[Chronic complications in type 1 diabetes mellitus. Analysis of a cohort of 291 patients with a mean evolution time of 15 years]. / Complicaciones crónicas en la diabetes mellitus tipo 1. Análisis de una cohorte de 291 pacientes con un tiempo medio de evolución de 15 años.

BACKGROUND AND OBJECTIVE: Micro- and macrovascular complications are the main cause of morbidity and mortality in type 1 diabetes mellitus (T1D). Given the scarcity of data on the subject in our population, we have analyzed the prevalence of vascular complications and possible risk factors in a cohort of T1D patients. PATIENTS AND METHODS: A cross-sectional study including patients aged 18 and over diagnosed of T1D with at least 6 months' evolution, seen in the Hospital del Mar, Barcelona and Hospital de Granollers during 2008 was carried out. RESULTS: We recruited 291 patients (166 men) with a mean age of 38 years and a T1D duration of 15.3 years. There was one or more diabetes-related vascular complications in 110 (37.8%) patients. Of these, 104 (35.7%) had microvascular complications, 22 (7.6%) macrovascular, and 16 (5.5%) both. Patients with microvascular complications had a higher prevalence of tobacco use (57% smokers Vs. 47.5%, P<.05), dyslipidemia (65.4% Vs. 28.3%, P <.05), hypertension (43.3% Vs. 23.5%, P <.05) and metabolic syndrome (41.3% Vs. 18.7%, P<.001). Moreover, they were older, had a longer duration of diabetes and higher values of glycosylated hemoglobin, triglycerides and systolic blood pressure. In the logistic regression analysis, diabetes duration (OR: 1.19 [95%CI: 1.07-1.32], P=.002), glycosylated hemoglobin levels (OR: 3.33 [95%CI: 1.58-7.03], P=.002) and the absence of metabolic syndrome (OR: 0.04 [95% CI:0.002-0.72], P=.03) showed an independent association with microangiopathy. Patients with T1D and macroangiopathy had longer diabetes duration (23.3±12.6 years Vs. 14.7±10.9 years, in patients without complications, P <.001), higher prevalence of metabolic syndrome (50% Vs. 24.9%, in patients without complications, P=.011) and were more frequently receiving lipid lowering treatment (59.1% Vs. 27.1%, in patients without complications, P=.002). In the multiple regression model, only diabetes duration (OR: 1.047 [95% CI: 1.01-1.09], P=.019) remained independently associated with macroangiopathy. CONCLUSIONS: More than 1/3 of the T1D patients suffered a diabetes-related complication, mainly microvascular, at the time of the study. Diabetes duration and metabolic syndrome are the two mostly strongly related factors to chronic complications of DM1.

Two Kunitz-type inhibitors with activity against trypsin and papain from Pithecellobium dumosum seeds: purification, characterization, and activity towards pest insect digestive enzyme.

Two trypsin inhibitors (called PdKI-3.1 and PdKI-3.2) were purified from the seeds of the Pithecellobium dumosum tree. Inhibitors were obtained by TCA precipitation, affinity chromatography on Trypsin-Sepharose and reversed-phase-HPLC. SDS-PAGE analysis with or without reducing agent showed that they are a single polypeptide chain, and MALDI-TOF analysis determined molecular masses of 19696.96 and 19696.36 Da, respectively. The N-terminal sequence of both inhibitors showed strong identity to the Kunitz family trypsin inhibitors. They were stable over a wide pH (2-9) and temperature (37 to 100 degrees C) range. These inhibitors reduced over 84% of trypsin activity with inhibition constant (Ki) of 4.20 x 10(-8) and 2.88 x 10(-8) M, and also moderately inhibited papain activity, a cysteine proteinase. PdKI-3.1 and PdKI-3.2 mainly inhibited digestive enzymes from Plodia interpunctella, Zabrotes subfasciatus and Ceratitis capitata guts. Results show that both inhibitors are members of the Kunitz-inhibitor family and that they affect the digestive enzyme larvae of diverse orders, indicating a potential insect antifeedant.

