Riboflavin in development and cell fate.

Riboflavin (7,8-dimethyl-10-ribitylisoalloxazine; vitamin B2) is a water-soluble vitamin, cofactor derivatives of which (FAD, FMN) act as electron acceptors in the oxidative metabolism of carbohydrate, amino acids and fatty acids and which in the reduced state can donate electrons to complex II of the electron transport chain. This means that riboflavin is essential for energy generation in the aerobic cell, through oxidative phosphorylation. The classic effects of riboflavin deficiency on growth and development have generally been explained in terms of these functions. However, research also suggests that riboflavin may have specific functions associated with cell fate determination, which would have implications for growth and development. In particular, riboflavin depletion interferes with the normal progression of the cell cycle, probably through effects on the expression of regulatory genes, exerted at both the transcriptional and proteomic level.

Molluscicidal activity of some marine substances against the snail Biomphalaria glabrata (Mollusca, Planorbidae).

Freshwater snails of the genus Biomphalaria play a major role as intermediate hosts of Schistosoma mansoni, the etiologic agent of schistosomiasis. While Biomphalaria spp. control by molluscicides is one of the main strategies to reduce the snail population in infected areas, there are few effective molluscicides commercially available. Natural products may be considered as potentially useful and safe molluscicides. We have evaluated the molluscicidal activity of 12 extracts from ten marine organisms on adult and embryonic stages of Biomphalaria glabrata. Only extracts of the red algae Liagora farinosa and of the sponge Amphimedon viridis presented molluscicidal activity. Lethal concentration (LC)(50) values obtained were 120 µg/mL for L. farinosa CH(2)Cl(2) extract (apolar fraction) and 20 µg/mL for A. viridis extract and halitoxin. The polar alga fraction and halitoxin had no effect on B. glabrata embryos. The algae apolar fraction was active on B. glabrata in all embryonic development stages, with LC(50) values for blastulae at 42 µg/mL, gastrulae at 124 µg/mL, trochophore at 180 µg/mL, and veliger at 222 µg/mL. This is the first report of extracts from marine organisms which presented molluscicidal activity.

Molluscicidal and ovicidal activities of plant extracts of the Piperaceae on Biomphalaria glabrata (Say, 1818).

Schistosomiasis is a tropical disease caused by Schistosoma and occurs in 54 countries, mainly in South America, the Caribbean region, Africa and the eastern Mediterranean. Currently, 5 to 6 million Brazilian people are infected and 30,000 are under infection risk. Typical of poor regions, this disease is associated with the lack of basic sanitation and very frequently to the use of contaminated water in agriculture, housework and leisure. One of the most efficient methods of controlling the disease is application of molluscicides to eliminate or to reduce the population of the intermediate host snail Biomphalaria glabrata. Studies on molluscicidal activity of plant extracts have been stimulated by issues such as environmental preservation, high cost and recurrent resistance of snails to synthetic molluscicides. The aim of this study was to determine the molluscicide action of extracts from Piperaceae species on adult and embryonic stages of B. glabrata. Fifteen extracts from 13 Piperaceae species were obtained from stems, leaves and roots. Toxicity of extracts was evaluated against snails at two different concentrations (500 and 100 ppm) and those causing 100% mortality at 100 ppm concentration were selected to obtain the LC90 (lethal concentration of 90% mortality). Piper aduncum, P. crassinervium, P. cuyabanum, P. diospyrifolium and P. hostmannianum gave 100% mortality of adult snails at concentrations ranging from 10 to 60 ppm. These extracts were also assayed on embryonic stages of B. glabrata and those from P. cuyabanum and P. hostmannianum showed 100% ovicidal action at 20 ppm.

Body image dissatisfaction and eating symptoms in mothers of adolescents with eating disorders.

