Using ESTs database to predict and validate single polymorphisms at the HLA system.

We propose a bioinformatics pipeline in which we use an ESTs database to predict and validate single-nucleotide polymorphisms (SNPs) directly linked to gene-coding regions at the HLA class I genes (HLA-A, HLA-B and HLA-C). Annotation originated from our analysis revealed various classes of possible new variations that may indicate possible new alleles. Thus, bioinformatics pipelines seem to be useful approaches to help screening for novel genetic variations at the HLA panel, and further analysis will foster this aim to provide celerity at the massive analysis of data currently generated in large-scale high-throughput experiments.

Relationship between ischemia/reperfusion injury and the stimulus of fibrogenesis in an experimental model: comparison among different preservation solutions.

BACKGROUND AND AIMS: Orthotopic liver transplantation (OLT) has been the standard treatment for end-stage acute and chronic liver disease. Ischemia-reperfusion (I/R) injury is one of the major causes of poor graft function early after OLT, and adversely influencing graft and patient survivals. It is unknown whether I/R injury influences liver fibrogenesis. MATERIALS AND METHODS: Livers from 25 adult male Wistar rats were randomly assigned into 5 experimental groups according to the preservation solution: saline solution (SS); University of Wisconsin (UW) solution; Fructose 1, 6-biphosphate (FBP); S-Nitroso-N-Acetylcysteine (SNAC): or UW+SNAC (SNAC+UW). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic dehydrogenase (LDH) were determined in preservation solution samples at 2, 4, and 6 hours. After 6 hours of cold ischemia, ex situ reperfusion was applied to the liver for 15 minutes. Serum AST, ALT, LDH, and renin levels were determined. Fresh liver slices were processed for histological studies, determination of thiobarbituric acid reactive substances, catalase, and glutathione, and expression of TGF-ß1 and angiotensin II AT1 receptor. RESULTS: AST was significantly lower during cold storage with UW than with the older media (P=.001); ALT was lower in the FBP group (P=.023) and LDH was lower in the FBP and SNAC groups (P=.007). After reperfusion, serum AST, ALT, LDH, and TBARS showed no significant differences among the groups. Catalase was significantly lower in the SS and FBP groups (P=.008 and P=.006, respectively). Compared with UW, glutathione concentrations were significantly higher in SS, FBP, and SNAC 200 (P=.004). Renin levels were significantly lower in the FBP group (P=.022). No histological signs of preservation injury were observed in the hepatic sample. No expressions were detected of TGF-ß1 or AT1 receptor. CONCLUSION: In this experimental model of early reperfusion injury, preservation changes related to higher levels of renin, which suggest its role in fibrogenesis. FBP was associated with lower renin levels than other solutions including UW.

Fructose 1-6 bisphosphate versus University of Wisconsin solution for rat liver preservation: does FBP prevent early mitochondrial injury?

BACKGROUND: Fructose 1,6-biphosphate (FBP) has been shown to exert therapeutic effects in models of ischemia-reperfusion in organs other than the liver. This study compared FBP and University of Wisconsin (UW) solution during cold storage and reperfusion, among mitochondria of adult male Wistar rat livers. METHODS: Adult male Wistar rats were assigned to two groups according to the preservation solution used; UW or FBP Aspartate transaminase (AST), alanine transferase (ALT); and lactic dehydrogenase (LDH) were measured in samples of the storage solution obtained at 2, 4 and 6 hours of preservation. After 6 hours of cold storage, we reperfused the liver, taking blood samples to measure AST, ALT, LDH, and throbarbituric acid reactive substances (TBARS). Hepatic fragments were processed for histologic analysis; for determinations of TBARS, catalase, and nitric oxide as well as for mitochondrial evaluation by infrared spectroscopy. RESULTS: During cold preservation, levels of AST and LDH in the storage solution were lower among the FBP group, but after reperfusion, serum levels of AST, ALT, and LDH were higher in this group, as was catalase activity. TBARS and nitric oxide were comparable between the groups. In the UW group there was a higher amide I/amide II ratio than in the FBP group, suggesting an abnormal protein structure of the mitochondrial membrane. No signs of preservation injury were observed in any liver biopsy, but sinusoidal congestion was present in livers preserved with FBP. CONCLUSION: FBP showed a protective effect for preservation during cold storage seeming to protect the mitochondrial membrane although it did not prevent reperfusion injury.