Efeitos de diferentes intervalos de recuperação no desempenho muscular isocinético em idosos / Effects of different rest intervals on isokinetic muscle performance among older adults

OBJETIVO: Comparar os efeitos de diferentes intervalos de recuperação (IR) entre séries de contração isocinética no desempenho muscular em idosos. MÉTODOS: Vinte idosos voluntários (66,9±3,9 anos, 76,1±13,4kg, 169±5,2cm) foram submetidos a três sessões de exercício resistido isocinético unilateral da musculatura extensora do joelho com diferentes IR (1 minuto, 2 minutos, e 3 minutos) na velocidade de 60°•s-1. Cada sessão consistia em três séries de 10 repetições durante as quais foram avaliados o pico de torque (PT), trabalho total (TT) e índice de fadiga (IF). ANOVA fatorial para medidas repetidas, com a análise de Post hoc por Bonferroni, foi utilizada para identificar possíveis diferenças entre os IR. O nível de significância estatístico utilizado foi de p<0,05. RESULTADOS: Não foram observadas diferenças no desempenho muscular entre as 1as séries nos diferentes IR (p>0,05). Apesar de ter sido observado menor desempenho muscular na 3ª série em todos os IR, maiores quedas no PT, TT e IF foram observadas quando adotado o IR de 1minuto (p<0,05). CONCLUSÃO: Os resultados mostraram que a variável IR exerce importante influência sobre o desempenho muscular isocinético em idosos, principalmente a partir da 3ª série, o que sugere incrementos no IR à medida que séries subsequentes forem executadas durante a mesma sessão de exercícios.

OBJECTIVE: To compare the effects of different rest intervals (RI) between sets of isokinetic contractions on muscle performance in older adults. METHODS: Twenty older participants (66.9±3.9 years; 76.1±13.4kg; 169±5.2cm) underwent three sessions of unilateral isokinetic training for the knee extensor muscles, with different RI (1 minute, 2 minutes and 3 minutes) at an angular velocity of 60°•s-1. Each session consisted of three sets of 10 repetitions, during which the peak torque (PT), total work (TW) and fatigue index (FI) were evaluated. Factorial ANOVA for repeated measurements, with Bonferroni post-hoc analysis, was used to identify possible differences between the RI. The statistical significance level was set at p<0.05. RESULTS: No differences in muscle performance during the first sets were observed between the different RI (p>0.05). Although muscle performance was lower during the third sets with all RI, the greatest decreases in PT, TW and FI occurred with the 1 minute RI (p<0.05). CONCLUSIONS: The results showed that the RI variable has an important influence on isokinetic muscle performance in older adults, particularly from the third sets onwards, which suggests that RI should be increased as successive sets are performed within the same exercise session.

Major digestive carbohydrase during larval development of meal moth, Plodia interpunctella (Lepidoptera: pyralidae).

The digestive system of P. interpunctella was characterized during its larval development to determination of carbohydrases using disaccharides (sucrose and maltose) and polysaccharides (starch and inulin) as substrate. At 6(th) instar larval, Invertase>alpha-amylase> maltase activities peaks were observed. Invertase was fractionated with acetone and isolated. The Invertase was 485.5 fold purified by Sephacryl S-200 and DEAE-Sephadex. Its kinetic parameters were K(m) of 6.6 mM, V(max) of 0.48, pH optimum of 5.5 and temperature optimum of 30 degrees C. This enzyme was activated by CaCl(2) and inhibited by EDTA. When analyzed by SDS-PAGE it showed one band of M(r) 34 kDa. The understanding of the digestive system of P. interpunctella could be a key step in the design of bioinsecticides.

Identification of a Kunitz-type proteinase inhibitor from Pithecellobium dumosum seeds with insecticidal properties and double activity.

A trypsin inhibitor, PdKI, was purified from Pithecellobium dumosum seeds by TCA precipitation, trypsin-sepharose chromatography, and reversed-phase-HPLC. PdKI was purified 217.6-fold and recovered 4.7%. SDS-PAGE showed that PdKI is a single polypeptide chain of 18.9 kDa and 19.7 kDa by MALDI-TOF. The inhibition on trypsin was stable in the pH range 2-10 and at a temperature of 50 degrees C. The Ki values were 3.56 x 10(-8)and 7.61 x 10(-7) M with competitive and noncompetitive inhibition mechanisms for trypsin and papain, respectively. The N-terminal sequence identified with members of Kunitz-type inhibitors from the Mimosoideae and Caesalpinoideae subfamilies. PdKI was effective against digestive proteinase from Zabrotes subfasciatus, Ceratitis capitata, Plodia interpunctella, Alabama argillaceae, and Callosobruchus maculatus, with 69, 66, 44, 38, and 29% inhibition, respectively. Results support that PdKI is a member of the Kunitz inhibitor family and its insecticidal properties indicate a potent insect antifeedant.

