Myenteric nitrergic neurons along the rat esophagus: evidence for regional and strain differences in age-related changes

Several studies have suggested an age-related reduction in the number of myenteric neurons in the lower gastrointestinal (GI) tract linked to changes in GI neuromuscular functions. The present study, combining protein gene product 9.5 immunostaining and NADPH-diaphorase histochemistry, aimed at quantifying the proportion of nitrergic neurons compared to the overall number of enteric neurons in the esophagus of young (3-4.5 months) and aged (18-20 months) Sprague-Dawley and Wistar rats. In both strains, the neuron numbers per ganglion in the cervical region were almost twice as high as in the other esophageal regions. Irrespective of age or strain, the esophagus harbored a very high proportion of intrinsic nitrergic neurons (greater than approximately 65%). Both strains showed with aging an overall neuronal loss of approximately 27%. While a significant increase (young: 64-71%; aged: 82-89%) was observed in all esophageal regions in the Wistar strain, the proportion of nitrergic neurons remained stable with aging in the Sprague-Dawley strain (range: 72-82%). In conclusion, the age-related reduction in the overall number of myenteric, nitrergic, and non-nitrergic neurons observed in the rat esophagus, appears to be highly region- and strain-dependent. Therefore, a protective mechanism against neuronal cell loss, selectively present in specific (nitrergic) enteric subpopulations, as suggested in earlier reports, cannot be put forward as a general phenomenon throughout the entire GI tract.     

Anti-beta2-glycoprotein I antibodies recognizing platelet factor 4-heparin complex in antiphospholipid syndrome in patient substantiated with mouse model

The antiphospholipid syndrome is defined by the presence of antiphospholipid antibodies associated with arterial and/or venous thrombosis, and recurrent abortion accompanied often by thrombocytopenia. These antibodies are heterogeneous and react against phospholipid-binding proteins such as beta2-glycoprotein I (beta2GPI) and prothrombin. The recognition of anti-beta2-glycoprotein I (anti-beta2GPI) by platelet factor 4-heparin complex (PF4-Hc) has been previously evoked and partially confirmed by the present inhibition studies. Further, the anti-beta2-glycoprotein I antibodies were purified from a patient with primary antiphospholipid syndrome using Affi-gel-10-beta2GPI immunoaffinity chromatography. The purified anti-beta2GPI IgM as well as patient serum equally recognized PF4-Hc in ELISA mode. In order to substantiate this data and to better understand we studied an animal model using mouse active immunization with the purified human anti-beta2GPI. The mice showed a significant decrease in their platelet count. In addition the ELISA responses of the immunized mice sera were positive against both beta2GPI and PF4-Hc, substantiating the double recognition. Despite many previous reported animal model studies, this is the first time we have shown the specific recognition of anti-beta2GPI antibodies by PF4-Hc, the results in the induced mice correlating the data observed with some patients.     

Quantification of tagitinin C in Tithonia diversifolia by reversed-phase high-performance liquid chromatography

A simple, rapid and reliable reversed-phase high-performance liquid chromatographic method for the determination of tagitinin C, an anti-plasmodial sesquiterpene lactone isolated from the aerial parts of Tithonia diversifolia, has been developed. The assay has been used to quantify tagitinin C in various extracts of the aerial parts of T. diversifolia.     

Effects of predation pressure on the feeding behaviour of the serpa tetra Hyphessobrycon eques (Ostariophysi, Characidae)

The indirect, non-lethal results of predation, such as reduction in feeding time or restraint in seeking sexual partners and/or natural resources, have a drastic effect on prey populations. In this study, we investigated the behaviour of two serpa tetras Hyphessobrycon eques groups, one wild and the other born and raised in captivity, to evaluate how their feeding behaviour is affected by the avian predator, the rufescent tiger-heron Trigrisoma lineatum (using a taxidermy specimen). For a total of 133 observation hours, the feeding behaviour of each fish group was observed according to the absence (control) or presence (treatments) of a predator near the aquarium surface. The results showed that the presence of a predator on the surface inhibits the feeding behaviour of H. eques. The differences observed between the groups are probably due to the fishes experiences with predators. Our results suggest that fish-eating birds may affect prey populations in streams and ponds perhaps more through non-lethal effects, on feeding behaviour for example, than directly through death rates.     

