Lectins as markers of rumen epithelial cell differentiation

Summary Lectins of different carbohydrate specificities (GNA (Galanthus nivalis), con A (Canavalia ensiformis), VFL (Vicia faba), PSL (Pisum sativum), LCA (Lens culinaris), PNA (Arachis hypogaea; with or without prior neuraminidase treatment), WGA (Triticum vulgare), SBA (Glycine max), UEA-I (Ulex europaeus), LPA (Limulus polyphemus), BS-I B₄ (Bandeiraea simplicifolia, isolectin B₄)) were explored for use as differentiation markers of rumen epithelial cellsin vivo andin vitro. Lectins specific for mannose (GNA), mannose/glucose (con A, VFL, PSL and LCA),N-acetylglucosamine (WGA) or forN-acetylneuraminic acid (LPA) reacted generally with all types of rumen epithelial cell from both rumen tissue and cell culture. They were, therefore, not suitable markers of epithelial differentiation. SBA was unsuitable because, although it reacted with both tissue and cultured rumen epithelial cells, it was also bound to non-stratified areas of primary rumen epithelial cell cultures. Both BS-I B₄ and PNA (after neuraminidase treatment) had to be ruled out because they did not react with differentiated rumen tissue epithelial cells, although they did bind to both stratified and non-stratified cultured cells. In contrast, UEA-I reacted strongly with differentiated rumen epithelial cells both from rumen tissue and cell cultures and therefore appears to be a good general marker for rumen epithelial cell differentiation.     

Paracuaria hispanica n. sp. (Nematoda: Acuariidae), a stomach parasite of the pyrenean desman Galemys pyrenaicus Geoffr. (Insectivora: Talpidae), with a redefinition of the genus Paracuaria Rao, 1951

Adult and fourth-stage larvae of Paracuaria hispanica n. sp., from the stomach of the Pyrenean desman Galemys pyrenaicus Geoffroy (Insectivora: Talpidae) in northern and central Spain, are described. The new species differs from the other members of the genus Paracuaria (P. adunca and P. soricis), among other morphological details, in its smaller body and spicule sizes, the presence of a cuticular ring around the tip of the female tail, and the existence of lateral alae running longitudinally along its body from the cervical region to the tail. In view of the latter feature, the genus Paracuaria is redefined. The fourth stage larva of the new species is distinguished from that of P. adunca by its monocuspid deirids. P. hispanica occurred in 45% of the 20 host specimens examined.     

A minichromosome-like structure in wheat embryos

A crude nuclear fraction of resting wheat embryos was used as the source of putative plant minichromosomes: unique DNA sequences the size of genes and flanked by telomere-type repeats. Preliminary separation of low-molecular-weight DNA species from chromosomal DNA (Hirt's method), velocity sedimentation, and isopycnic centrifugation were followed by PCR amplification of minichromosome-like sequences. The most abundant PCR product was cloned and sequenced. In addition to telomeric repeats (defined by a PCR primer), which were the expected sequences, the linear DNA molecule (637 pb) contained an ARS-like element, RAP1-binding site, and two relatively long ORFs. The whole sequence seems to represent a naturally occurring plant minichromosome.     

Interactions between nitrate and ammonium during uptake by carob seedlings and the effect of the form of earlier nitrogen nutrition

Seedlings of carob ( Ceratonia siliqua L. cv. Mulata) were used in two sets of experiments in order to evaluate; (1) the reciprocal effects of each nitrogen form on net uptake of nitrate and ammonium, and (2) the effect of earlier nitrogen nutrition on ammonium versus nitrate uptake. In the former group of experiments we studied the kinetics of nitrate and ammonium uptake as well as the interference of each of the two forms with net uptake of ammonium and nitrate by both nitrogen depleted and nitrogen fed carob seedlings. On the whole, nitrogen depletion led to increase in both affinity and V_max of the system for both forms of nitrogen, at the same time as the effects of nitrate on uptake of ammonium and vice versa were concentration dependent. In the second group of experiments the effects of earlier nitrogen nutrition on nitrate and ammonium uptake were characterized, and in this case we observed that: (a) if only one form of N was supplied, ammonium was taken up in greater amounts than nitrate; (b) the presence of ammonium enhanced nitrate uptake; (c) ammonium uptake was inhibited by nitrate; (d) there was a significant effect of the earlier nitrogen nutrition on the response of the plants to a different nitrogen source. The latter was evident mainly as regards ammonium uptake by plants grown in ammonium nitrate. The interactions between nitrate and ammonium uptake systems are discussed on the basis of the adaptation to the nitrogen source during early growth.     

Polymorphism of the tumor necrosis factor beta gene in systemic lupus erythematosus: TNFB-MHC haplotypes

We investigated the Nco I restriction fragment length polymorphism (RFLP) of the tumor necrosis factor beta (TNFB) gene in 173 patients with systemic lupus erythematosus (SLE), 192 unrelated healthy controls, and eleven panel families, all of German origin. The phenotype frequency of the TNFB*1 allele was significantly increased in patients compared to controls (63.6% vs 47.1%, RR = 1.96, p <0.002). The results of a two-point haplotype statistical analysis between TNFB and HLA alleles show that there is linkage disequilibrium between TNFB*1 and HLA-A1, Cw7, B8, DR3, DQ2, and C4A DE. The frequency of TNFB*1 was compared in SLE patients and controls in the presence or absence of each of these alleles. TNFB*1 is increased in patients over controls only in the presence of the mentioned alleles. Therefore, the whole haplotype A1, Cw7, B8, TNFB*1, C4A DE, DR3, DQ2 is increased in patients and it cannot be determined which of the genes carried by this haplotype is responsible for the susceptibility to SLE. In addition, two-locus associations were analyzed in 192 unrelated healthy controls for TNFB and class I alleles typed by serology, and for TNFB and class II alleles typed by polymerase chain reaction/oligonucleotide probes. We found positive linkage disequilibrium between TNFB*1 and the following alleles: HLA-A24, HLA-B8, DRB1*0301, DRB1*1104, DRB1*1302, DQA1*0501, DQB1*0201, DQB1*0604, and DPB1*0101. TNFB*2 is associated with HLA-B7, DRB1*1501, and DQB1*0602.This study was supported by grants from the Federal Ministry of Research and Technology (BMFT/DFVLR, 01 VM 8608/9), the German Academic Exchange Service (DAAD, 322/501/014/0), and SFB (217).This work is part of the doctoral thesis of M. P. Bettinotti.     