The purpose of the present study was to assess body dissatisfaction and eating symptoms in mothers of eating disorder (ED) female patients and to compare results with those of a control group. The case group consisted of 35 mothers of female adolescents (aged between 10 and 17 yrs) diagnosed with ED who attended the Interdisciplinary Project for Care, Teaching and Research on Eating Disorders in Childhood and Adolescence (PROTAD) at Clínicas Hospital Institute of Psychiatry of the Universidade de São Paulo Medical School. Demographic and socioeconomic data were collected. Eating symptoms were assessed using the Eating Attitudes Test (EAT-26) and body image was assessed by the Body Image Questionnaire (BSQ) and Stunkard Figure Rating Scale (FRS). The case group was compared to a control group consisting of 35 mothers of female adolescents (between 10 and 17 years) who attended a private school in the city of São Paulo, southeastern Brazil. With regard to EAT, BSQ and FRS scores, we found no statistically significant differences between the two groups. However, we found a positive correlation between BMI and BSQ scores in the control group (but not in the case group) and a positive correlation between EAT and FRS scores in the case group (but not in the control group). It appears to be advantageous to assess body image by combining more than one scale to evaluate additional components of the construct.

Polymerization Stress and Gap Formation of Self-adhesive, Bulk-fill and Flowable Composite Resins.

CLINICAL RELEVANCE: Bulk-fill materials show a similar or better performance than control flowable materials regarding interfacial integrity. However, some self-adhesive composites need improvements to achieve competitive performance. SUMMARY: Objective: This laboratory study compared the polymerization stress and gap formation of self-adhesive, bulk-fill and control flowable composites. The degree of conversion (DC) and post-gel shrinkage were also assessed.Methods: Two self-adhesive (Vertise Flow and Fusio Liquid Dentin), two bulk-fill (Tetric N-Flow Bulk-Fill and Filtek Bulk-Fill Flowable Restorative), and two control flowable (Z350 XT Flowable Restorative and Tetric N-Flow) composites were evaluated. Polymerization stress (PS) was determined in a universal testing machine (n=5). Gap formation was evaluated by scanning electron microscopy in class I restorations (n=6). DC was measured by Fourier transform infrared spectroscopy (n=3). Post-gel volumetric shrinkage (VS) was measured using the strain gauge method (n=5). Data were submitted to one-way analysis of variance or a Kruskal-Wallis test (α=0.05).Results: Vertise Flow and Fusio Liquid Dentin presented the highest interfacial gap (27%±5% and 21%±6%, respectively), which was associated with their highest PS (4.1±0.8 MPa and 3.5±0.6 MPa, respectively) and DC (63%±2% and 60%±2%, respectively) in spite of the lowest VS (1.0%±0.2% and 1.0%±0.3%, respectively). Tetric N-Flow Bulk-Fill and Filtek Bulk-Fill Flowable Restorative presented similar PS (2.9± 0.3 MPa and 2.4±0.2 MPa, respectively) to both control materials. However, the Tetric N-Flow Bulk-Fill showed the lowest gap (7%±2%) and the highest DC (64.3%±0.4%), and the Filtek Bulk-fill presented a marginal gap (17.8%±3.4%) and a DC (54.5%±2.7%) similar to the control materials. The VS values of both bulk-fill materials were similar to those of Tetric N-Flow and lower than that of Z350 XT Flowable Restorative.Conclusions: Bulk-fill composites showed either similar or significantly lower interfacial gaps and PS than the control flowable composites. The self-adhesive composites showed a significantly higher gap percentage and PS than the control and bulk-fill materials.

Characterization of an established cell line from human renal carcinoma.

A cell line, designated as OUR-10, has been established from a renal carcinoma in a Japanese woman. This cell line forms monolayers of polygonal epithelial cells with scattered round or dendritic cells and exhibits multilayering. With electron microscopy, differentiated surface structures that resemble the microvilli characteristic of renal carcinomas can be seen even at the 60th transfer. The cells have a hypodiploid karyotype with modal numbers of 39 and 40. No marker chromosomes were seen, but definite nonrandom loss of three chromosomes in Group D and one in Group E were recognized. The doubling time was estimated as approximately 32 hr in exponentially growing cultures, and the cells formed colonies in soft agar with an average efficiency of 25%. Heterotransplantation into the cheek pouch of immunosuppressed hamsters produced tumors that were histologically similar to the original cancerous tissue. The electrophoretic mobility of gamma-glutamyl transpeptidase extracted from the cells coincided with that of a novel isozyme found in human renal carcinoma tissue, and the genetic phenotype of the glucose-6-phosphate dehydrogenase was proved to be the B phenotype. The antigenic structure of HLA was determined as HLA-A2, 11; B5, 40, which was the same as that of peripheral blood lymphocytes of the woman with renal carcinoma.

