Xanthine oxido-reductase activity in ischemic human and rat intestine

We measured time course and extent of xanthine dehydrogenase (XD) to xanthine oxidase (XO) conversion in ischemic human and rat intestine. To model normothermic no-flow ischemia, we incubated fresh biopsies for 0, 2, 4, 8 and 16 h. At [Formula: See Text] XO was less in humans than in rats [Formula: See Text] while XD was essentially the same [Formula: See Text] After 16 h incubation at 37°C, there was no appreciable XD-to-XO conversion and no change in neither XO nor XD activity in human intestine. In contrast, the rat intestine had [Formula: See Text] ratio doubled in the first 2 h and then maintained that value until [Formula: See Text] In conclusion, no XO-to-XD conversion was appreciable after 16 h no-flow normothermic ischemia in human intestine; in contrast, XO activity in rats increased sharply after the onset of ischemia. An immunohistochemical labelling study shows that, whereas [Formula: See Text] expression in liver tissue is localised in both hepatocytes and endothelial cells, in the intestine that expression is mostly localised in epithelial cells. We conclude that XO may be considered as a major source of reactive oxygen species in rats but not in humans.     

ENTH/ANTH domains expand to the Golgi

Clathrin-coated vesicles (CCVs) play important roles in nutrient uptake, downregulation of signaling receptors, pathogen invasion and biogenesis of endosomes and lysosomes. Although detailed models for endocytic CCV formation have emerged, the process of CCV formation at the Golgi and endosomes has been less clear. Key to endocytic CCV formation are proteins containing related phosphoinositide-binding ENTH and ANTH domains. Now, recent studies have identified novel ENTH/ANTH proteins that participate in CCV-mediated traffic between the trans-Golgi Network (TGN) and endosomes and have defined a molecular basis for interaction with AP-1 and GGA adaptors in clathrin coats of the TGN/endosomes. Thus, ENTH/ANTH domain proteins appear to be universal elements in nucleation of clathrin coats.     

Pharmacological blockade of mGlu2/3 metabotropic glutamate receptors reduces cell proliferation in cultured human glioma cells

Glial cell proliferation in culture is under the control of metabotropic glutamate (mGlu) receptors. We have examined whether this control extends to human glioma cells. Primary cultures were prepared from surgically removed human glioblastomas. RT-PCR combined with western blot analysis showed that most of the cultures (eight out of 11) expressed group-II mGlu receptors. In two selected cultures (MZC-12 and FCN-9), the mGlu2/3 receptor antagonist, LY341495, slowed cell proliferation when applied to the growth medium from the second day after plating. This effect was reversible because linear cell growth was restored after washing out the drug. LY341495 reduced glioma cell proliferation at concentrations lower than 100 nm, which are considered as selective for mGlu2/3 receptors. In addition, its action was mimicked by the putative mGlu2/3 receptor antagonist (2S)-alpha-ethylglutamate. The anti-proliferative effect of LY341495 was confirmed by measuring [methyl-3H]-thymidine incorporation in cultures arrested in G0 phase of the cell cycle and then stimulated to proliferate by the addition of 10% fetal calf serum or 100 ng/mL of epidermal growth factor (EGF). In cultures treated with EGF, LY341495 was also able to reduce the stimulation of the mitogen-activated protein kinase (MAPK) pathway, as well as the induction of cyclin D1. Both effects, as well as decreased [methyl-3H]-thymidine incorporation, were partially reduced by co-addition of the potent mGlu2/3 receptor agonist, LY379268. We conclude that activation of group-II mGlu receptors supports the growth of human glioma cells in culture and that antagonists of these receptors should be tested for their ability to reduce tumour growth in vivo.     

Low levels of STRP variability are not universal in American Indians

Data related to 15 short tandem repeat polymorphisms (STRPs) are reported for five Brazilian Indian populations, and a set of them compared with results previously reported for Asian, neo-Brazilian, North American, Iberian, and African populations. The low variability observed for these markers among the Suruí Indians is confirmed, but the other populations show variability levels that are similar to those found elsewhere. Previous suggestions of population bottlenecks in the prehistorical colonization of the New World were not confirmed. On the other hand, STRPs again showed to be good markers for the establishment of population relationships.     

Characterization of Paracoccidioides brasiliensis atypical isolates by random amplified polymorphic DNA analysis

Two atypical Paracoccidioides brasiliensis strains (yeast form at room temperature) have been isolated from chronically infected patients living in Brazil. Different random primers were used to characterize these isolates and compare them to typical strains. The RAPD patterns allowed the differentiation of all the selected isolates. Their genetic distance ranged from 5% to 80% of non-shared bands depending on the strains and the primer used. The RAPD data were used to build a Wagner phenogram, which showed two major branched with more than 56% of genetic distance separating them. No significant difference was observed between the atypical isolates and the others suggesting that specific genes are involved in the dimorphism phenomenon.     