Cytosolic 5′-nucleotidase/nucleoside phosphotransferase: A nucleoside analog activating enzyme?

Nucleoside phosphotransferase acting on inosine and deoxyinosine has been partially purified from cultured Chinese hamster lung fibroblasts (V79). The activity is associated with a cytosolic 5′-nucleotidase acting on IMP and deoxyIMP. The transfer of the phosphate group from IMP to inosine catalyzed by this enzyme was activated by ATP and 2,3-bisphosphoglycerate. Inosine, deoxyinosine, guanosine, deoxyguanosine, and the nucleoside analogs 2′,3′-dideoxyinosine and 8-azaguanosine are substrates, while adenosine and deoxyadenosine are not. IMP, deoxyIMP, GMP, and deoxyGMP are the best phosphate donors. The cytosolic 5′-nucleotidase/phosphotransferase substrate, 8-azaguanosine, was found to be very toxic for cultured fibroblasts (LD₅₀ = 0.32 μM). Mutants resistant to either 8-azaguanosine and the correspondent base 8-azaguanine were isolated and characterized. Our results indicated that the 8-azaguanosine-resistant cells were lacking both cytosolic 5′-nucleotidase and hypoxanthine-guanine phosphoribosyltransferase, while 8-azaguanine resistant cells were lacking only the latter enzyme. Despite this observation, both mutants displayed 8-azaguanosine resistance, thus indicating that cytosolic 5′-nucleotidase is not essential for the activation of this nucleoside analog.     

Fine structure of the spermatozoa of Crassostrea gigas (Mollusca,Bivalvia)

We describe sperm ultrastructure and acrosome differentiation during spermiogenesis in Crassostrea gigas (Mollusca Bivalvia). The sperm cell is a uniflagellated cell of the primitive type. The head region contains a rounded or conical nucleus surmounted by small acrosome. This organelle consists of a membrane-bound acrosomal granule, the contents of which have a homogeneous density, except in the anterior region, which is positive for PTA. The acrosome also surrounds the perforatorium, which includes oriented fibrillar elements: this is the axial body. The middle piece contains four mitochondria encircling two perpendicular centrioles. The distal centriole is provided with a system of mechanical fixation to the plasma membrane, consisting of nine fibers in radial arrangement. The tail flagellum, about 50 m?m long, contains the usual microtubular axoneme. © 1993 Wiley-Liss, Inc.     

Female reproductive fluid concentrations affect sperm performance of alternative male phenotypes in an external fertilizer

There is growing evidence that the female reproductive fluid (FRF) plays an important role in cryptic female choice through its differential effect on the performance of sperm from different males. In a natural spawning event, the male(s) may release ejaculate closer or further away from the spawning female. If the relative spatial proximity of competing males reflects the female pre-mating preference towards those males, then favoured males will encounter higher concentrations of FRF than unpreferred males. Despite this being a common situation in many external fertilizers, whether different concentrations of FRF can differentially influence the sperm performance of distinct male phenotypes (favoured and unfavoured by the female) remains to be elucidated. Here, we tested this hypothesis using the grass goby (Zosterisessor ophiocephalus), a fish with distinct territorial-sneaker reproductive tactics and female pre-mating preference towards territorial males, that consequently mate in an advantaged position and whose sperm experience higher concentrations of FRF. Our findings revealed a differential concentration-dependent effect of FRF over sneaker and territorial sperm motility only at low concentrations (i.e. at the distance where sneakers typically ejaculate), with increasing FRF concentrations (i.e. close to the eggs) similarly boosting the sperm performance of both sneaker and territorial males. The ability to release sperm close to the eggs is a prerogative of territorials, but FRF can likewise advantage the sperm of those sneakers that are able to get closer, allowing flexibility in the direction of female post-mating choice.     

Fine-scale mapping of physicochemical and microbial landscapes of the coral skeleton

The coral skeleton harbours a diverse community of bacteria and microeukaryotes exposed to light, O₂ and pH gradients, but how such physicochemical gradients affect the coral skeleton microbiome remains unclear. In this study, we employed chemical imaging of O₂ and pH, hyperspectral reflectance imaging and spatially resolved taxonomic and inferred functional microbiome characterization to explore links between the skeleton microenvironment and microbiome in the reef-building corals Porites lutea and Paragoniastrea benhami. The physicochemical environment was more stable in the deep skeleton, and the diversity and evenness of the bacterial community increased with skeletal depth, suggesting that the microbiome was stratified along the physicochemical gradients. The bulk of the coral skeleton was in a low O₂ habitat, whereas pH varied from pH 6–9 with depth. Physicochemical gradients of O₂ and pH of the coral skeleton explained the β-diversity of the bacterial communities, and skeletal layers that showed O₂ peaks had a higher relative abundance of endolithic algae, reflecting a link between the abiotic environment and the microbiome composition. Our study links the physicochemical, microbial and functional landscapes of the coral skeleton and provides new insights into the involvement of skeletal microbes in the coral holobiont metabolism.