All in an afternoon: mixed breeding system in one-day lasting flowers of Hypericum elodes L. (Hypericaceae)

Knowledge about mixed mating systems can improve our understanding of the evolutionary dynamics of reproductive systems. Here we report a study of the floral and reproductive biology of Hypericum elodes, an Atlantic-European soft-water pools specialist which shows a floral architecture consistent with both self- and cross-pollination. Controlled pollination experiments were performed in a natural population during three consecutive years. Marked flowers were monitored until fruit production, and laboratory germination experiments were conducted with the seeds produced. Plants were self-compatible (SCI>0.75), however, compared with selfing, cross-pollination enhanced fruit-set, seed-set and seedling growth, but not seed germination. Inbreeding depression (δ) was mild in the pre-dispersal stages (δ = 0.22 for fruit set, 0.18 for seed set and 0.13 for seed mass), low for germination percentage (δ = 0.003) and mild for seedling growth (δ = 0.23). The breeding system of H. elodes promotes outcrossing and assures reproductive success by means of competitive autogamy. Our results suggest a mixed mating strategy for the studied population, characterized by mild inbreeding depression (cumulative δ = 0.57), highlighting the benefit of this reproductive mode in unpredictable habitat, as the typical shallow-water meadows where H. elodes grows.     

A one-day double-labelling technique for tissue specimens: Immunogold-silver staining forin situ hybridization combined with alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunohistochemistry for antigens

Summary An improved technique is described that addresses the problems of sensitivity, specificity, the use of hazardous radioactive equipment and time consumption in immunohistochemical labelling and double labelling ofin situ hybridization of tissue specimens. It consists of a two-step protocol in which digoxigenin-uridine triphosphate (UTP) labelled riboprobes in thein situ hybridization step are visualized by the immunogold-silver staining method, and double labelling of tissue antigens is achieved by the application of an alkaline phosphatase-anti-alkaline phosphatase staining step. We tested this protocol using snap-frozen tissue sections of synovial tissue from patients with rheumatoid arthritis. The target mRNA was detected by perforin or cathepsin D riboprobes, the double labelling was performed using anti-collagen type IV and alpha-smooth muscle actin antibodies. It is concluded that, in comparison with an established three-to four-day double-labelling protocol used in many laboratories, this one-day combination is currently the most rapid assay of reliable quality for double labelling ofin situ hybridization products and tissue antigens.     

Further studies on the feasibility of one-day Salmonella detection by enzyme-linked immunosorbent assay.

A model system previously developed for the rapid detection of Salmonella typhimurium in foods was improved and extended to many other Salmonella serotypes. The original protocol, which consisted of an overnight nonselective culture followed by a specific enzyme-linked immunosorbent assay (ELISA), was modified and improved. A sandwich ELISA which used polyclonal antibodies for the capture stage and a cocktail of monoclonal antibodies for the detector stage was developed. The assay recognized a wide range of Salmonella serotypes; S. enteritidis, the most important serotype in the United Kingdom had a detection limit in the ELISA of about 4 x 10(2) cells ml-1. The cultural stage prior to the ELISA was either a single nonselective broth (incubated for 28 h) or a preenrichment broth (incubated for 7 h) plus a selective broth (incubated for 21 h). Antibodies which bind to cells grown in the unfavorable conditions of a selective medium were selected. It was concluded that, in the future, the shortened protocols for the detection of Salmonella spp. in foods described here will be of considerable value.     

API Listeria, a new and promising one-day system to identify Listeria isolates.

API Listeria is a new 10-test strip for 24-h biochemical identification of Listeria isolates. With this commercial system, 85% of 646 Listeria strains, including atypical isolates selected for this study, were recognized at the species and subspecies level without a complementary test. A new test differentiates Listeria monocytogenes from L. innocua on the basis of the absence of arylamidase from the former. With this system, 97.7% (252 of 258) of the L. monocytogenes strains tested were correctly identified and differentiated from 99.4% (175 of 176) of the L. innocua strains also tested. Gram-positive bacteria other than Listeria spp. gave quite different biochemical patterns. This system considerably reduced the time needed for conventional identification, since results were available within 18 to 24 h.     

API Listeria, a new and promising one-day system to identify Listeria isolates.

API Listeria is a new 10-test strip for 24-h biochemical identification of Listeria isolates. With this commercial system, 85% of 646 Listeria strains, including atypical isolates selected for this study, were recognized at the species and subspecies level without a complementary test. A new test differentiates Listeria monocytogenes from L. innocua on the basis of the absence of arylamidase from the former. With this system, 97.7% (252 of 258) of the L. monocytogenes strains tested were correctly identified and differentiated from 99.4% (175 of 176) of the L. innocua strains also tested. Gram-positive bacteria other than Listeria spp. gave quite different biochemical patterns. This system considerably reduced the time needed for conventional identification, since results were available within 18 to 24 h.     

Lateralization of housekeeping genes in the brain of one-day old chicks

Real-time quantitative PCR is an exceptionally sensitive method that can detect even very small differences in gene expression and, as such, it is essential to use suitable reference genes. Domestic chickens are used in a wide range of studies including neurobiology, behavior, ecology and disease transmission. In recent avian gene expression experiments, 18S (18S ribosomal RNA), beta actin (ACTB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have frequently been used; however, there is not enough evidence that these reference genes are suitable for all types of experiments. There is considerable evidence for lateralization in numerous learning tasks and for differences in the functional contribution of the two brain hemispheres. Therefore, the purpose of this study was to identify a set of reference genes for chick brain region called an intermediate medial mesopallium (IMM), which is connected with memory formation in the chick brain, whilst also taking into consideration the differences between the left and right hemispheres. This study evaluated the expression stability of eleven candidate housekeeping genes in the IMM region of the 1-day old chick brain. In our experimental system, the most reliable results were given by the NormFinder algorithm. The results show for the first time that ACTB, commonly used as an avian reference gene, is not suitable for investigation of gene expression in the chick brain and that brain lateralization exact selection of different reference gens for each hemisphere. For memory process investigations using tasks in one-day old chicks the most effective reference genes for the left hemisphere were HMBS and SDHA, and for the right hemisphere the most effective was RPL19.     