Composition and decomposition of internal models in motor learning under altered kinematic and dynamic environments.

The learning process of reaching movements was examined under novel environments whose kinematic and dynamic properties were altered. We used a kinematic transformation (visuomotor rotation), a dynamic transformation (viscous curl field), and a combination of these transformations. When the subjects learned the combined transformation, reaching errors were smaller if the subject first learned the separate kinematic and dynamic transformations. Reaching errors under the kinematic (but not the dynamic) transformation were smaller if subjects first learned the combined transformation. These results suggest that the brain learns multiple internal models to compensate for each transformation and has some ability to combine and decompose these internal models as called for by the occasion.

Molecular cloning and expression in Escherichia coli of a cDNA clone encoding luciferase of a firefly, Luciola lateralis.

We have cloned a cDNA encoding Luciola lateralis (a common firefly in Japan) luciferase from a cDNA library of lantern poly(A)+ RNA, using a cDNA of L. cruciata (another common firefly in Japan) luciferase as a probe. The primary structure of L. lateralis luciferase deduced from the nucleotide sequence was shown to consist of 548 amino acids with a molecular weight of 60,132. Sequence comparison indicates that L. lateralis luciferase has significant sequence identity (94%) to L. cruciata luciferase, and that it has less sequence similarity (67%) to Photinus pyralis (a North American firefly) luciferase. The isolated cDNA clone, when introduced into Escherichia coli, directed the synthesis of enzymatically active luciferase under the control of the lacZ promoter.

Purification and characterization of luciferases from fireflies, Luciola cruciata and Luciola lateralis.

Luciferases of Luciola cruciata and Luciola lateralis, LcL and LlL, were purified to homogeneity by ammonium sulfate precipitation, gel-filtration column chromatography, and hydroxyapatite HPLC. The molecular masses of the enzymes determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were both 62 kDa, almost identical to that of Photinus pyralis (PpL). LcL was found to be similar to PpL in thermal stability, pH stability, and the wavelength of maximum light intensity. LlL was superior to LcL and PpL in thermal and pH stability, and the reaction catalyzed by LlL emits green light with a peak intensity at 552 nm, which is 10 nm shorter in wavelength than those of PpL and LcL.

Elucidation of the thermal stability of the neutral proteinase II from Aspergillus oryzae.

The neutral proteinase II from Aspergillus oryzae (NpII) is a zinc proteinase with three intramolecular disulfide bonds. NpII is most unstable after 10 min at about 75 degrees C, but regains stability beyond this temperature and is relatively stable at 100 degrees C. We analyzed the thermal stability of wild-type NpII and apo NpII. The results suggested that NpII unfolds reversibly upon incubation up to 100 degrees C, and that the irreversible inactivation observed is mainly due to autoproteolysis. To further understand the stability, a mutant NpII (Cys78-->Ala) lacking one of the disulfide bonds, was produced in a heterologous yeast expression system. The mutant NpII showed a similar stability profile, but the most unstable temperature and the most catalytically active temperature decreased to the same extent (around 10 degrees C), confirming that autoproteolysis is the main cause of the irreversible inactivation. Several lines of evidence presented in this study demonstrated that the thermal stability of o++NpII is attributed to reversible thermal unfolding and autoproteolysis.

Nucleotide sequence of the phosphotransacetylase gene of Escherichia coli strain K12.

The phosphotransacetylase gene (pta) from Escherichia coli strain K-12 1100 was identified in a cloned fragment of chromosomal DNA (Yamamoto-Otake, H., Matsuyama, A. and Nakano, A. (1990) Appl. Microbiol. Biotechnol. 33, 680-682). Overexpression in E. coli confirmed the presence of the pta gene within the cloned fragment. DNA sequence analysis of the cloned pta gene indicates that the predicted phosphotransacetylase polypeptide chain is 713 amino acids in length. The carboxyterminal region of the E. coli phosphotransacetylase shows 42.6% sequence identity with the corresponding enzyme from Methanosarcina thermophila (142 out of 333 residues in corresponding positions are identical). Several short regions of high sequence identity may be structurally or functionally important for enzymic activity.