Fructose-1,6-bisphosphate reduces inflammatory pain-like behaviour in mice: role of adenosine acting on A1 receptors.

BACKGROUND AND PURPOSE: D-Fructose-1,6-bisphosphate (FBP) is an intermediate in the glycolytic pathway, exerting pharmacological actions on inflammation by inhibiting cytokine production or interfering with adenosine production. Here, the possible antinociceptive effect of FBP and its mechanism of action in the carrageenin paw inflammation model in mice were addressed, focusing on the two mechanisms described above. EXPERIMENTAL APPROACH: Mechanical hyperalgesia (decrease in the nociceptive threshold) was evaluated by the electronic pressure-metre test; cytokine levels were measured by elisa and adenosine was determined by high performance liquid chromatography. KEY RESULTS: Pretreatment of mice with FBP reduced hyperalgesia induced by intraplantar injection of carrageenin (up to 54%), tumour necrosis factor alpha (40%), interleukin-1 beta (46%), CXCL1 (33%), prostaglandin E(2) (41%) or dopamine (55%). However, FBP treatment did not alter carrageenin-induced cytokine (tumour necrosis factor alpha and interleukin-1 beta) or chemokine (CXCL1) production. On the other hand, the antinociceptive effect of FBP was prevented by systemic and intraplantar treatment with an adenosine A(1) receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine), suggesting that the FBP effect is mediated by peripheral adenosine acting on A(1) receptors. Giving FBP to mice increased adenosine levels in plasma, and adenosine treatment of paw inflammation presented a similar antinociceptive mechanism to that of FBP. CONCLUSIONS AND IMPLICATIONS: In addition to anti-inflammatory action, FBP also presents an antinociceptive effect upon inflammatory hyperalgesia. Its mechanism of action seems dependent on adenosine production but not on modulation of hyperalgesic cytokine/chemokine production. In turn, adenosine acts peripherally on its A(1) receptor inhibiting hyperalgesia. FBP may have possible therapeutic applications in reducing inflammatory pain.

Hepatic reperfusion in rats: a new model with portal arterialization in studying early ischemia-reperfusion injury.

Isolated liver perfusion has been used to evaluate the beneficial effects of several agents. In the present study, we developed a model using a recipient rat to reperfuse harvested livers in an ex situ, in vivo recirculating system. A total of 25 reperfusion procedures using adult male Wistar rats as donors and recipients were done. The preservation of the livers was performed with University of Wisconsin solution for 6 hours. Thereafter, the liver was reperfused with blood from another rat. We believe that the model presented herein offers an alternative method to evaluate early hepatocellular damage or hepatic microcirculation.

Analyses of the temperature and pH effects on the complexation of magnesium and calcium in human blood plasma: an approach using artificial neural networks.

The temperature and pH effects on the equilibrium of a blood plasma model have been studied on the basis of artificial neural networks. The proposed blood plasma was modeled considering two important metals, calcium and magnesium, and six ligands, namely, alanate, carbonate, citrate, glycinate, histidinate and succinate. A large data set has been used to simulate different concentrations of magnesium and calcium as a function of temperature and pH and these data were used for training the neural network. The proposed model allowed different types of analyses, such as the effects of pH on calcium and magnesium concentrations, the competition between calcium and magnesium for ligands and the effects of temperature on calcium and magnesium concentrations. The model developed was also used to predict how the variation of calcium concentration can affect magnesium concentrations. A comparison of neural network predictions against experimental data produced errors of about 3%. Moreover, in agreement with experimental measurements (Wang et al. in Arch. Pathol. 126:947-950, 2002; Heining et al. in Scand. J. Clin. Lab. Invest. 43:709-714, 1983), the artificial neural network predicted that calcium and magnesium concentrations decrease when pH increases. Similarly, the magnesium concentrations are less sensitive than calcium concentrations to pH changes. It is also found that both calcium and magnesium concentrations decrease when the temperature increases. Finally, the theoretical model also predicted that an increase of calcium concentrations will lead to an increase of magnesium concentration almost at the same rate. These results suggest that artificial neural networks can be efficiently applied as a complementary tool for studying metal ion complexation, with especial attention to the blood plasma analysis.

