Purification and characterization of hyaluronic acid from the mollusc bivalve Mytilus galloprovincialis

Hyaluronan (hyaluronic acid, HA) was for the first time extracted, purified and characterized from the species of mollusc bivalve Mytilus galloprovincialis. HA was characterized by agarose-gel electrophoresis, 13C-NMR, HPLC and normal polarity capillary electrophoresis by evaluating the unsaturated disaccharide, DeltaDiHA (Delta-hexuronic acid-N-acetyl-glucosamine) after treatment with chondroitin ABC lyase, and by separating Delta-tetrasaccharide and Delta-hexasaccharide generated by the specific action of hyaluronate lyase from Streptomyces hyalurolyticus. The weight average molecular weight (M(w)) was found to be about 200 kDa as determined by HPSEC. HA from M. galloprovincialis was not able to interact with aggrecan from bovine cartilage to form high molecular mass aggregate and also had a very low specific viscosity, but it showed the same capacity to inhibit cell proliferation (50 microg per 10(3) human fibroblasts inhibit cell proliferation by about 50%) than high molecular mass HA. HA of M. galloprovincialis could have a physiological role in the regulation of cell functions.     

Milligram-scale preparation and purification of oligosaccharides of defined length possessing the structure of chondroitin from defructosylated capsular polysaccharide K4

Escherichia coli K4 bacterium synthesizes a nonsulfated capsule polysaccharide (K4) composed of a repeating disaccharide subunit of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid residues. The K4 polyanion is easily defructosylated under acid conditions with no fragmentation of the polymer to produce a polysaccharide having a repeated disaccharide unit of chondroitin consisting of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) (K4d). K4 and K4d were depolymerized by partial digestion with testicular hyaluronidase and separated into uniform-size oligosaccharides from 4-mers to 16-mers by preparative anion-exchange chromatography after removal of the hyaluronidase. The purity and size of each oligosaccharide was confirmed by using anion-exchange HPLC, HPSEC analysis, and FACE. Mg-scale K4d oligosaccharides were obtained from 50 mg K4d starting material. Under the conditions used to degrade the K4 polysaccharide by testicular hyaluronidase, fructose is slowly liberated forming the defructosylated K4. As a consequence, a mixture of uniform- size K4 and K4d oligosaccharide species, from approximately 4- to 20-mers, are generated and size-separated by anion-exchange chromatography. These pure, uniform-size, and large ranges of K4d oligosaccharides having the structure of a chondroitin, -->4)-GlcUA-beta(1-->3)GalNAc-beta(1-->, will be available for investigating important biological functions of this polymer.     

Whole body and skeletal muscle glutamine metabolism in healthy subjects

We measured glutamine kinetics using L-[5-15N]glutamine and L-[ring-2H5]phenylalanine infusions in healthy subjects in the postabsorptive state and during ingestion of an amino acid mixture that included glutamine, alone or with additional glucose. Ingestion of the amino acid mixture increased arterial glutamine concentrations by approximately 20% (not by 30%; P < 0.05), irrespective of the presence or absence of glucose. Muscle free glutamine concentrations remained unchanged during ingestion of amino acids alone but decreased from 21.0 +/- 1.0 to 16.4 +/- 1.6 mmol/l (P < 0.05) during simultaneous ingestion of glucose due to a decrease in intramuscular release from protein breakdown and glutamine synthesis (0.82 +/- 0.10 vs. 0.59 +/- 0.06 micromol x 100 ml leg(-1) x min(-1); P < 0.05). In both protocols, muscle glutamine inward and outward transport and muscle glutamine utilization for protein synthesis increased during amino acid ingestion; leg glutamine net balance remained unchanged. In summary, ingestion of an amino acid mixture that includes glutamine increases glutamine availability and uptake by skeletal muscle in healthy subjects without causing an increase in the intramuscular free glutamine pool. Simultaneous ingestion of glucose diminishes the intramuscular glutamine concentration despite increased glutamine availability in the blood due to decreased glutamine production.     