Noninvasive imaging of peripherally injected Alzheimer's disease type synthetic A beta amyloid in vivo

The pathological hallmark of Alzheimer's disease (AD) is accumulation in the brain of amyloid composed of the 40-mer peptide A beta. Many fundamental questions about the biology of (AD) remain unanswered because there is currently no method of quantifying A beta amyloid in vivo. A noninvasive method of detecting and quantifying A beta amyloid in vivo would have wide application for the premortem diagnosis of AD and the efficient evaluation of candidate therapeutics aimed at inhibiting the formation and growth of A beta amyloid. Taking advantage of the extraordinarily high affinity of A beta for itself, we have synthesized an N'-terminal diethylenetriaminepentaacetic acid (DTPA) derivative of A beta possessing the kinetic activity and specificity for A beta amyloid desired of a probe to be used for noninvasive imaging. DTPA-A beta(3-40) is readily labeled with (111)InOAc(3) to yield a stable probe with exquisite specificity for naturally occurring and synthetic A beta amyloid in vitro. Moreover, (111)In-DTPA-A beta(3-40), administered intravascularly can specifically deposit onto and label previously injected synthetic A beta amyloid and be imaged in vivo with a gamma camera. The present results demonstrate the design, synthesis, and use of an A beta amyloid-specific probe and methods for its use as a noninvasive imaging agent. In vivo imaging of A beta amyloid represents an important step toward the development of biochemically based objective tools for the assessment of progression of AD and efficacy of potential therapeutics.     

Trypanosoma cruzi heat-shock protein-70 kDa,alone or fused to the parasite KMP11 antigen,induces functional maturation of murine dendritic cells

We analyse the effect of Trypanosoma cruzi heat-shock protein-70 (HSP70) on the maturation of murine dendritic cells (DC)generated from bone marrow precursor cells. The results obtained show that HSP70, both alone and fused to the KMP11 antigen, as well as a HSP70 fragment, is capable of maturing murine DC. Mature DC have enhanced expression of IL12, TNF-alpha cytokines, costimulation molecules and activation markers, showing a clear increase in the allostimulatory capacity. These findings suggest that T. cruzi HSP70 may be a very useful vehicle for developing DC-based immunoprophylaxis and therapy against infections.     

Melusin,a muscle-specific integrin beta1-interacting protein,is required to prevent cardiac failure in response to chronic pressure overload

Cardiac hypertrophy is an adaptive response to a variety of mechanical and hormonal stimuli, and represents an early event in the clinical course leading to heart failure. By gene inactivation, we demonstrate here a crucial role of melusin, a muscle-specific protein that interacts with the integrin beta1 cytoplasmic domain, in the hypertrophic response to mechanical overload. Melusin-null mice showed normal cardiac structure and function in physiological conditions, but when subjected to pressure overload--a condition that induces a hypertrophic response in wild-type controls--they developed an abnormal cardiac remodeling that evolved into dilated cardiomyopathy and contractile dysfunction. In contrast, the hypertrophic response was identical in wild-type and melusin-null mice after chronic administration of angiotensin II or phenylephrine at doses that do not increase blood pressure--that is, in the absence of cardiac biomechanical stress. Analysis of intracellular signaling events induced by pressure overload indicated that phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) was specifically blunted in melusin-null hearts. Thus, melusin prevents cardiac dilation during chronic pressure overload by specifically sensing mechanical stress.     

Association of the low-density lipoprotein receptor gene with obesity in Native American populations

Five low-density lipoprotein receptor gene (LDLR) restriction fragment length polymorphisms (RFLPs: TaqI, intron 4; HincII, exon 12; AvaII, exon 13; MspI and NcoI, exon 18) were investigated in 131 individuals from five Brazilian Indian tribes. All markers were polymorphic in this ethnic group. In the whole sample of Amerindians, 13 (41%) of the 32 expected haplotypes were identified, but only three were shared by all tribes. The Xavante, Suruí, Zoró, and Gavi?o tribes, who had been studied for anthropometry, were grouped according to their genotypes, and the corresponding mean values were examined. Significant associations were observed between HincII *H-, AvaII *A+, MspI *M-, and NcoI *N+ and the body mass index (BMI), triceps and subscapular skinfolds, and the arm fat index (AFI). Haplotypes were derived for these four RFLPs, and (*H-/*A+/*M-/*N+) haplotype carriers were compared with noncarriers of this haplotype with equally significant results for the three parameters (BMI, P=0.021; skinfold thickness, P<0.001; AFI, P=0.005). These results suggest that the LDLR gene has some influence over adipose tissue deposition.