Site‐specific interactions of neurotrophin‐3 and fibroblast growth factor (FGF2) in the embryonic development of the mouse cochlear nucleus

Neurotrophins and FGF2 contribute to formation of the cochlea, but their roles in cochlear nucleus development are unknown. The effects of these factors may differ in the cochlea and cochlear nucleus, which may influence each other's development. It is important to analyze the effects of these factors on cellular structures at well‐defined steps in the normal morphogenetic sequence. The present study used immunohistochemistry to localize factors in situ and to test hypotheses about their roles in an in vitro model. Specific antibody staining revealed that TrkC, the NT3 receptor, is present in neural precursors prior to embryonic day E11 until after birth. NT3 appeared in precursor cells during migration (E13–E15) and disappeared at birth. TrkC and NT3 occurred in the same structures, including growing axons, terminals, and their synaptic targets. Thus, NT3 tracks the migration routes and the morphogenetic sequences within a window defined by TrkC. In vitro, the cochlear nucleus anlage was explanted from E11 embryos. Cultures were divided into groups fed with defined medium, with or without FGF2, BDNF, and NT3 supplements, alone or in combinations, for 7 days. When neuroblasts migrated and differentiated, immunostaining was used for locating NT3 and TrkC in the morphogenetic sequence, bromodeoxyuridine for proliferation, and synaptic vesicle protein for synaptogenesis. By time‐lapse imaging and quantitative measures, the results support the hypothesis that FGF2 promotes proliferation and migration. NT3 interacts with FGF2 and BDNF to promote neurite outgrowth, fasciculation, and synapse formation. Factors and receptors localize to the structural sites undergoing critical changes. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

The role of enteric bacteria in the anerobic metabolism of 5-aminosalicylate

The primary (and inactive) enteric metabolite of 5-aminosalicylate is N-acetyl-5-amino-salicylate. Previous studies have demonstrated acetylation of this anti-inflammatory agent by intestinal and bacterial homogenates. To assess the contribution of anerobic bacteria to the N-acetylation in vivo, we have measured the production of N-acetyl-5-aminosalicylate in anerobic microculture. Our results indicate that enteric bacteria play a minor role in N-acetylation, but may contribute to the production of other metabolites of pharmacologic and toxicological interest.     

Mechanism of action of Clostridium perfringens enterotoxin. Effects on membrane permeability and amino acid transport in primary cultures of adult rat hepatocytes

Purified enterotoxin from the bacterium Clostridium perfringens rapidly decreased the hormonally induced uptake of α-aminoisobutyric acid in primary cultures of adult rat hepatocytes. At 5 min after toxin addition the decrease in α-aminoisobutyric acid uptake appeared not due to increased passive permeation (estimated with l-glucose) or to increased α-aminoisobutyric acid efflux. When short uptake assay times were employed a depression of α-aminoisobutyric acid influx was observed in toxin-treated hepatocytes. The depression of α-aminoisobutyric acid influx was correlated with a rapid increase in intracellular Na⁺ (estimated using ²²Na⁺) apparently effected by membrane damage. In contrast, the uptake of cycloleucine in the presence of unlabeled α-aminoisobutyric acid (assay for Na⁺-independent amino acid uptake) by hepatocytes treated with toxin for 5 min was decreased to only a small extent or not at all depending upon experimental design. At later times, C. perfringens enterotoxin increased the exodus of l-glucose, $\text{3-O-}\text{methylglucose}$ and α-aminoisobutyric acid from pre-loaded cells indicating that the toxin effects progressive membrane damage. When enterotoxin was removed by repeated washing after 5–20 min the decay of α-aminoisobutyric acid uptake ceased and appeared to undergo recovery towards the hormonally induced control level. The degree of recovery of α-aminoisobutyric acid uptake was inverse to the length of time of exposure to toxin. Adding at 10 min specific rabbit antiserum against C. perfringens enterotoxin without medium change also reversed the effect of toxin on increased intracellular ²²Na⁺, and on the exodus (from preloaded cells) of α-aminoisobutyric acid, L-glucose, and $\text{3-O-}\text{methylglucose}$.     

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the “angio-follicular” units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.     

The cooperativity phenomena in a pigment-protein complex of light-harvesting antenna revealed by picosecond absorbance difference spectroscopy

A model of the cooperative changes in optical properties of light-harvesting bacteriochlorophyll molecules of complex B890 in response to the absorption of light quanta is proposed. According to the model, each antenna chromophore may persist in either of two optically non-excited states, R and T. The occurrence of at least one excitation per complex causes all optically non-excited chromophores of the complex to be converted from state R to state T. The theory is shown to be in good agreement with experimental ‘light curves’ (ΔAvs intensity of picosecond excitation pulse) for the ‘minor’ and ‘major’ signals of light-harvesting bacteriochlorophylls of complex B890 from Chromatium minutissimum.     

NleB/SseKs ortholog effectors as a general bacterial monoglycosyltransferase for eukaryotic proteins

Protein glycosylation is the most common post-translational modification as more than 50% of all human proteins are glycosylated. Pathogenic bacteria glycosylation allows adhesion to host cells and manipulates eukaryotic functions. A variety of acceptor proteins in bacterial glycosylation was recently discovered. Especially NleB/SseKs type III effectors unexpectedly glycosylate a poor nucleophile arginine. Other pathogenic toxins modify the unusual tyrosine, as well as canonical serine/threonine residues. And a huge diversity is found in target proteins; Rho/Ras families, death domains and moreover themselves for autoglycosylation. However, in spite of this acceptor diversity, all their sugar donors are only UDP-Glc/-GlcNAc and structural alignments as liganded show their catalytic cores are geometrically conserved, where DRY and DXD motives and W residues equally position to hold the sugar donors and to π-π bind with a uridine ring, respectively. Therefore, bacterial glycosyltransferases have a key for carbohydrate research problems concerning the sugar donors and target proteins recognition.     

Statistical optimization of bacterial cellulose production by Leifsonia soli and its physico-chemical characterization

Bacterial cellulose has multiple applications in various industries such as food, biomedical, textile due to its uniqueness of being a better bio-compatible coating agent, binding material, etc. In this study, optimization of the culture medium for producing BC from Leifsonia soli was carried out by selecting different parameters. Five significant factors such as maltose, pH, incubation days, soy whey and calcium chloride were estimated through ANOVA based response surface methodology. Maximum cellulose production (5.97 g/L) was obtained where maltose 1 % (w/v) supplemented with 0.8 % (v/v) soy whey and calcium chloride 0.8 % (w/v) at pH 6.5 for 7 days of incubation. In addition, assurance of cellulose production from bacteria was done by using High-performance liquid chromatography analysis. Further, the structure and purity of obtained cellulose were examined by SEM and elemental analysis where it was observed that the sample holds the value of carbon 44.1 ± 0.20 % and hydrogen 6.2 ± 0.3 %, respectively. This study concludes that the addition of maltose and soy whey could be used as carbon, nitrogen sources and calcium chloride was used as an additive for the bacterial cellulose production compared to the Hestrin Schramm medium. In addition, the calculated water holding capacity of the sample was found to be 73 %.