Matching strategies for genetic association studies in structured populations

Association studies in populations that are genetically heterogeneous can yield large numbers of spurious associations if population subgroups are unequally represented among cases and controls. This problem is particularly acute for studies involving pooled genotyping of very large numbers of single-nucleotide-polymorphism (SNP) markers, because most methods for analysis of association in structured populations require individual genotyping data. In this study, we present several strategies for matching case and control pools to have similar genetic compositions, based on ancestry information inferred from genotype data for approximately 300 SNPs tiled on an oligonucleotide-based genotyping array. We also discuss methods for measuring the impact of population stratification on an association study. Results for an admixed population and a phenotype strongly confounded with ancestry show that these simple matching strategies can effectively mitigate the impact of population stratification.     

First production of wild hemmer (<Emphasis Type="Italic">Triticum turgidum</Emphasis> ssp. dicoccoides) transgenic plants

Plant genetic transformation and regeneration has become a valuable research tool for functional genomics. A successful transformation event involves the transfer of the target gene into a suitable explant, the integration and expression of the transgene into the host genome and the regeneration of the fertile transgenic plants from the transformed tissues. Wheat is considered as a recalcitrant species even if many efforts have been done in recent years to improve transformation efficiency. The transformation of its progenitors has never been attempted, even though the possibility to transform wild hemmer represents a valuable tool to evaluate structural and functional variability occurring in wild hemmer and explaining its higher adaptation to abiotic stresses. In this paper we report, as far as we know, for the first time, the microparticle transformation of immature embryos of the wild hemmer Triticum dicoccoides with the Tapgip1 gene. The transformation method was successfully transferred from durum wheat and several transgenic lines were obtained. Its application for the exploitation of wheat progenitors for molecular breeding is of great relevance for genomic and functional genomics studies. This result, indeed, opens new perspectives in complementation studies for the comprehension of durum and bread wheat adaptation mechanisms to stresses.     

GATA repeats in the genome of Asellus aquaticus (Crustacea,Isopoda)

A 500 bp fragment of Drosophila genomic DNA containing 37 copies of the tetranucleotide GATA was used to probe, by Southern DNA blotting and in situ hybridization, two natural populations of the isopod crustacean Asellus aquaticus collected from the Sarno and Tiber rivers. This species does not have a recognizable sex chromosome pair. In a number of males from the Sarno population chromomycin A₃ staining reveals a heteromorphic chromosome pair. The heterochromosome has two blocks of heterochromatin. After digestion of genomic DNA with six restriction endonucleases and hybridization with the GATA probe, the two populations exhibit different fragment length patterns. No sex-linked pattern was observed in either population. In situ hybridization to chromosomes of males and females from the Sarno population does not reveal any sex-specific pattern of labelling and indicates a scattered distribution of GATA sequences on most chromosomes with some areas of preferential concentration. The heterochromatic arcas of the male heterochromosome are not labelled.by E.R. Schmidt     

Intravenously infused substance P enhances basal and growth hormone (GH) releasing hormone-stimulated GH secretion in normal men.

The effect of synthetic substance P (SP), infused intravenously (IV) in doses of 0.5, 1, or 1.5 pmol/kg-1/min-1 over 60 min, on GH secretion was evaluated in seven healthy men. Substance P tests and a control test with normal saline were randomly performed at weekly intervals. No untoward side effects or changes in blood pressure were observed during SP infusions. Serum GH concentrations did not change when normal saline, the lowest dose, or the middle dose of SP were infused. In contrast, GH levels rose significantly when the highest dose of SP was given, with a mean peak two times higher than baseline. Further studies were performed to test the possible influence of SP on the GH response to GH-RH. For this purpose, seven other healthy men were tested with GH-RH (1 micrograms/kg body weight in an IV bolus) during saline or SP (1.5 pmol/Kg-1/min-1 x 60 min) infusion. The GH-RH induced a significant GH rise, with a mean peak seven times higher than baseline. When subjects were infused with SP, the GH response to GH-RH was greatly enhanced, with a mean peak 12 times higher than baseline. These results demonstrate for the first time in humans that the systemic infusion of SP stimulates GH secretion, and suggest that SP might interact with GH-RH in the stimulation of GH secretion.