The calcium-modulated proteins, S100A1 and S100B, as potential regulators of the dynamics of type III intermediate filaments

The Ca2+-modulated, dimeric proteins of the EF-hand (helix-loop-helix) type, S100A1 and S100B, that have been shown to inhibit microtubule (MT) protein assembly and to promote MT disassembly, interact with the type III intermediate filament (IF) subunits, desmin and glial fibrillary acidic protein (GFAP), with a stoichiometry of 2 mol of IF subunit/mol of S100A1 or S100B dimer and an affinity of 0.5-1.0 µM in the presence of a few micromolar concentrations of Ca2+. Binding of S100A1 and S100B results in inhibition of desmin and GFAP assemblies into IFs and stimulation of the disassembly of preformed desmin and GFAP IFs. S100A1 and S100B interact with a stretch of residues in the N-terminal (head) domain of desmin and GFAP, thereby blocking the head-to-tail process of IF elongation. The C-terminal extension of S100A1 (and, likely, S100B) represents a critical part of the site that recognizes desmin and GFAP. S100B is localized to IFs within cells, suggesting that it might have a role in remodeling IFs upon elevation of cytosolic Ca2+ concentration by avoiding excess IF assembly and/or promoting IF disassembly in vivo. S100A1, that is not localized to IFs, might also play a role in the regulation of IF dynamics by binding to and sequestering unassembled IF subunits. Together, these observations suggest that S100A1 and S100B may be regarded as Ca2+-dependent regulators of the state of assembly of two important elements of the cytoskeleton, IFs and MTs, and, potentially, of MT- and IF-based activities.

Frequency of islet cell autoantibodies (IA-2 and GAD) in young Brazilian type 1 diabetes patients

Type 1 diabetes, as an autoimmune disease, presents several islet cell-specific autoantibodies such as islet cell antibody (ICA), anti-insulin, anti-glutamic acid decarboxylase (GAD) and the antibody (Ab) against tyrosine phosphatase (PTP)-like protein known as ICA-512 (IA-2). In order to determine the frequency of the anti-GAD and anti-IA-2 autoantibodies in Brazilian type 1 diabetes patients we studied 35 diabetes mellitus (DM) type 1 patients with recent-onset disease (£12 months) and 37 type 1 diabetes patients with long-duration diabetes (>12 months) who were compared to 12 children with normal fasting glucose. Anti-GAD65 and anti-IA-2 autoantibodies were detected with commercial immunoprecipitation assays. The frequency of positive results in recent-onset DM type 1 patients was 80.0% for GADAb, 62.9% for IA-2Ab and 82.9% for GADAb and/or IA-2Ab. The long-duration type 1 diabetes subjects presented frequencies of 54.1% for GADAb and IA-2Ab, and 67.5% for GAD and/or IA-2 antibodies. The control group showed no positive cases. Anti-GAD and IA-2 assays showed a high frequency of positivity in these Brazilian type 1 diabetes patients, who presented the same prevalence as a Caucasian population.

Transforming growth factor beta activity in urine of patients with type 2 diabetes and diabetic nephropathy

Diabetic nephropathy (DN) is characterized structurally by progressive mesangial deposition of extracellular matrix (ECM). Transforming growth factor-ß (TGF-ß) is considered to be one of the major cytokines involved in the regulation of ECM synthesis and degradation. Several studies suggest that an increase in urinary TGF-ß levels may reflect an enhanced production of this polypeptide by the kidney cells. We evaluated TGF-ß in occasional urine samples from 14 normal individuals and 23 patients with type 2 diabetes (13 with persistent proteinuria >500 mg/24 h, DN, 6 with microalbuminuria, DMMA, and 4 with normal urinary albumin excretion, DMN) by enzyme immunoassay. An increase in the rate of urinary TGF-ß excretion (pg/mg UCreat.) was observed in patients with DN (296.07 ± 330.77) (P<0.001) compared to normal individuals (17.04 ± 18.56) (Kruskal-Wallis nonparametric analysis of variance); however, this increase was not observed in patients with DMMA (25.13 ± 11.30) or in DMN (18.16 ± 11.82). There was a positive correlation between the rate of urinary TGF-ß excretion and proteinuria (r = 0.70, a = 0.05) (Pearson's analysis), one of the parameters of disease progression.