Performance of bean bruchids Callosobruchus maculatus and Zabrotes subfasciatus (Coleoptera: Bruchidae) reared on resistant (IT81D-1045) and susceptible (Epace 10) Vigna unguiculata seeds: relationship with trypsin inhibitor and vicilin excretion.

Callosobruchus maculatus (Cm) and Zabrotes subfasciatus (Zs) were reared on resistant (IT81D-1045) and on susceptible (Epace 10) cowpea seeds. The emergence of adult insects, total developmental period (TDP) and excretion of trypsin inhibitor and vicilin were determined for both bruchid populations. Parameter evaluation showed that the Zs populations emerged from both seeds had no significant differences in emergence and TDP. The Cm population raised from resistant seeds had lower emergence (5.6+/-1.3%) and delayed TDP (46+/-1.25 days) than those emerged from susceptible seeds. The excretion of defense proteins showed that Zs reared in resistant seeds excreted 1.7 times more trypsin inhibitor, but this did not affect emergence or TDP. Furthermore, Cm population emerged from resistant seeds excreted 7 times higher vicilin and 0.4 times less trypsin inhibitor than that emerged from susceptible seeds. These results indicate that vicilins from resistant seeds are involved to significantly longer TDP (46 days) and also drastic reduction of insect emergence ( approximately 5%) of C. maculatus.

A serine proteinase inhibitor isolated from Tamarindus indica seeds and its effects on the release of human neutrophil elastase.

Proteinaceous inhibitors with high inhibitory activities against human neutrophil elastase (HNE) were found in seeds of the Tamarind tree (Tamarindus indica). A serine proteinase inhibitor denoted PG50 was purified using ammonium sulphate and acetone precipitation followed by Sephacryl S-300 and Sephadex G-50 gel filtration chromatographies. Inhibitor PG50 showed a Mr of 14.9 K on Sephadex G-50 calibrated column and a Mr of 11.6 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PG50 had selective activity while cysteine proteinases (papain and bromelain) and serine proteinases (porcine pancreatic elastase and bovine chymotrypsin) were not inhibited, it was strongly effective against serine proteinases such as bovine trypsin and isolated human neutrophil elastase. The IC50 value was determined to be 55.96 microg.mL-1. PG50 showed neither cytotoxic nor haemolytic activity on human blood cells. After pre-incubation of PG50 with cytochalasin B, the exocytosis of elastase was initiated using PAF and fMLP. PG50 exhibited different inhibition on elastase release by PAF, at 44.6% and on release by fMLP, at 28.4%. These results showed that PG50 preferentially affected elastase release by PAF stimuli and this may indicate selective inhibition on PAF receptors.

Genetic diversity among Mycobacterium bovis isolates: a preliminary study of strains from animal and human sources.

Mycobacterium bovis has the broadest host range of species in the Mycobacterium tuberculosis complex and is responsible for disease in humans and diverse animal species. We report on genotypic differences at multiple loci among 13 isolates derived from a range of human and animal infections. All isolates were classified as M. bovis by phenotypic analysis but could be subdivided into five distinct genotypes based on polymorphisms at the pncA and oxyR loci, the status of the RD5 deletion region, and the spoligotype pattern. These findings suggest the existence of a spectrum of strains with genotypic characteristics between those of M. tuberculosis and M. bovis.

Vicilins (7S storage globulins) of cowpea (Vigna unguiculata) seeds bind to chitinous structures of the midgut of Callosobruchus maculatus (Coleoptera: Bruchidae) larvae.