Effect of fructose-1,6-bisphosphate in the cold storage solution after 12 and 36 hours of rat liver preservation.

Fructose-1,6-bisphosphate (FBP) has been reported to have a protective effect on liver injury following ischemic/reperfusion periods because it maintains ATP levels during cold preservation. In the present study, we evaluated the effects of addition of FBP to storage solutions for cold liver preservation during 12 or 36 hours. Adult male Wistar rats were randomly divided into three experimental groups. The hepatic perfusion and preservation were performed with these solutions: UW; UW plus 10 mmol/L FBP; and FBP 10 mmol/L (FBPS) alone. The biochemical measurements of AST and ALT were performed on samples of the cold storage solution after 12- or 36-hour preservation. UW and FBPS solutions showed similar preservation grades at 12 hours. Addition of 10 mmol/L of FBP to UW solution induced liver injury and a poor preservation grade during 12 or 36 hours. UW solution was better than FBPS after 36 hours preservation. UW solution continues to offer a superior performance for liver preservation during long times; however, FBPS may be an alternative for short cold preservation times.

Protective effect of fructose-1,6-bisphosphate in the cold storage solution for liver preservation in rat hepatic transplantation.

Fructose-1,6-bisphosphate (FBP) has been reported to have a protective effect on liver injury following ischemic/reperfusion periods. FBP maintains ATP levels and thereby cellular energy metabolism, which is important to the liver during cold preservation. In the present study, we evaluated the effects of FBP on the composition of storage solutions for cold liver preservation. Adult male Wistar rats were randomly divided into three experimental groups. Hepatic perfusion and preservation were performed with UW, UW plus 10 mmol/L FBP (UWM), and FBP 10 mmol/L (FBPS) alone solutions. Biochemical measurements of AST, ALT, and TBARS were performed on samples of the cold storage solution at 0, 12, 18, and 24 hours preservation. FBPS and UW solutions showed similar preservation grades during 18 hours. Addition of 10 mmol/L of FBP to UW solution induced liver injury and a poor preservation grade. FBP appears to protect the liver from injury caused by free radicals when the preservation time is less than 18 hours. Therefore, FBP may exert a protective effect for the preservation of livers during cold storage, and could represent an important component of new cold storage solutions.

Analysis of a novel functional polymorphism within the promoter region of the serotonin transporter gene (5-HTT) in Brazilian patients affected by bipolar disorder and schizophrenia.

It has been suggested that the serotonin transporter (5-hydroxytryptamine-transporter or 5-HTT) may be involved in the pathogenesis of affective disorders. Recently, Collier et al. (1996) found that the frequency of the low-activity short variant (s) of the 5-HTT-linked polymorphic region (5-HTTLPR) was higher among patients with affective disorders than in normal controls. However, since the observed level of significance was not high, they suggest that these findings should be replicated in independent samples. We have analyzed 86 unrelated patients (47 with bipolar disorder and 39 with schizophrenia) and 98 normal controls from the Brazilian population for the 5-HTTLPR. Statistical analysis revealed that the genotypes (LL, Ls, ss) as well as the estimated allele frequencies (L,s) did not differ significantly among the three studied groups or between bipolar and normal controls. In addition, although not statistically significant, the genotype ss in our sample was less frequent among our bipolar patients than in our normal controls (12.8% versus 16.3%) which is the opposite of what was found by Collier et al. (24% versus 18%) in the European study. Although it will be important to extend the present analysis in a larger sample, our preliminary results suggest that the 5-HTTLPR does not seem to play a major role in the genetics of bipolar and schizophrenic disorders at least in this group of Brazilian psychiatric patients.

Fructose 1,6-bisphosphate protects against D-galactosamine toxicity in isolated rat hepatocytes.