Biodiversity of exopolysaccharides produced by Streptococcus thermophilus strains is reflected in their production and their molecular and functional characteristics

Twenty-six lactic acid bacterium strains isolated from European dairy products were identified as Streptococcus thermophilus and characterized by bacterial growth and exopolysaccharide (EPS)-producing capacity in milk and enriched milk medium. In addition, the acidification rates of the different strains were compared with their milk clotting behaviors. The majority of the strains grew better when yeast extract and peptone were added to the milk medium, although the presence of interfering glucomannans was shown, making this medium unsuitable for EPS screening. EPS production was found to be strain dependent, with the majority of the strains producing between 20 and 100 mg of polymer dry mass per liter of fermented milk medium. Furthermore, no straightforward relationship between the apparent viscosity and EPS production could be detected in fermented milk medium. An analysis of the molecular masses of the isolated EPS by gel permeation chromatography revealed a large variety, ranging from 10 to >2,000 kDa. A distinction could be made between high-molecular-mass EPS (>1,000 kDa) and low-molecular-mass EPS (<1,000 kDa). Based on the molecular size of the EPS, three groups of EPS-producing strains were distinguished. Monomer analysis of the EPS by high-performance anion-exchange chromatography with amperometric detection was demonstrated to be a fast and simple method. All of the EPS from the S. thermophilus strains tested were classified into six groups according to their monomer compositions. Apart from galactose and glucose, other monomers, such as (N-acetyl)galactosamine, (N-acetyl)glucosamine, and rhamnose, were also found as repeating unit constituents. Three strains were found to produce EPS containing (N-acetyl)glucosamine, which to our knowledge was never found before in an EPS from S. thermophilus. Furthermore, within each group, differences in monomer ratios were observed, indicating possible novel EPS structures. Finally, large differences between the consistencies of EPS solutions from five different strains were assigned to differences in their molecular masses and structures.     

Structure of gene coding for the fruit body-specific hydrophobin Fbh1 of the edible basidiomycete Pleurotus ostreatus

Two fruit body-specific hydrophobins (Fbh1 and POH1) have been identified in two different strains of the edible basidiomycete Pleurotus ostreatus. Comparison of their nucleotide and amino acid sequences yielded similarity values (59% and 66%, respectively) smaller than those found for alleles of the same hydrophobin gene but higher than those found for different hydrophobin genes in P. ostreatus var. florida (Pe?as et al 2002). In this paper, we have addressed the question of Fbh1 and POH1 allelism by studying the structure of the gene fbh1 and by a classical genetic analysis to compare it with that of POH1. The structure of both genes is similar, as revealed by the similarity of their promoters and leader peptide sequences and by the conserved position of their introns. Furthermore, the allelism analysis revealed that both genes segregated as alleles when present in the same hybrid. These results suggest an allelic condition for POH1 and fbh1 and stress the importance of the similarity of fbh1/POH1 promoter and leader sequences. Furthermore, we have identified various microsatellite-like regions in this gene that can be used for strain and species typing in the future.     

Reconstitution of lethally irradiated mice by cells isolated from adipose tissue

It is suggested that hematopoietic stem cells (HSC) could be found in several tissues of mesodermic origin. Among these, adipose tissue can expand throughout adult life and its expansion is not only due to mature adipocyte hypertrophy but also to the presence of precursor cells in stroma-vascular fraction (SVF). Here we report that transplantation of cells isolated from mice adipose tissue can efficiently rescue lethally irradiated mice and results in a reconstitution of major hematopoietic lineages. Donor cells can be detected in blood and in hematopoietic tissues of recipient mice. Adipose tissue contains a significant percentage of CD34, CD45 positive cells, and SVF cells were able to give rise to hematopoietic colonies in methylcellulose. We demonstrate the presence of hematopoietic progenitors in adipose tissue by phenotypic and functional characteristics. Thus adipose tissue could be considered as an important and convenient source of cells able to support hematopoiesis.     