Ultrastructure of synaptosomes from one-day old and adult rat brain stem

Summary The ultrastructure and protein content of the five subfractions of the crude mitochondrial fraction from the brain stem of the 1-day old and adult rat was examined. The morphological composition of the subfractions after fixation in glutaraldehyde and osmiumtetroxide in the adult rat brain stem resembled that previously reported for the whole brain; synaptosomes sedimented in a sucrose gradient in subfractions C and D. In the 1-day old rat, mature synaptosomes were found in subfractions A, B, C and D; E contained mainly free mitochondria. 80–95% of the processes in the adult and 10–30% in the 1-day old rat contained synaptic vesicles which were of four types: (1) small agranular vesicles (2) large dense core vesicles (3) large agranular vesicles (4) coated vesicles. Pre- and postsynaptic membrane thickenings were demonstrated in many nerve-ending particles. In the subfractions of the 1-day old rat the protein content was one half and the distribution resembled that in the adult. Evidently nerve endings develop faster in the brain stem than in cortical areas; a serotoninor adrenergic origin of the early synaptosomes is suggested.This study was supported by a grant from the Paulo Foundation.     

Clonidine (Dixarit) for menopausal flushing.

Clonidine, 0.05 mg twice daily, was evaluated in a multicentre, randomized, placebo-controlled, double-blind crossover study in 66 patients who had had menopausal flushing for less than 1 year. Although the placebo effect was substantial, clonidine reduced the frequency of attacks significantly more than did placebo. In three of the four trials the patients'' comparisons of symptoms before and after crossover indicated significantly greater improvement when the crossover was from placebo to clonidine rather than the reverse. The frequency, severity and duration of attacks were reduced by clonidine in 78%, 89% and 88% of the patients respectively, and by placebo in 50%, 53% and 50%. Side effects were minimal and their pattern was the same for clonidine as for placebo. Clonidine''s action as a peripheral vascular stabilizer makes it potentially useful for the treatment of menopausal flushing. It would be prudent to include clonidine at the beginning of treatment so that its efficacy could be assessed in each individual. Its use would enhance the effects of the usual management of the menopausal syndrome, which consists of explanation, reassurance and, sometimes, the use of tranquillizers. Clonidine is a symptomatic medication that makes flushing more tolerable and should reduce the number of patients who would otherwise be exposed to the risks of estrogens.     

Proposal of Microsquama subgen. nov. for Nephroselmis pyriformis (Carter) Ettl (Nephroselmidophyceae,Chlorophyta)

A new subgenus, Microsquama, is proposed for Nephroselmis pyriformis based on its phylogenetic position and some ultrastructural characters, such as the absence of large stellate scales. Other species of the genus are classified in the subgenus Nephroselmis.     

The erection of Leporidotaenia n. g. (Cestoda: Anoplocephalidae) for Anoplocephaloides spp. parasitising Leporidae (Lagomorpha)

Leporidotaenia n. g. is proposed for four cestode species parasitising Leporidae (Lagomorpha) which were previously considered as members of the genus Anoplocephaloides. The new genus is differentiated from Anoplocephaloides by the presence of a spinose covering on the tegument, the protrusible genital atrium, the massive cirrus-sac with highly developed musculature, the presence of a retractor muscle associated with the cirrus-sac, the posterior position of vaginal pore relative to the male orifice, the cirrus armed with long spines and its occurrence only in hosts of the family Leporidae (Lagomorpha). The genus includes: L. romerolagi (type-species), L. floresbarroetae, L. pseudowimerosa and L. wimerosa. the species (excluding L. floresbarroetae) are redescribed and figured and a differential key is presented. An attempt is made to explain the range of Leporidotaenia spp. in Central America and the western Palaearctic on the basis of the palaeozoogeography of rabbits and hares.     

Requirements for alpha(5)beta(1) integrin-mediated retraction of fibronectin-fibrin matrices.

Retraction of the blood clot by nucleated cells contributes both to hemostasis and to tissue remodeling. Although plasma fibronectin (FN) is a key component of the clot, its role in clot retraction is unclear. In this report, we demonstrate that the incorporation of FN into fibrin matrices significantly improves clot retraction by nucleated cells expressing the integrin alpha(5)beta(1). Further, we show that FN-fibrin clots support increased cell spreading when compared with fibrin matrices. To determine the structural requirements for FN in this process, recombinant FN monomers deficient in ligand binding or fibrin cross-linking were incorporated into fibrin clots. We show that recombinant FN monomers support clot retraction by Chinese hamster ovary cells expressing the integrin alpha(5)beta(1). This process depends on both the Arg-Gly-Asp (RGD) and the synergy cell-binding sites and on covalent FN-fibrin binding, demonstrating that cross-linking within the clot is important for cell-FN interactions. These data show that alpha(5)beta(1) can bind to FN within a clot to promote clot retraction and support cell shape change. This provides strong evidence that alpha(5)beta(1)-FN interactions may contribute to the cellular events required for wound contraction.