the outgrowth of new dendrites. Here, we show that Neurogenin 3 is a proneural gene that is negatively regulated by Homologue of enhancer of split 1/5. It also influences dendrite morphology. Hence, a deficit of Notch or NGF/p75NTR activation can lead to the production of high levels of Neurogenin 3, which stimulates the outgrowth of new dendrites. Conversely, activation of either Notch or p75NTR depressed Neurogenin 3 expression, which not only decreased the number of dendrites but also favoured inhibitory (GABAergic) synaptogenesis, thereby diminishing the ratios of excitatory/inhibitory inputs. NGF also augmented the levels of mRNA encoding the vesicular inhibitory amino acid transporter, but it did not affect the fraction of GAD65/67-positive neurones. Conversely, overexpression of Neurogenin 3 largely reduced the number of inhibitory synaptic contacts and, consequently, produced a strong increase in the ratios of excitatory/inhibitory synaptic terminals. Our results reveal a hitherto unknown contribution of NGF/p75NTR to dendritic and synaptic plasticity through Neurogenin 3 signalling.  相似文献   
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