Specific enhancement of acylase I and acylpeptide hydrolase activities by the corresponding N-acetylated substrates in primary rat hepatocyte cultures

The specific effects of N-acetyl-L-methionine on acylase I activity and of both N-acetyl-L-alanyl-L-alanine and N-acetyl-L-methionyl-L-alanine on N-acylpeptide hydrolase activity were investigated in primary rat hepatocyte cultures. Each of the above two substrates is known to be much more rapidly hydrolyzed than other derivatives of the same type under optimum enzyme assay conditions. After a two-day incubation of the substrates in the presence of primary rat hepatocyte cultures, the N-acetylaminoacid was found to specifically induce an increase in the acylase I activity, whereas the two N-acetylated peptides increased the acylpeptide hydrolase activity in the soluble 100,000 x g fraction from the culture medium. No change in any of the enzyme activities could be detected during the same period of time when the medium was not supplemented with N-acetylated substrates. In addition, the acylase I activity showed a dose dependent response when the N-acetyl-L-methionine concentration increased from 10 fold to 50 fold. It is therefore suggested that the efficient hydrolysis of each type of substrate that occured in the 48 h hepatocyte cell cultures was due to the increase observed in the overall activity of the corresponding enzymes. The ratio of acylpeptide hydrolase to that of acylase I increased considerably when the medium was supplemented with N-acetylpeptides, and decreased in the presence of the N-acetylaminoacid.     

Limited seed dispersal and microspatial population structure of an agamospermous grass of West African savannahs, Hyparrhenia diplandra (Poaceae)

We studied the microspatial population structure of the perennial tussock grass, Hyparrhenia diplandra (Poaceae), a facultative agamospermous species of West African savannahs. The microspatial population structure of H. diplandra was investigated by choosing two 100-m(2); quadrats at random from which all individuals were mapped. The genotype of every individual was determined using two highly polymorphic microsatellite loci. A chloroplast locus was also used to investigate the role of seed dispersal on the genetic structure of populations. The genetic diversity index (0.85) was high for a clonal species. Significant genetic differentiation over short distances was detected by F statistics, and spatial autocorrelation analyses within both quadrats showed significant isolation-by-distance patterns, both with the cytoplasmic locus and the nuclear loci. Some clones formed large patches (up to 5 m in diameter) whereas others were more scattered. However, the genetic differentiation between quadrats was much higher when studied with the cytoplasmic locus than with the nuclear loci, indicating that gene flow via pollen, but not seeds, may frequently occur between quadrats. The maintenance of genetic diversity in this facultative agamospermous species most likely results from several factors, such as low seed dispersal ability, nonnegligible gene flow through pollen, and selective pressures induced by regularly occurring fires in this ecosystem.     

A novel type II secretion system in Pseudomonas aeruginosa

The genome sequence of Pseudomonas aeruginosa strain PAO1 has been determined to facilitate postgenomic studies aimed at understanding the capacity of adaptation of this ubiquitous opportunistic pathogen. P. aeruginosa produces toxins and hydrolytic enzymes that are secreted via the type II secretory pathway using the Xcp machinery or 'secreton'. In this study, we characterized a novel gene cluster, called hxc for homologous to xcp. Characterization of an hxcR mutant, grown in phosphate-limiting medium, revealed the absence of a 40 kDa protein found in the culture supernatant of wild-type or xcp derivative mutant strains. The protein corresponded to the alkaline phosphatase L-AP, renamed LapA, which is secreted in an xcp-independent but hxc-dependent manner. Finally, we showed that expression of the hxc gene cluster is under phosphate regulation. This is the first report of the existence of two functional type II secretory pathways within the same organism, which could be related to the high adaptation potential of P. aeruginosa.     

Detection and Identification by PCR of a Highly Virulent Phylogenetic Subgroup among Extraintestinal Pathogenic Escherichia coli B2 Strains

Closely related Escherichia coli B2 strains O1:K1, O2:K1, O18:K1, and O45:K1 constitute a major subgroup causing extraintestinal infections. A DNA pathoarray analysis was used to develop a PCR specific for this subgroup that was included in the multiplex phylogenetic-grouping PCR method. Our PCR may serve to identify this virulent subgroup among different ecological niches.     

A hybrid micro-macroevolutionary approach to gene tree reconstruction.

