Molecular analysis of PKU haplotypes in the population of southern Poland

Summary Out of a population of 138598 infants born in southern Poland between 1987 and 1989, and screened for phenylketonuria (PKU), 28 cases were ascertained giving an incidence of 1 in 5000. DNA from 22 of these probands and their parents was isolated and eight polymorphic restriction sites were analyzed within the phenylalanine hydroxylase gene region. Twenty-one different haplotypes (HT) were revealed, five of them representing new categories. The most common haplotypes among those carrying normal alleles were: HT1 (27.3%) and HT4 (11.4%). Within the group of haplotypes with mutant alleles the most frequent was HT2 (56.8%), whereas the frequency of this haplotype in other European populations, such as French, Danish and German, ranged from 12% to 24%. HT3, being the most common in Danish (38%), and relatively frequent in the other western European (13–14%) populations, appeared to be very rare in our sample (2.3%). The mutation of codon 408 (exon 12, CT, ArgTrp), which has been described to be tightly linked to HT2, was tested on amplified DNA by dot-blot hybridization. This mutation was found in 25 out of 44 proband chromosomes. In one case it was linked to HT5, in the remaining 24 to HT2. Our results confirm molecular heterogeneity of PKU haplotypes, as well as their significant interpopulation variation.     

Clinical pharmacology of the new COMT inhibitor CGP 28 014

CGP 28 014 is a specific inhibitor of catechol-O-methyltransferase (COMT) in vivo. In humans, the inhibition was assessed by measuring urinary excretion of isoquinolines and with the levodopa test. Following administration of CGP 28 014, urinary excretion of isoquinolines was significantly increased. In rats, CGP 28 014 reduced plasma and striatal concentrations of 3-O-methyldopa (30MD) in a dose-dependent manner. Acute and subchronic administration of CGP 28 014 alone or in combination with the peripherally acting decarboxylase inhibitor benserazide decreased plasma 30MD as an index of COMT inhibition by about 50%. There seems to be a close relationship between the time-course of plasma concentrations of CGP 28 014 and the extent of COMT inhibition assessed by the 30MD/DOPA ratio in plasma.     

Darwin's ‘mystery of mysteries’: the role of sexual selection in plant speciation

Sexual selection is considered one of the key processes that contribute to the emergence of new species. While the connection between sexual selection and speciation has been supported by comparative studies, the mechanisms that mediate this connection remain unresolved, especially in plants. Similarly, it is not clear how speciation processes within plant populations translate into large-scale speciation dynamics. Here, we review the mechanisms through which sexual selection, pollination, and mate choice unfold and interact, and how they may ultimately produce reproductive isolation in plants. We also overview reproductive strategies that might influence sexual selection in plants and illustrate how functional traits might connect speciation at the population level (population differentiation, evolution of reproductive barriers; i.e. microevolution) with evolution above the species level (macroevolution). We also identify outstanding questions in the field, and suitable data and tools for their resolution. Altogether, this effort motivates further research focused on plants, which might potentially broaden our general understanding of speciation by sexual selection, a major concept in evolutionary biology.     

Characterization of sheep hemopexin glycovariants

The hemopexin phenotype HpxB1 isolated from sheep serum, yields three major bands when subjected to starch gel and/or polyacrylamide gel electrophoresis which are here designated as subcomponents HpxB1-I, HpxB1-II and HpxB1-III. Electrospray mass spectrometric analysis of samples of the isolated subcomponents prepared by ion exchange chromatography showed that each was composed of three glycoproteins and that the major difference between the subcomponents was due to their constituent glycoproteins possessing different numbers of sialic acid residues. Combined analysis of the ESI-MS data and of the overall carbohydrate compositional data obtained by colorimetric procedures, leads to the composition of the glycan of each glycoprotein, and a combined methylation and 400 MHz H-NMR analysis of the alkaline cleaved glycans identified them as being of only the biantennaryN-acetyllactosamine type. Taking into account the molecular mass, the carbohydrate content and structure it may be concluded that each of the constituent glycoproteins contain fiveN-glycosidically linked glycans.Abbreviations HpxB1 hemopexin phenotype B1 - Man mannose - Gal galactose - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - GlcNAc-ol N-acetylglucosaminitol     

Fusion of hamster and pig zona-free eggs stimulated by boar and guinea pig sperm at fertilization in vitro

In the course of in vitro fertilization of zona-free hamster and pig eggs by boar and guinea-pig spermatozoa it was observed that homologous and heterologous eggs fused together, forming cell hybrids between two or more cells. The fusogenic activity was attributed to spermatozoa and this was the hypothesis tested. The fusogenic activity (coinciding with sperm penetration activity) was dependent on the duration of sperm preincubation, which may be regarded as capacitation in vitro. Fusion occurred only after 3 hr of sperm preincubation and a narrow optimum was detected at 4–4.5 hr. Fusion of eggs was also dependent on sperm concentration. A relatively high proportion of fusions was observed at a sperm concentration of 4.0 × 10⁴ per ml and an optimum was attained at a concentration of 5.0 × 10⁵ per ml. The first fusions were observed at 90 min after semination. After 3 hr more than a half of the eggs reacted, and by 20 hr of incubation 80% of ova were fused. The fusability of eggs was tested and found to occur at 14 hr after ovulation. The fusion process was also studied using transmission electron microscopy. It is supposed that the process of egg fusion may be caused either by a similar mechanism to sperm-egg fusion, or by products released during the sperm acrosome reaction.     