Residual ß-cell function and microvascular complications in type 1 diabetic patients

To determine the influence of residual ß-cell function on retinopathy and microalbuminuria we measured basal C-peptide in 50 type 1 diabetic outpatients aged 24.96 ± 7.14 years, with a duration of diabetes of 9.1 ± 6.2 years. Forty-three patients (86%) with low C-peptide (<0.74 ng/ml) had longer duration of diabetes than 7 patients (14%) with high C-peptide (³0.74 ng/ml) (9 (2-34) vs 3 (1-10) years, P = 0.01) and a tendency to high glycated hemoglobin (HBA1) (8.8 (6-17.9) vs 7.7 (6.9-8.7)%, P = 0.08). Nine patients (18%) had microalbuminuria (two out of three overnight urine samples with an albumin excretion rate (AER) ³20 and <200 µg/min) and 13 (26%) had background retinopathy. No association was found between low C-peptide, microalbuminuria and retinopathy and no difference in basal C-peptide was observed between microalbuminuric and normoalbuminuric patients (0.4 ± 0.5 vs 0.19 ± 0.22 ng/ml, P = 0.61) and between patients with or without retinopathy (0.4 ± 0.6 vs 0.2 ± 0.3 ng/ml, P = 0.43). Multiple regression analysis showed that duration of diabetes (r = 0.30, r2 = 0.09, P = 0.031) followed by HBA1 (r = 0.41, r2 = 0.17, P = 0.01) influenced basal C-peptide, and this duration of diabetes was the only variable affecting AER (r = 0.40, r2 = 0.16, P = 0.004). In our sample of type 1 diabetic patients residual ß-cell function was not associated with microalbuminuria or retinopathy.

The pathogenesis of tropical spastic paraparesis/human T-cell leukemia type I-associated myelopathy

Tropical spastic paraparesis/human T-cell leukemia type I-associated myelopathy (TSP/HAM) is caused by a human T-cell leukemia virus type I (HTLV-I) after a long incubation period. TSP/HAM is characterized by a chronic progressive paraparesis with sphincter disturbances, no/mild sensory loss, the absence of spinal cord compression and seropositivity for HTLV-I antibodies. The pathogenesis of this entity is not completely known and involves a multivariable phenomenon of immune system activation against the presence of HTLV-I antigens, leading to an inflammatory process and demyelination, mainly in the thoracic spinal cord. The current hypothesis about the pathogenesis of TSP/HAM is: 1) presence of HTLV-I antigens in the lumbar spinal cord, noted by an increased DNA HTLV-I load; 2) CTL either with their lytic functions or release/production of soluble factors, such as CC-chemokines, cytokines, and adhesion molecules; 3) the presence of Tax gene expression that activates T-cell proliferation or induces an inflammatory process in the spinal cord; 4) the presence of B cells with neutralizing antibody production, or complement activation by an immune complex phenomenon, and 5) lower IL-2 and IFN-gamma production and increased IL-10, indicating drive to a cytokine type 2 pattern in the TSP/HAM subjects and the existence of a genetic background such as some HLA haplotypes. All of these factors should be implicated in TSP/HAM and further studies are necessary to investigate their role in the development of TSP/HAM.

Gliclazide and bedtime insulin are more efficient than insulin alone for type 2 diabetic patients with sulfonylurea secondary failure

To determine the effects of combined therapy of gliclazide and bedtime insulin on glycemic control and C-peptide secretion, we studied 25 patients with type 2 diabetes and sulfonylurea secondary failure, aged 56.8 ± 8.3 years, with a duration of diabetes of 10.6 ± 6.6 years, fasting plasma glucose of 277.3 ± 64.6 mg/dl and a body mass index of 27.4 ± 4.8 kg/m². Patients were submitted to three therapeutic regimens lasting 2 months each: 320 mg gliclazide (phase 1), 320 mg gliclazide and bedtime NPH insulin (phase 2), and insulin (phase 3). At the end of each period, glycemic and C-peptide curves in response to a mixed meal were determined. During combined therapy, there was a decrease in all glycemic curve values (P<0.01). Twelve patients (48%) reached fasting plasma glucose <140 mg/dl with a significant weight gain of 64.8 kg (43.1-98.8) vs 66.7 kg (42.8-101.4) (P<0.05), with no increase in C-peptide secretion or decrease in HbA1. C-Peptide glucose score (C-peptide/glucose x 100) increased from 0.9 (0.2-2.1) to 1.3 (0.2-4.7) during combined therapy (P<0.01). Despite a 50% increase in insulin doses in phase 3 (12 U (9-30) vs 18 U (11-60); P<0.01) only 3 patients who responded to combined therapy maintained fasting plasma glucose <140 mg/dl (P<0.02). A tendency to a higher absolute increase in C-peptide (0.99 (0.15-2.5) vs 0.6 (0-2.15); P = 0.08) and C-peptide incremental area (2.47 (0.22-6.2) vs 1.2 (0-3.35); P = 0.07) was observed among responders. We conclude that combined therapy resulted in a better glucose response to a mixed meal than insulin alone and should be tried in type 2 diabetic patients before starting insulin monotherapy, despite difficulties in predicting the response.