Gene family evolution is determined by microevolutionary processes (e.g., point mutations) and macroevolutionary processes (e.g., gene duplication and loss), yet macroevolutionary considerations are rarely incorporated into gene phylogeny reconstruction methods. We present a dynamic program to find the most parsimonious gene family tree with respect to a macroevolutionary optimization criterion, the weighted sum of the number of gene duplications and losses. The existence of a polynomial delay algorithm for duplication/loss phylogeny reconstruction stands in contrast to most formulations of phylogeny reconstruction, which are NP-complete. We next extend this result to obtain a two-phase method for gene tree reconstruction that takes both micro- and macroevolution into account. In the first phase, a gene tree is constructed from sequence data, using any of the previously known algorithms for gene phylogeny construction. In the second phase, the tree is refined by rearranging regions of the tree that do not have strong support in the sequence data to minimize the duplication/lost cost. Components of the tree with strong support are left intact. This hybrid approach incorporates both micro- and macroevolutionary considerations, yet its computational requirements are modest in practice because the two-phase approach constrains the search space. Our hybrid algorithm can also be used to resolve nonbinary nodes in a multifurcating gene tree. We have implemented these algorithms in a software tool, NOTUNG 2.0, that can be used as a unified framework for gene tree reconstruction or as an exploratory analysis tool that can be applied post hoc to any rooted tree with bootstrap values. The NOTUNG 2.0 graphical user interface can be used to visualize alternate duplication/loss histories, root trees according to duplication and loss parsimony, manipulate and annotate gene trees, and estimate gene duplication times. It also offers a command line option that enables high-throughput analysis of a large number of trees.     

Neurosecretory systems in decapod Crustacea

Summary The sinus glands of the Brachyura are composed of swollen nerve fiber endings, which store and release secretory material synthesized within cells of the eyestalks, the brain and probably the thoracic ganglionic mass. Removal of the sinus glands from the land crab, Gecarcinus lateralis, does not induce molt, because sinus glands are reservoirs, not sources, of molt-inhibiting hormone. Increase in respiratory rate and fall in respiratory quotient, which follow eyestalk removal and signify the approach of molt, do not occur after sinus gland removal.From recent studies on the eyestalks and brain of the crayfish, Cambarus virilis, it is clear that morphologically the neurosecretory system of this crayfish is similar to that of the land crab. There is a marked resemblance in arrangement of neurosecretory cells and in pathways followed by the fibers, the endings of which form the sinus glands. Maps of eyestalks and brain of Cambarus and Gecarcinus emphasize this fundamental likeness between an astacuran and a brachyuran. Regions in which neurosecretory cells are found have been numbered so that these maps may guide cytological and physiological study on these species and on other decapod Crustacea.This paper was delivered at the Symposium on Neurosecretion held May 11–16, 1953, at the Stazione Zoologica, Naples, Italy. An abstract of this paper. together with summaries of other papers presented at the Symposium, is appearing in the Pubblicazioni della Stazione Zoologica di Napoli.The preparation of this paper was aided by a National Science Foundation (USA.) grant to the first author and by the use of the American Table at the Naples Zoological Station.     

Diet deficient in alpha-linolenic acid alters fatty acid composition and enzymatic properties of Na+, K+-ATPase isoenzymes of brain membranes in the adult rat

The effects of dietary (n-6)/(n-3) polyunsaturated fatty acid balance on fatty acid composition, ouabain inhibition, and Na(+) dependence of Na(+), K(+)-ATPase isoenzymes of whole brain membranes were studied in 60-day-old rats fed over two generations a diet either devoid of alpha-linolenic acid [18:3(n-3)] (sunflower oil diet) or rich in 18:3(n-3) (soybean oil diet). In the brain membranes, the sunflower oil diet led to a dramatic decrease in docosahexaenoic acid [22:6(n-3)] membrane content. The activities of Na(+), K(+)-ATPase isoenzymes were discriminated on the basis of their differential affinities for ouabain and their sensitivity to sodium concentration. The ouabain titration curve of Na(+), K(+)-ATPase activity displayed three inhibitory processes with markedly different affinity [i.e., low (alpha1), high (alpha2), and very high (alpha3)] for brain membranes of rats fed the sunflower oil diet, whereas the brain membranes of rats fed the soybean oil diet exhibited only two inhibitory processes, low (alpha1) and high (alpha2' = alpha2 + alpha3). Regardless of the diet, on the basis of the Na(+) dependence of Na(+), K(+)-ATPase activity, three isoenzymes were found: alpha1 form displaying an affinity 1.5- to 2-fold higher that of than alpha2 and 3-fold higher that of alpha3. In rats fed the sunflower oil diet, alpha2 isoenzyme exhibited higher affinity for sodium (Ka = 8.8 mmol/L) than that of rats fed the soybean oil diet (Ka = 11.7 mmol/L). These results suggest that the membrane lipid environment modulates the functional properties of Na(+), K(+)-ATPase isoenzymes of high ouabain affinity (alpha2).     