Pharmacological sensitivity of ATP release triggered by photoliberation of inositol-1,4,5-trisphosphate and zero extracellular calcium in brain endothelial cells

Recently, ATP has gained much interest as an extracellular messenger involved in the communication of calcium signals between cells. The mechanism of ATP release is, however, still a matter of debate. In the present study we investigated the possible contribution of connexin hemichannels or ion channels in the release of ATP in GP8, a rat brain endothelial cell line. Release of ATP was triggered by photoactivation of InsP(3) or by reducing the extracellular calcium concentration. Both trigger protocols induced ATP release significantly above baseline. InsP(3)-triggered ATP release was completely blocked by alpha-glycyrrhetinic acid (alpha-GA), the connexin mimetic peptides gap 26 and 27, and the trivalent ions gadolinium and lanthanum. ATP release triggered by zero calcium was, in addition to these substances, also blocked by flufenamic acid (FFA), niflumic acid, and NPPB. Gap 27 selectively blocked zero calcium-triggered ATP release in connexin-43 transfected HeLa cells, while having no effect in wild-type and connexin-32 transfected cells. Of all the agents used, only alpha-GA, FFA and NPPB significantly reduced gap junctional coupling. In conclusion, InsP(3) and zero calcium-triggered ATP release show major similarities but also some differences in their sensitivity to the agents applied. It is suggested that both stimuli trigger ATP release through the same mechanism, which is connexin-dependent, permeable in both directions, potently blocked by connexin mimetic peptides, and consistent with the opening of connexin hemichannels.     

Inhibition of alpha, betaI, delta, eta, and zeta protein kinase C isoforms by xanthonolignoids

The effect of the xanthonolignoids trans-(+/-)-kielcorin C, cis-(+/-)-kielcorin C, trans-(+/-)-kielcorin D, trans-(+/-)-isokielcorin D and trans-(+/-)-kielcorin E on isoforms alpha, betaI, delta, eta and zeta of protein kinase C (PKC) was studied using the yeast phenotypic assay. All the compounds tested revealed an effect compatible with PKC inhibition, similar to that exhibited by the well established PKC inhibitor chelerythrine, and with differences in their potency towards the distinct isoforms tested, being, in general, potent inhibitors of the atypical PKC isoform (PKC-zeta). PKC inhibition caused by these kielcorins was confirmed using an in vitro kinase assay. The present study constitutes the first attempt to unravel the molecular mechanism of kielcorins activity, and shows that xanthonolignoids are a promising group of compounds to investigate for isoform selective PKC inhibitors.     

Differential cytoplasmic and whole cellular expression of histone H1 within the avian bursa of Fabricius

Bursal anti-steroidogenic peptide (BASP), purified from the chicken bursa of Fabricius (BF), has been previously demonstrated to be a potent and efficacious inhibitor of steroid hormone biosynthesis from chicken ovarian, and both mammalian and avian adrenal cells in vitro. Other studies have demonstrated that BASP can markedly reduce avian and mammalian mitogen-stimulated lymphocyte proliferation. Recent studies have indicated that BASP has a structural and functional relationship with histone H1. Immunohistochemical studies using a monoclonal antibody, which is known to recognize a common histone H1 epitope from several plant and animal species identified the protein within the cytoplasm and nucleus of distinct cells within both the cortex and medulla of all BF follicles. Additionally, epithelial cells within the BF expressed the protein strongly in the cytoplasm with reduced nuclear staining. In contrast, the same antibody did not recognize the protein in thymus of the same animals. The differential expression of histone H1 immunoreactivity within selected cells of the BF may support a previous proposed role of histone H1 in extranuclear and extracellular signaling in chickens and possibly other species.