Etofibrate but not controlled-release niacin decreases LDL cholesterol and lipoprotein (a) in type IIb dyslipidemic subjects

Etofibrate is a hybrid drug which combines niacin with clofibrate. After contact with plasma hydrolases, both constituents are gradually released in a controlled-release manner. In this study, we compared the effects of etofibrate and controlled-release niacin on lipid profile and plasma lipoprotein (a) (Lp(a)) levels of patients with triglyceride levels of 200 to 400 mg/dl, total cholesterol above 240 mg/dl and Lp(a) above 40 mg/dl. These patients were randomly assigned to a double-blind 16-week treatment period with etofibrate (500 mg twice daily, N = 14) or niacin (500 mg twice daily, N = 11). In both treatment groups total cholesterol, VLDL cholesterol and triglycerides were equally reduced and high-density lipoprotein cholesterol was increased. Etofibrate, but not niacin, reduced Lp(a) by 26% and low-density lipoprotein (LDL) cholesterol by 23%. The hybrid compound etofibrate produced a more effective reduction in plasma LDL cholesterol and Lp(a) levels than controlled-release niacin in type IIb dyslipidemic subjects.

Role of Tamm-Horsfall protein in the binding and in vivo phagocytosis of type 1 fimbriated Escherichia coli by mouse peritoneal macrophages

Tamm-Horsfall glycoprotein (THP) contains manno-oligosaccharides that are recognized by type 1 fimbriae (F1) of Escherichia coli. In the present study, we examined the in vivo phagocytic activity of mouse peritoneal macrophages after treatment of bacteria with THP. At low THP concentrations (12.5 µg/ml and 50 µg/ml) no significant difference was observed in the phagocytosis of E. coli F1+. However, at high THP concentrations (500 µg/ml and 1250 µg/ml) we obtained a reduction of bacterial phagocytosis by mouse peritoneal macrophages.

Comparison of methods for urinary albumin determination in patients with type 1 diabetes

We tested the correlation of the albumin-to-creatinine ratio (A/C) in an early-morning urine sample, measured with a commercial kit (DCA 2000®), with the conventional immunoturbidimetric determination in the laboratory and with overnight albumin excretion rate (reference method). Fifty-five type 1 diabetic adolescents had their first-morning urine collected on the 1st and 8th day of the period. Urinary albumin and creatinine were determined immediately using the DCA 2000® kit. Samples were also stored for laboratory analysis. To evaluate the correlation between early-morning urinary A/C ratio and overnight albumin excretion rate, 16 subjects had a timed overnight urine collection. A/C ratios determined with the DCA 2000® kit and by the laboratory method were 13.1 ± 20.5 and 20.4 ± 46.3 mg/g, respectively. A/C results by both methods proved to be strongly correlated (r = 0.98, P<0.001). DCA 2000®-determined A/C showed 50% sensitivity and 100% specificity when compared to the reference method. Spot urinary A/C of the subset of 16 subjects significantly correlated with their overnight albumin excretion rate (r = 0.98, P<0.001). Intraindividual variation ranged from 17 to 32% and from 9 to 63% for A/C and overnight albumin excretion rate, respectively. In conclusion, an early-morning specimen should be used instead of timed overnight urine and the A/C ratio is an accurate, reliable and easily determined parameter for the screening of diabetic nephropathy. Immediate measurement of the A/C ratio is feasible using the DCA 2000® kit. Intraindividual variability indicates the need for repeated determinations to confirm microalbuminuria and the diagnosis of incipient diabetic nephropathy.

Genetic characterization of dengue virus type 3 isolates in the State of Rio de Janeiro, 2001

The genetic characterization of dengue virus type 3 (DEN-3) strains isolated from autochthonous cases in the State of Rio de Janeiro, Brazil, in 2001 is presented. Restriction site-specific (RSS)-PCR performed on 22 strains classified the Brazilian DEN-3 viruses as subtype C, a subtype that contains viruses from Sri Lanka, India, Africa and recent isolates from Central America. Nucleic acid sequencing (positions 278 to 2550) of one DEN-3 strain confirmed the origin of these strains, since genotype III - classified by sequencing - and RSS-PCR subtype C are correlated. This genetic subtype has been associated with hemorrhagic dengue epidemics and the information provided here could be useful to implement appropriate prevention and control measures.

