Simultaneous detection of submicrogram quantities of hyaluronic acid and dermatan sulfate on agarose-gel by sequential staining with toluidine blue and Stains-All

A new discontinuous agarose-gel electrophoresis in 0.05 M HCl/0.04 M barium acetate combined with the highly sensitive visualization technique using toluidine blue/Stains-All has been developed for the simultaneous assaying of hyaluronic acid (HA) and dermatan sulfate (DS) with a detection limit at submicrogram level greater than other conventional procedures. Furthermore, this procedure also separates and reveals chondroitin sulfate (CS). The densitometric analysis of bands resulted in a linear response between 0.01 and 0.5 microg of glycosaminoglycans (GAGs) with correlation coefficients greater than approximately 0.94. Hyaluronic acid and dermatan sulfate extracted and purified from the abdominal skin of six rats were separated and quantified in comparison with the evaluation made by treatment of chondroitin ABC lyase and separation of Delta-disaccharides from hyaluronic acid (DeltadiHA) and dermatan sulfate/chondroitin sulfate (Deltadi4s and Deltadi6s) by HPLC. The total amount of rat skin polysaccharides (hyaluronic acid and dermatan sulfate) was 1.24+/-0.26 microg/mg of tissue by discontinuous agarose-gel electrophoresis and 1.20+/-0.33 microg/mg by HPLC with hyaluronic acid and dermatan sulfate percentages of 50.32+/-2.38 and 49.66+/-2.53, respectively. The analyses also confirmed that hyaluronic acid and dermatan sulfate are the main rat abdominal skin polysaccharides with chondroitin sulfate present in trace amounts. This new agarose-gel electrophoresis could be particularly useful in the study of the distribution of glycosaminoglycans in the skin from different body sites of animals and normal human subjects and may be of importance in understanding the changes that occur in the skin, especially the metabolism of extracellular matrix constituents, in connective tissue disorders.     

Treatment of rabbit neutrophils with phorbol esters results in increased ADP-ribosylation catalyzed by pertussis toxin and inhibition of the GTPase stimulated by fMet-Leu-Phe

The effects of pretreatment of rabbit neutrophils with phorbol 12-myristate 13-acetate on the ability of pertussis toxin to catalyze ADP-ribosylation and of fMet-Leu-Phe to activate a high-affinity GTPase in these cell homogenates were examined. The addition of phorbol 12-myristate 13-acetate, but not 4 alpha-phorbol 12,13-didecanoate, to intact cells was found to stimulate by more than 100% the pertussis toxin-dependent ribosylation of a 41 kDa protein (either the alpha-subunit of the 'inhibitory' guanine nucleotide-binding protein N or a closely analogous protein) and to inhibit by more than 60% the activation by fMet-Leu-Phe of the GTPase of the neutrophil homogenates. The addition of fMet-Leu-Phe to intact cells increases the ADP-ribosylation catalyzed by pertussis toxin of the 41 kDa protein. On the other hand, the exposure of neutrophil homogenates to fMet-Leu-Phe results in a decreased level of ADP-ribosylation. This decreased ribosylation reflects a dissociation of the GTP-binding protein oligomer that is not followed by association, possibly because of the release of the alpha-subunit into the suspending media. The implications of these results for the understanding of the mechanism of inhibition of cell responsiveness by phorbol esters and the heterologous desensitization phenomenon are discussed. Prominent among these are the possibilities that (i) the rate of dissociation of the Ni oligomer is affected by the degree of its phosphorylation by protein kinase C, and/or (ii) the dissociated phosphorylated alpha-subunit (the 41 kDa protein) is functionally less active than its dephosphorylated couterpart.     

Abnormal arginine-vasopressin responses to metoclopramide and insulin-induced hypoglycemia in type I diabetes mellitus

Alterations in the control of arginine-vasopressin (AVP) secretion have been described in type I diabetes mellitus. In order to gain a better insight into this problem, we examined whether insulin-dependent diabetics in good metabolic conditions and without diabetic complications had an abnormal AVP responsiveness to metoclopramide (MCP), an AVP-stimulating agent with a central site of action. In addition, we tested the AVP response to insulin-induced hypoglycemia in the same subjects. Twenty insulin-dependent diabetic men without neuropathy or other diabetic complications were divided into two groups according to the duration of their illness (10 patients who had been diabetic for less than 10 years, group 1, and 10 patients who had been diabetic for more than 10 years, group 2). Eleven age- and weight-matched normal men participated as controls. All groups were tested with MCP (20 mg in an intravenous bolus) and, on a different occasion, with insulin-induced (0.15 IU/kg) hypoglycemia. Experiments started after optimization of the metabolic status of the diabetic men by 3 days of treatment with continuous subcutaneous insulin infusion. Basal concentrations of AVP were similar in all groups (diabetics of group 1: 2.2 +/- 0.2 pmol/l, mean +/- SE; group 2: 2.3 +/- 0.2 pmol/l; normal controls: 2.2 +/- 0.2 pmol/l). Administration of MCP induced a striking elevation of plasma AVP levels in the normal controls and in the diabetic subjects of groups 1 and 2. All subjects showed a mean peak response at 15 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genomic organization and chromosomal localization of the mouse IKBKAP gene.