Incubation of hepatocytes with D-galactosamine (GalN) produced a dose-dependent alteration in cell viability and a fall in ATP and fructose 2,6-bisphosphate (Fru-2,6-P2) levels. The reduction in Fru-2,6-P2 can be explained by changes in the substrates or modulators of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase, because neither the adenosine 3',5'-cyclic monophosphate level nor the activity ratio of the enzyme was modified. Microcalorimetric measurements showed that GalN produced an exothermic peak followed by a progressive decrease in heat dissipation. Simultaneous administration of GalN and fructose 1,6-bisphosphate (Fru-1,6-P2) significantly increased cell viability, and concentrations of ATP and Fru-2,6-P2 and led to stable heat production. In the presence of Fru-1,6-P2 alone, hepatocytes kept ATP and Fru-2,6-P2 levels constant, whereas they increased the oxygen uptake-to-heat output ratio. Our results suggest that GalN initiates the hepatotoxic effect by means of an energy-dissipating interaction, produced before its metabolism and presumably at the membrane level, whereas Fru-1,6-P2 protects the cells against this injury in a way that prevents the initial interaction and increases the metabolic efficiency of the cell.

HPLC analysis of hexosamine phosphates in biological samples.

Galactosamine is quickly metabolized to galactosamine 1-phosphate in rats treated with this compound. An HPLC method to quantify hexosamine phosphates in biological samples is described, modified from the o-phthaldialdehyde amino acid analysis procedure. o-Phthaldialdehyde derivatives of hexosamines and hexosamine-phosphates can be eluted from a reverse-phase column at different retention times, with a total analysis time of 30 min and without overlapping with free amino acids at physiological concentrations. The standard curves are linear between 1 and 40 nmol. This simple method is more selective and sensitive than previous enzymatic analyses of hexosamine phosphorylation.

Effect of galactosamine on hepatic carbohydrate metabolism: protective role of fructose 1,6-bisphosphate.

Intraperitoneal administration of galactosamine (400 mg/kg body wt) to rats results in reversible liver cell injury that is related to a dose-dependent depletion of uridine phosphates by formation of UDP-sugar derivatives. This damage was monitored through changes in serum enzymatic activities that increased after the first 6 hr of drug administration. Glycemia and serum albumin remained stable during liver injury, whereas cholesterol and triglycerides decreased. To maintain plasma glucose concentration, the hepatic carbohydrate metabolism was greatly altered. Glycogen dropped during the first hours, remaining low for up to 48 hr. Fructose 2,6-bisphosphate and ATP levels decreased even faster than glycogen, with lactate following a similar diminution and being restored in parallel with both metabolites. The reduction in fructose 2,6-bisphosphate can be explained by changes in the substrates or modulators of the 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase, because neither the cyclic AMP levels nor the activity ratio of the enzyme were modified. Simultaneous administration of galactosamine and fructose 1,6-bisphosphate (2 gm/kg) prevented liver cell death, as monitored by serum enzyme activities. Furthermore, the bisphosphorylated metabolite had protective effects on the changes in liver calcium content and ATP and fructose 2,6-bisphosphate concentrations. In contrast, fructose, fructose-1-phosphate and fructose-6-phosphate had no significant protection. Fructose 1,6-bisphosphate might decrease galactosamine toxicity by increasing fructose 2,6-bisphosphate and ATP levels, the changes in both metabolites probably being related. The significance of these findings with respect to the mechanism of galactosamine-induced liver injury is also discussed.

[Wilm's tumor in children: review of 38 cases]. / Tumor de Wilms' na criança: revisão de 38 casos.

Analysis of the incidence of the Nephroblastoma and of its clinic, echographic and radiologic aspects. Review of 38 cases handled in the Pediatric Surgery Service of Santa Maria Hospital between January 1960 and January 1987. In addition to the distribution by age and stadiation, the results were studied in 3 groupings corresponding to successive therapeutic phases along this period. The survival is more favourable in the etary group under 3 years old and does not differ substantially in stages I and II. In the analysis of the prognostic to the therapeutic sequences, there was an evident improvement of survival from 0% before 1966 (Surgery and Radiotherapy) to 46.15% in the second phase, 1966-76, with the introduction of Chemotherapy (Actinomycina D). In the last phase after 1977, using the therapeutic scheme of National Wilms Tumour Study (NWTS) survival reached 80.95%. Trying to correlate the prognostic with the histologic type, it turns out to be difficult to make a criterious characterization in the cases submitted to pre-operative Radio and/or Chemotherapy. The advantage of this therapeutic attitude in parallel with the faulaification of the histologic type and the Tumour stadiation is hereby discussed.