The presence of chitin in midgut structures of Callosobruchus maculatus larvae was shown by chemical and immunocytochemical methods. Detection by Western blotting of cowpea (Vigna unguiculata) seed vicilins (7S storage proteins) bound to these structures suggested that C. maculatus-susceptible vicilins presented less staining when compared to C. maculatus-resistant vicilins. Storage proteins present in the microvilli in the larval midgut of the bruchid were recognized by immunolabeling of vicilins in the appropriate sections with immunogold conjugates. These labeling sites coincided with the sites labeled by an anti-chitin antibody. These results, taken together with those previously published showing that the lower rates of hydrolysis of variant vicilins from C. maculatus-resistant seeds by the insect's midgut proteinases and those showing that vicilins bind to chitin matrices, may explain the detrimental effects of variant vicilins on the development of C. maculatus larvae.

Vicilins (7S storage globulins) of cowpea (Vigna unguiculata) seeds bind to chitinous structures of the midgut of Callosobruchus maculatus (Coleoptera: Bruchidae) larvae

The presence of chitin in midgut structures of Callosobruchus maculatus larvae was shown by chemical and immunocytochemical methods. Detection by Western blotting of cowpea (Vigna unguiculata) seed vicilins (7S storage proteins) bound to these structures suggested that C. maculatus-susceptible vicilins presented less staining when compared to C. maculatus-resistant vicilins. Storage proteins present in the microvilli in the larval midgut of the bruchid were recognized by immunolabeling of vicilins in the appropriate sections with immunogold conjugates. These labeling sites coincided with the sites labeled by an anti-chitin antibody. These results, taken together with those previously published showing that the lower rates of hydrolysis of variant vicilins from C. maculatus-resistant seeds by the insect's midgut proteinases and those showing that vicilins bind to chitin matrices, may explain the detrimental effects of variant vicilins on the development of C. maculatus larvae

Lima bean (Phaseolus lunatus) seed coat phaseolin is detrimental to the cowpea weevil (Callosobruchus maculatus).

The presence of phaseolin (a vicilin-like 7S storage globulin) peptides in the seed coat of the legume Phaseolus lunatus L. (lima bean) was demonstrated by N-terminal amino acid sequencing. Utilizing an artificial seed system assay we showed that phaseolin, isolated from both cotyledon and testa tissues of P. lunatus, is detrimental to the nonhost bruchid Callosobruchus maculatus (F) (cowpea weevil) with ED50 of 1.7 and 3.5%, respectively. The level of phaseolin in the seed coat (16.7%) was found to be sufficient to deter larval development of this bruchid. The expression of a C. maculatus-detrimental protein in the testa of nonhost seeds suggests that the protein may have played a significant role in the evolutionary adaptation of bruchids to legume seeds.

Lima bean (Phaseolus lunatus) seed coat phaseolin is detrimental to the cowpea weevil (Callosobruchus maculatus)

The presence of phaseolin (a vicilin-like 7S storage globulin) peptides in the seed coat of the legume Phaseolus lunatus L. (lima bean) was demonstrated by N-terminal amino acid sequencing. Utilizing an artificial seed system assay we showed that phaseolin, isolated from both cotyledon and testa tissues of P. lunatus, is detrimental to the nonhost bruchid Callosobruchus maculatus (F) (cowpea weevil) with ED50 of 1.7 and 3.5 percent, respectively. The level of phaseolin in the seed coat (16.7 percent) was found to be sufficient to deter larval development of this bruchid. The expression of a C. maculatus-detrimental protein in the testa of nonhost seeds suggests that the protein may have played a significant role in the evolutionary adaptation of bruchids to legume seeds

Fungal infection in single and bilateral lung transplantation: report of 17 cases in 42 consecutive recipients

We reviewed the frequency and type of fungal infections in patients undergoing lung transplantation at Pavilhäo Pereira Filho Hospital between may 1989 and january 1995. Among the 42 lung transplant recipients studies, 17 ( 40 por cento ) had histologic diagnosis of fungal infection: aspergillosis 8 casos, candidosis 6 cases, and aspergillosis combined with candidosis 3 cases. Among all fungal infections, 47 por cento ( 8 of 17 cases ) occurred in the first month after transplantation, and 82 por cento occurred within the first six months after the transplant. The diagnosis was done during life in candidosis in 77 por cento of patients by transbronchial biopsy. However, aspergillosis was revealed in autopsy material in 82 por cento of cases. Most of the patients with candidosis had a local infection in the transplanted lung. On the contrary, in aspergillosis patients 88 por cento had the mycosis in the native lung

Chitin-binding proteins from cowpea (Vigna unguiculata) seeds.