Bone mineral density in young women of the city of São Paulo, Brazil: correlation with both collagen type I alpha 1 gene polymorphism and clinical aspects

Osteoporosis is a multifactorial disease with great impact on morbidity and mortality mainly in postmenopausal women. Although it is recognized that factors related to life-style and habits may influence bone mass formation leading to greater or lower bone mass, more than 85% of the variation in bone mineral density (BMD) is genetically determined. The collagen type I alpha 1 (COLIA1) gene is a possible risk factor for osteoporosis. We studied a population of 220 young women from the city of São Paulo, Brazil, with respect to BMD and its correlation with both COLIA1 genotype and clinical aspects. The distribution of COLIA1 genotype SS, Ss and ss in the population studied was 73.6, 24.1 and 2.3%, respectively. No association between these genotypes and femoral or lumbar spine BMD was detected. There was a positive association between lumbar spine BMD and weight (P<0.0001), height (P<0.0156), and body mass index (BMI) (P<0.0156), and a negative association with age at menarche (P<0.0026). There was also a positive association between femoral BMD and weight (P<0.0001), height (P<0.0001), and BMI (P<0.0001), and a negative correlation with family history for osteoporosis (P<0.041). There was no association between the presence of allele s and reduced BMD. We conclude that a family history of osteoporosis and age at menarche are factors that may influence bone mass in our population.

Intravenous dipyrone for the acute treatment of episodic tension-type headache: A randomized, placebo-controlled, double-blind study

Acute headaches are responsible for a significant percentage of the case load at primary care units and emergency rooms in Brazil. Dipyrone (metamizol) is easily available in these settings, being the most frequently used drug. We conducted a randomized, placebo-controlled, double-blind study to assess the effect of dipyrone in the acute treatment of episodic tension-type headache. Sixty patients were randomized to receive placebo (intravenous injection of 10 ml saline) or 1 g dipyrone in 10 ml saline. We used seven parameters of analgesic evaluation. The patients receiving dipyrone showed a statistically significant improvement (P<0.05) of pain compared to placebo up to 30 min after drug administration. The therapeutic gain was 30% in 30 min and 40% in 60 min. The number of patients needed to be treated for at least one to have benefit was 3.3 in 30 min and 2.2 in 60 min. There were statistically significant reductions in the recurrence (dipyrone = 25%, placebo = 50%) and use of rescue medication (dipyrone = 20%, placebo = 47.6%) for the dipyrone group. Intravenous dipyrone is an effective drug for the relief of pain in tension-type headache and its use is justified in the emergency room setting.

Sec61alpha synthesis is enhanced during translocation of nascent chains of collagen type IV in F9 teratocarcinoma cells after retinoic acid treatment

Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (ER) membrane through a protein-conducting channel called translocon. Sec61alpha, a multispanning membrane translocon protein, has been implicated as being essential for translocation of polypeptide chains into the cisterns of the ER. Sec61alpha forms a protein complex with collagen and Hsp47, an ER-resident heat shock protein that binds specifically to collagen. However, it is not known whether Sec61alpha is ubiquitously produced in collagen-producing F9 teratocarcinoma cells or under heat shock treatment. Furthermore, the production and utilization of Sec61alpha may depend on the stage of cell differentiation. Cultured F9 teratocarcinoma cells are capable of differentiation in response to low concentrations of retinoic acid. This differentiation results in loss of tumorigenicity. Mouse F9 cells were grown in culture medium at 37ºC and 43ºC (heat shock treatment) treated or not with retinoic acid, and labeled in certain instances with 35S-methionine. Membrane-bound polysomes of procollagen IV were then isolated. Immunoprecipitation and Western blot analysis were performed using polyclonal antibodies against collagen IV, Hsp47 and Sec61alpha. Under retinoic acid-untreated conditions, F9 cells produced undetectable amounts of Sec61alpha. Sec61alpha, Hsp47 and type IV collagen levels were increased after retinoic acid treatment. Heat shock treatment did not alter Sec61alpha levels, suggesting that Sec61alpha production is probably not affected by heat shock. These data indicate that the enhanced production of Sec61alpha in retinoic acid-induced F9 teratocarcinoma cells parallels the increased synthesis of Hsp47 and collagen type IV.