The autosomal recessive disorder familial dysautonomia (FD) has recently been demonstrated to be caused by mutations in the IKBKAP gene, so named because an initial report suggested that it encoded an IkappaB kinase complex associated protein (IKAP). Two mutations in IKBKAP have been reported to cause FD. The major mutation is a T-->C transition in the donor splice site of intron 20 and the minor mutation is a missense mutation in exon 19 that disrupts a consensus serine/threonine kinase phosphorylation site. We have characterized the cDNA sequences of the mouse, rat and rabbit IKBKAP-encoded mRNAs and determined the genomic organization and chromosomal location of mouse IKBKAP. There is significant homology in the amino acid sequence of IKAP across species and the serine/threonine kinase phosphorylation site altered in the minor FD mutation of IKAP is conserved. The mouse and human IKBKAP genes exhibit significant conservation of their genomic organization and the intron 20 donor splice site sequence, altered in the major FD mutation, is conserved in the human and mouse genes. Mouse IKBKAP is located on the central portion of chromosome 4 and maps to a region in which there is conserved linkage homology between the human and mouse genomes. The homologies observed in the human and mouse sequences should allow, through the process of homologous recombination, for the generation of mice that bear the IKBKAP mutations present in individuals with FD. The characterization of such mice should provide significant information regarding the pathophysiology of FD.     

Structural characterization of chondroitin sulfate from sturgeon bone

Chondroitin sulfate (CS) was purified for the first time from the bones of sturgeon and analyzed to evaluate its structure and properties. A single polysaccharide was extracted from sturgeon bone in a concentration of 0.28-0.34% for dry tissue and characterized as CS. By means of specific chondroitinases and HPLC separation of generated unsaturated repeating disaccharides, this polymer was found to be composed of ∼55% of disaccharide monosulfated in position 6 of the GalNAc, ∼38% of disaccharide monosulfated in position 4 of the GalNAc, and ∼7% of nonsulfated disaccharide. The charge density was 0.93 and the ratio of 4:6 sulfated residues was equal to 0.69, a value confirmed by ¹³C NMR experiments. Chondroitinase B confirmed that the purified sturgeon CS contained mainly GlcA (>99.5%) as uronic acid. PAGE analysis showed a CS having a high molecular mass with an average value of 39,880 according to HPSEC values producing a weight average molecular weight (Mw) of 37,500. On the basis of the data collected, it is reasonable to assume that CS isolated from sturgeon bone might be potentially useful for scientific and pharmacological applications, making this bony fish, which is generally discarded after ovary collection, a useful source of this polymer. Finally, this newly identified source of CS would enable the production of this macromolecule having a particular repeating disaccharide composition, structure, and biological properties.     

Effect of insulin on human skeletal muscle protein synthesis is modulated by insulin-induced changes in muscle blood flow and amino acid availability

Insulin promotes muscle anabolism, but it is still unclear whether it stimulates muscle protein synthesis in humans. We hypothesized that insulin can increase muscle protein synthesis only if it increases muscle amino acid availability. We measured muscle protein and amino acid metabolism using stable-isotope methodologies in 19 young healthy subjects at baseline and during insulin infusion in one leg at low (LD, 0.05), intermediate (ID, 0.15), or high (HD, 0.30 mUxmin(-1)x100 ml(-1)) doses. Insulin was infused locally to induce muscle hyperinsulinemia within the physiological range while minimizing the systemic effects. Protein and amino acid kinetics across the leg were assessed using stable isotopes and muscle biopsies. The LD did not affect phenylalanine delivery to the muscle (-9 +/- 18% change over baseline), muscle protein synthesis (16 +/- 26%), breakdown, or net balance. The ID increased (P < 0.05) phenylalanine delivery (+63 +/- 38%), muscle protein synthesis (+157 +/- 54%), and net protein balance, with no change in breakdown. The HD did not change phenylalanine delivery (+12 +/- 11%) or muscle protein synthesis (+9 +/- 19%), and reduced muscle protein breakdown (-17 +/- 15%), thus improving net muscle protein balance but to a lesser degree than the ID. Changes in muscle protein synthesis were strongly associated with changes in muscle blood flow and phenylalanine delivery and availability. In conclusion, physiological hyperinsulinemia promotes muscle protein synthesis as long as it concomitantly increases muscle blood flow, amino acid delivery and availability.     