Phylogenetic scale in ecology and evolution

Aim

Many important patterns and processes vary across the phylogeny and depend on phylogenetic scale. Nonetheless, phylogenetic scale has never been formally conceptualized, and its potential remains largely unexplored. Here, we formalize the concept of phylogenetic scale, review how phylogenetic scale has been considered across multiple fields and provide practical guidelines for the use of phylogenetic scale to address a range of biological questions.

Innovation

We summarize how phylogenetic scale has been treated in macroevolution, community ecology, biogeography and macroecology, illustrating how it can inform, and possibly resolve, some of the longstanding controversies in these fields. To promote the concept empirically, we define phylogenetic grain and extent, scale dependence, scaling and the domains of phylogenetic scale. We illustrate how existing phylogenetic data and statistical tools can be used to investigate the effects of scale on a variety of well‐known patterns and processes, including diversification rates, community structure, niche conservatism or species‐abundance distributions.

Main conclusions

Explicit consideration of phylogenetic scale can provide new and more complete insight into many longstanding questions across multiple fields (macroevolution, community ecology, biogeography and macroecology). Building on the existing resources and isolated efforts across fields, future research centred on phylogenetic scale might enrich our understanding of the processes that together, but over different scales, shape the diversity of life.     

Mixed and non-cognate SNARE complexes. Characterization of assembly and biophysical properties.

Assembly of soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) proteins between two opposing membranes is thought to be the key event that initiates membrane fusion. Many new SNARE proteins have recently been localized to distinct intracellular compartments, supporting the view that sets of specific SNAREs are specialized for distinct trafficking steps. We have now investigated whether other SNAREs can form complexes with components of the synaptic SNARE complex including synaptobrevin/VAMP 2, SNAP-25, and syntaxin 1. When the Q-SNAREs syntaxin 2, 3, and 4, and the R-SNARE endobrevin/VAMP 8 were used in various combinations, heat-resistant complexes were formed. Limited proteolysis revealed that these complexes contained a protease-resistant core similar to that of the synaptic complex. All complexes were disassembled by the ATPase N-ethylmaleimide-sensitive fusion protein and its cofactor alpha-SNAP. Circular dichroism spectroscopy showed that major conformational changes occur during assembly, which are associated with induction of structure from unstructured monomers. Furthermore, no preference for synaptobrevin was observed during the assembly of the synaptic complex when endobrevin/VAMP 8 was present in equal concentrations. We conclude that cognate and non-cognate SNARE complexes are very similar with respect to biophysical properties, assembly, and disassembly, suggesting that specificity of membrane fusion in intracellular membrane traffic is not due to intrinsic specificity of SNARE pairing.     

Serotonin and its metabolites reduce oxidative stress in murine RAW264.7 macrophages and prevent inflammation

In this study, we focused on comparing the effects of serotonin and its metabolites on the functions of RAW264.7 cells (emphasis on oxidative burst and production of nitric oxide and cytokines), thereby expanding the scope of existing knowledge with advent of novel findings in this field. Changes in production of reactive oxygen species (ROS) by RAW264.7 cells after treatment with serotonin, N-acetylserotonin and melatonin were determined using the chemiluminescence (CL) assay. To exclude the direct scavenging effects of the studied compounds on the CL response, the antioxidant properties of all respective compounds were measured using TRAP and amperometrical method. Nitric oxide (NO) production was measured by Griess reagent and inducible NO synthase (iNOS) expression by Western blot. Cytokine production was assessed using the Mouse Cytokine Panel A Array kit and ELISA. We showed that all tested compounds were able to reduce oxidative stress, as well as inhibit production of inflammatory cytokines by macrophages. Of the tested compounds, serotonin and N-acetylserotonin were markedly better antioxidants than melatonin. In comparison, other effects of tested compounds were very similar. It can be concluded that antioxidant capacity of tested compounds is a major advantage in the early stages of inflammation. Since plasma concentrations of N-acetylserotonin and melatonin are lower than serotonin, it can be deduced that serotonin plays a key role in modulation of inflammation and the regulatory functions of immune cells, while also protecting cells against oxidative stress.