Nested clade and phylogeographic analyses of the chub, Leuciscus cephalus (Teleostei, cyprinidae), in Greece: implications for Balkan Peninsula biogeography

Phylogenetic relationships among Greek populations of the chub, Leuciscus cephalus, were investigated using 600 bp of the cytochrome b gene. The aim of this study was to test the assumption that the main difference in ichthyological composition between both sides of the Balkan Peninsula is directly linked to differences in the dispersion mechanisms used by fish in order to extend their distribution range. Phylogenetic and nested clade analyses clearly showed that populations in Greece are significantly differentiated. Greek populations were found to descend from three lineages in three geographical provinces: Western, Central, and Eastern Greece. The chub reached Western Greece at the beginning of the Pleistocene and Eastern Greece during the mid-Pleistocene. Chub dispersion occurred mainly by river confluence due to sea level lowering and river capture in Western Greece and sea dispersal with low-salinity conditions within the Aegean Sea in Eastern Greece. However, in Central Greece, the original mtDNA lineage has presumably been lost owing to a genetic introgression following a second invasion from the Danube during the final stage of the last glaciation. This study provides new elements for a better understanding of the composition of the contemporary ichthyofauna in Greece and highlights possible evolutionary mechanisms responsible for the high endemism rate in the Western Greek biogeographic province.     

Differential rates of glycoprotein secretion by isolated rat hepatocytes studied in terms of concanavalin A binding.

Using a concanavalin-A-based method which respects cell function, we have shown that the kinetics of glycoprotein secretion appear to depend on the nature of the oligosaccharide moiety. In 37 degrees C pulse/chase experiments using freshly isolated normal rat hepatocytes, we found that except for transferrin, whose rate of secretion was independent of its concanavalin A reactivity, the secretion of the concanavalin-A-retained forms of alpha 1 acid glycoprotein, T-kininogen, alpha 1 protease inhibitor and alpha 1 inhibitor III was slower than that of the concanavalin-A-non-retained forms. When hepatocytes were incubated at 20 degrees C, secretion was blocked with the accumulation of mainly endoglycosidase-H-sensitive forms. The secretion kinetics of the concanavalin-A-differentiated forms were still different when the temperature was shifted back to 37 degrees C. The divergence between the secretion rates of the concanavalin-A-differentiated forms would appear to be due to a late event in intracellular protein trafficking, which may depend on the sugar content and/or the number of carbohydrate chains of the glycoproteins.     

ADAPTIVE SEX ALLOCATION IN A SIMULTANEOUS HERMAPHRODITE

When applied to hermaphrodite organisms, the local mate competition hypothesis predicts an increase of the ratio of sperm to ova produced as the number of mates increases. Here we test this prediction using a hermaphroditic platyhelminth parasite (trematode), Echinostoma caproni. This worm inhabits the small intestine of vertebrates, inevitably inducing the formation of highly subdivided populations, a condition known to promote local mate competition. Moreover this echinostome exhibits an unrestricted mating pattern involving both selfing and outcrossing as well as multiple fertilizations. We quantified the investment in reproductive organs by estimations of testes, cirrus sac, ovary, and egg size and fecundity when echinostomes were isolated alone, in pairs, or in groups of 20 worms. Adult body size was also recorded as a covariate. When mating group size increases (singles, pairs, or groups) we observed a significant increase in resource allocation to male function in addition to a significant decrease in ovary size. Smaller ovaries do not seem to affect egg size, but do result in a reduction in fecundity. Finally, our results are in accordance with the expected theoretical relationship between male allocation and the number of potential mates given local mate competition.