The combination of lanthanum chloride and the calcimimetic calindol delays the progression of vascular smooth muscle cells calcification

Phosphate (Pi)-binders are commonly used in dialysis patients to control high Pi levels, that associated with vascular calcification (VC). The aim of this study was to investigate the effects of lanthanum chloride (LaCl(3)) on the progression of high Pi-induced VC, in rat vascular smooth muscle cells (VSMCs). Pi-induced Ca deposition was inhibited by LaCl(3), with a maximal effect at 100μM (59.0±2.5% inhibition). Furthermore, we studied the effects on VC of calcium sensing receptor (CaSR) agonists. Gadolinium chloride, neomycin, spermine, and the calcimimetic calindol significantly inhibited Pi-induced VC (55.9±2.2%, 37.3±4.7%, 30.2±5.7%, and 63.8±5.7%, respectively). To investigate the hypothesis that LaCl(3) reduces the progression of VC by interacting with the CaSR, we performed a concentration-response curve of LaCl(3) in presence of a sub-effective concentration of calindol (10nM). Interestingly, this curve was shifted to the left (IC(50) 9.6±2.6μM), compared to the curve in the presence of LaCl(3) alone (IC(50) 19.0±4.8μM). In conclusion, we demonstrated that lanthanum chloride effectively reduces the progression of high phosphate-induced vascular calcification. In addition, LaCl(3) cooperates with the calcimimetic calindol in decreasing Ca deposition in this in vitro model. These results suggest the potential role of lanthanum in the treatment of VC induced by high Pi.     

Iterative taxonomy reveals a new species of Trichomycterus Valenciennes 1832 (Siluriformes,Trichomycteridae) widespread in Rio Doce basin: a pseudocryptic of T. immaculatus

This paper reports on a new species of Trichomycterus from the Rio Doce basin. Unusually for new taxa in the genus during the past few decades, the new species is not narrowly endemic but instead widely distributed in its major drainage, the Rio Doce. The species has been collected and deposited in scientific collections for some years, but has been systematically misidentified as the more abundant Trichomycterus immaculatus or, to a lesser degree, as other morphologically similar species from south-eastern Brazil such as T. nigricans and T. pradensis. A combination of several morphological characteristics, such as vertebral number, pectoral-fin ray counts, pigmentation pattern and barcoding distance, were iteratively used and unambiguously distinguish the new species from all congeners. The present case reveals a pattern of diversity-discovery in which rare and narrowly endemic morphologically conspicuous species are discovered and described before visually inconspicuous taxa, even when the latter are more abundant and widespread. The morphological similarities among south-eastern Brazilian species with a uniform dark-grey color serve as basis for a brief discussion about the concepts of cryptic and pseudo-cryptic species in Trichomycterus and their consequences for potentially hidden diversity in the genus.     

Common fragile sites in colon cancer cell lines: role of mismatch repair, RAD51 and poly(ADP-ribose) polymerase-1

Common fragile sites (CFS) are specific chromosomal areas prone to form gaps and breaks when cells are exposed to stresses that affect DNA synthesis, such as exposure to aphidicolin (APC), an inhibitor of DNA polymerases. The APC-induced DNA damage is repaired primarily by homologous recombination (HR), and RAD51, one of the key players in HR, participates to CFS stability. Since another DNA repair pathway, the mismatch repair (MMR), is known to control HR, we examined the influence of both the MMR and HR DNA repair pathways on the extent of chromosomal damage and distribution of CFS provoked by APC and/or by RAD51 silencing in MMR-deficient and -proficient colon cancer cell lines (i.e., HCT-15 and HCT-15 transfected with hMSH6, or HCT-116 and HCT-116/3+6, in which a part of a chromosome 3 containing the wild-type hMLH1 allele was inserted). Here, we show that MMR-deficient cells are more sensitive to APC-induced chromosomal damage particularly at the CFS as compared to MMR-proficient cells, indicating an involvement of MMR in the control of CFS stability. The most expressed CFS is FRA16D in 16q23, an area containing the tumour suppressor gene WWOX often mutated in colon cancer. We also show that silencing of RAD51 provokes a higher number of breaks in MMR-proficient cells with respect to their MMR-deficient counterparts, likely as a consequence of the combined inhibitory effects of RAD51 silencing on HR and MMR-mediated suppression of HR. The RAD51 silencing causes a broader distribution of breaks at CFS than that observed with APC. Treatment with APC of RAD51-silenced cells further increases DNA breaks in MMR-proficient cells. The RNAi-mediated silencing of PARP-1 does not cause chromosomal breaks or affect the expression/distribution of CFS induced by APC. Our results indicate that MMR modulates colon cancer sensitivity to chromosomal breaks and CFS induced by APC and RAD51 silencing.