A sensitive high performance liquid chromatographic method for determining small amounts of glycosylproteins

We developed a simple isocratic high performance liquid chromatography (HPLC) method for the quantitative determination of 5-hydroxymethyl-2-furfuraldehyde (5-HMF) liberated by mild hydrolysis of small amounts of glycosyl proteins. The absorbance of hydrolysate components after HPLC separation was recorded at 280 nm. To detect substances possibly interfering with the 5-HMF peak we always recorded the ratio of the peak heights A280 nm/A254 nm which was a constant value of 4.4. For each sample the blank was obtained by reduction with NaBH4 before hydrolysis with oxalic acid 1 mol/l. The best NaBH4/protein ratio was found to be 4 mg/mg. With this method we measured the nonenzymatic glycosylation (glycation) as 5-HMF in samples with a protein concentration as low as 0.8 mg/ml. 5-HMF produced per milligram of protein was independent from protein concentration for a wide range (0.8-10 mg/ml). The mean coefficient of variation for within assay and between precision was 6.8 and 11.6%, respectively. The 5-HMF measured on plasma proteins from normal subjects (n = 7) was 0.16 +/- 0.04 nmol/mg. Protein from insulin-dependent diabetic patients was 0.31 +/- 0.07 nmol/mg. With this method we succeeded in detecting an excessive glycation of platelet membrane proteins in 13 type-I diabetic patients.     

Differential staining of catecholamines, 5-hydroxytryptamine and related compounds in aldehyde-fixed tissues

Summary Factors influencing the fixation of biogenic amines by solutions of glutaraldehyde, formaldehyde, acrolein and glyoxal as well as the influences of these aldehydes on the histochemical reactivity of biogenic amines, have been extensively investigated. Selective stainings of 5-hydroxytryptamine, noradrenaline, dopamine and related substances in many aldehyde-fixed tissues have been achieved by systematically applying some phenol methods such as argentaffin, ferric-ferricyanide, diazonium and thioindoxyl reactions, and some indole methods such as xanthydrol, dimethylaminobenzaldehyde, vanilline, salicilaldehyde and benzaldehyde tests.The list of the structures which react like 5-hydroxytryptamine in in vitro tests includes enterochromaffin cells, carcinoid tumours, rodent mast cells, thyroid C cells, a kind of cell found in the human carotid body and chemodectomas, and some cells secreting protein hormones. NA cells of the adrenal medulla, another kind of cell in the carotid body and chemodectomas, as well as ruminants' mast cells, reacted like catecholamines in in vitro tests.Supported by grant No. 115.1139.04715 from the Italian Consiglio Nazionale delle Ricerche.     

Effect of 17β-estradiol on calcium response to phytohaemagglutinin in human lymphocytes

Pre-treatment of human lymphocytes with 17-estradiol diminishes the increase in concentration of cytosolic free calcium after stimulation with phytohaemagglutinin. The effect is dependent on 17-estradiol concentration and on the preincubation time. The effect is not due to an interaction between 17-estradiol and phytohaemagglutinin, but appears to be a consequence of the binding of the hormone to the cell surface. The effect is specific for 17-estradiol, since the isomer and other steroid hormones (progesterone, testosterone, diethylstilbestrol and 5-androstan), have no effect. Since the effect of the 17-estradiol can be suppressed by treatment of lymphocytes with ouabain, it appears that the effect of estradiol on the rise of cytosolic calcium induced by phytohaemagglutinin is mediated by the (Na, K)-ATPase.     

Physiologic target of the Air-1 trans-activator revealed by stable transfection assay

RJ 2.2.5 is a human B cell mutant, derived from Raji cells, which has lost expression of major histocompatibility complex (MHC) class II genes because of a defect in the AIR1 locus function. The MHC class II-positive phenotype can be restored by introducing an active AIR1 locus or its mouse equivalent, Air-1. An example of the latter is the H4 cell hybrid, derived by somatic cell fusion between RJ 2.2.5 and mouse class II-positive spleen cells. H4 contains a single mouse chromosome, autosome 16, in which the Air-1 locus maps, and an entire RJ 2.2.5-derived genome. In the present study we show that the physiologic target of the Air-1 locus product is contained within a limited HLA-DRA promoter sequence of 300 base pairs, encompassing the crucial Y, X, and W cis-acting elements. A plasmid construct, pDRA300neo, containing the HLA-DRA promoter sequence which drives the expression of the neomycin resistance gene, has been stably integrated in the genome of the H4 hybrid. Transfectants selected in the presence of G418 retain mouse chromosome 16 and express the DR genes. On the other hand, transfectants grown in a non-selective medium segregate mouse chromosome 16; this is accompanied by a loss of DRA gene expression and G418 resistance, although pDRA300neo is still integrated in the genome. These results offer scope for using this experimental model to clone the Air-1 gene in a straightforward way. Correspondence to: R. S. Accolla.     

Meiotic recombination in an Irish family with beta-thalassaemia

Using the technique of allele-specific priming of the polymerase chain reaction (PCR), the C-T substitution in codon 39 was identified as the cause of -thalassaemia in an Irish family. Analysis of the restriction fragment length polymorphisms (RFLPs) in the -globin gene cluster established linkage of the -thalassaemia mutation to a particular -haplotype but indicated that a recombinational event had occurred in the paternal chromosome in the younger of two affected children. Non-paternity was excluded by DNA fingerprinting analysis with hypervariable minisatellite probes. This is the fourth case of recombination in the -globin gene cluster to be reported. The event has occurred 5 of the polymorphic RsaI site at position-550 bp upstream of the -globin gene mRNA Cap site, within the 9.1-kb region that has been shown to be a hot spot for recombination in the -globin gene cluster.     

Molecular characterization of Inonotus rickii /Ptychogaster cubensis isolates from different geographic provenances

Thirteen isolates of Inonotus rickii/Ptychogaster cubensis, from different geographic provenances, were analyzed by sequencing ITS1, ITS 2 and 5,8S ribosomal RNA region. A phylogenetic tree, also including sequences available in Genbank database, showed that the strains enclosed in this study fall into two well-separated groups, one formed by isolates from Florida (USA) and the other one by isolates from Europe, Argentina and China. Differences were also highlighted on the growth rate of mycelial cultures at different temperatures. In fact, although the tested isolates generally attained the best growth at 30°C, isolates from Europe seem well adapted to higher temperatures and went on growing at 40°C whilst the growth of isolates from Florida significantly decreased at 35°C. Since the teleomorph I. rickii was never detected in Florida, and in this study noticeable differences were detected by analysis of ITS region, the existence of two possible distinct species, not discriminated solely on the basis of morphological characters, could be suggested.     

8,000 years of climate,vegetation, fire and land-use dynamics in the thermo-mediterranean vegetation belt of northern Sardinia (Italy)

Vegetation History and Archaeobotany - Knowledge about the vegetation history of Sardinia, the second largest island of the Mediterranean, is scanty. Here, we present a new sedimentary record...