Vicilins (7S storage proteins) from cowpea (Vigna unguiculata) and other legume seeds were shown to bind to chitin, to regenerated chitin (fully acetylated chitin) and to chitosan (deacetylated chitin). Adsorbed vicilins were desorbed from these matrices by acetic and hydrochloric acids and by highly polymerized soluble chitosan. Proteins such as the lectin of common bean (PHA), soybean trypsin inhibitor (Kunitz), a beta-1,3-glucanase from cowpea seeds, bovine pancreatic alpha-chymotrypsin, chicken ovalbumin, serum albumin and rabbit gamma-globulin did not bind. The present result is the first description of vicilin binding to chitin but other proteins, such as wheat germ agglutinin (WGA), a lectin that contains the so called "chitin-binding domain", and a chitinase isolated from cowpea seeds, which are involved in the defense mechanisms of plants against insects and fungi, were also shown to bind to chitin as previously reported. The binding of vicilins to chitin is probably effected not through a "chitin-binding domain" because they do not share this sequence with the defense-related proteins cited above. We propose that this association of vicilins with chitin may be related to the effect of variant vicilins on the development of Callosobruchus maculatus (bruchid) in resistant cowpea seeds.

Chitin-binding proteins from cowpea (Vigna unguiculata) seeds

Vicilins (7S storage proteins) from cowpea (Vigna unguiculata) and other legume seeds were shown to bind to chitin, to regenerated chitin (fully acetylated chitin) and to chitosan (deacetylated chitin). Adsorbed vicilins were desorbed from these matrices by acetic and hydrochloric acids and by highly polymerized soluble chitosan. Proteins such as the lectin of common bean (PHA), soybean trypsin inhibitor (Kunitz), a beta-1,3-glucanase from cowpea seeds, bovine pancreatic alpha-chymotrypsin, chicken ovalbumin, serum albumin and rabbit-gamma- globulin did not bind. The present result is the first description of vicilin binding to chitin but other proteins, such as wheat germ agglutinin (WGA), a lectin that contains the so called "chitin-binding domain", and a chitinase isolated from cowpea seeds, which are involved in the defense mechanisms of plants against insects and fungi, were also shown to bind to chitin as previously reported. The binding of vicilins to chitin is probably effected not through a "chitin-binding domain" because they do not share this sequence with the defense-related proteins cited above. We propose that this association of vicilins with chitin may be related to the effect of variant vicilins on the development of Callosobruchus maculatus (bruchid) in resistant cowpea seeds.

Purification and properties of storage proteins (vicilins) from cowpea (Vigna unguiculata) seeds which are susceptible or resistant to the bruchid beetle Callosobruchus maculatus.

Vicilins (7S storage proteins) from seeds of cowpea (Vigna unguiculata) cultivars which are susceptible or resistant to the bruchid beetle C. maculatus were purified by size-exclusion and ion-exchange chromatography. The vicilins were partially characterized by polyacrylamide gel electrophoresis under both denaturing and nondenaturing conditions, by Western blotting and by amino acid analysis. The variant vicilins from C. maculatus-resistant seeds do not differ appreciably from vicilins from susceptible seeds by these criteria except that they are more strongly bound to DEAE-Sepharose, suggesting differences in charge between the various molecules.

Purification and properties of storage proteins (vicilins) from cowpea (Vigna unguiculata) seeds which are susceptible or resistant to the bruchid beethe Callosobruchus maculatus

Vicilins (7S storage proteins) from seeds of cowpea (Vigna unguiculata) cultivars which are susceptible or resistant to the bruchil veetle C. maculatus were purified by size-exclusion and ion-exchange chromatography. The vicilins were partially characterized by polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions, by Western blotting and by amino acid analysis. The variant vicilins from C. maculatus-resistant seeds do not differ appreciably from vicilins from susceptible seeds by these criteria except that they are more strongly bound to DEAE-Sepharose, suggesting differences in charge between the various molecules