Oxidative damage in selenium deficient hearts on perfusion with adriamycin: protective role of glutathione peroxidase system.

The protective effects of the glutathione peroxidase system against functional damage induced by perfusion of isolated hearts with adriamycin, an anthracycline antibiotic, were studied. We used selenium deficient rats, in which cardiac glutathione peroxidase activity was only 3% of control rats. Both contractile tension and coronary flow decreased during perfusion with the antibiotic. The degree of decline was significantly greater in the selenium deficient hearts than in the control hearts. The increase in malondialdehyde, a product of lipid peroxidation, induced by adriamycin perfusion was more evident in selenium deficient hearts, though the level of reduced glutathione was well maintained. Isolated mitochondrial function also decreased after aerobic adriamycin perfusion and the decrease was greater in selenium deficient rats. These observations indirectly suggest that the decrease in cardiac function induced by adriamycin is protected by the glutathione peroxidase system and that the decrease may be due, at least in part, to damage to the mitochondria caused by oxygen radicals generated by adriamycin.

Cloning and sequence analysis of cDNA for luciferase of a Japanese firefly, Luciola cruciata.

Luciferases of Japanese and North American fireflies act on a common substrate (luciferin) but the resulting lights emitted are of different colors. As a step toward an understanding of the molecular mechanism of the luciferase reaction, a cDNA clone (pGLf1) was isolated from a cDNA library of lantern poly(A)+RNA of the Japanese firefly, Luciola cruciata ('Genji-botaru' in Japanese), using a cDNA of North American firefly luciferase. The isolated 2-kb cDNA sequence was able to direct the synthesis of active luciferase in Escherichia coli under the control of the lac promoter. The primary structure of Genji firefly luciferase deduced from the nucleotide sequence was shown to consist of 548 amino acids (aa) with an Mr of 60,024. Homology between the amino acid sequences of the Genji and North American firefly luciferases was 67%, but a number of amino acid changes were found in the first 200 aa from the N terminus.

New technique for analysis of cardiac energetics using a modified Fenn equation.

In 12 dogs being supported by cardiopulmonary bypass, the relationship among myocardial oxygen consumption and four energy-consuming factors (basal metabolism, heart rate, tension development, and external work) was studied. Tension (internal work) in the left ventricular wall was evaluated by myocardial tissue pressure with a Mikro-Tip pressure transducer. In an empty beating heart with constant perfusion pressure, both systolic tissue pressure and developed tissue pressure represented the same characteristics as developed tension measured by other methods. As the heart rate was increased, the systolic tissue pressure and developed tissue pressure continued to increase stepwise (Bowditch effect) up to some stimulation rate, at which, however, a decrease began despite a further increase in heart rate (Woodworth effect). Significant regression was established between myocardial oxygen consumption and heart rate, tension (developed tissue pressure x heart rate), and external work (minute work): myocardial oxygen consumption = (9.05 x 10(-3) heart rate) + (1.95 x 10(-4) developed tissue pressure) x heart rate + (1.63 x 10(-3) minute work) + 1.42 (r = 0.7999), where activation energy = 9.05 x 10(-3) ml/100 gm per beat, tension-related energy = 1.95 x 10(-4) ml/100 gm per unit of internal work, energy for work = 1.63 ml/100 gm per unit of external work, and basal metabolism = 1.42 ml/min/100 gm. We concluded that myocardial tissue pressure is a good substitute for tension and that multiple regression with heart rate, tension, and external work (as by modified Fenn's equation) seems indispensable to predict myocardial oxygen tension in the whole heart.

Predisposing factors of renal dysfunction following total correction of tetralogy of Fallot in the adult.

A retrospective study of 21 adult patients with tetralogy of Fallot (TOF) was undertaken to determine the predisposing risk factors of renal dysfunction following total correction of the disease. Five of the 21 patients exhibited moderate-to-severe postoperative azotemia and an additional five exhibited mild azotemia. Significant risk factors for postoperative renal dysfunction found in routine preoperative examinations were as follows: arterial oxygen saturation of less than 90%, cardiothoracic ratio (CTR) of greater than 50%, mean electrical axis of the QRS complexes of greater than +120 degrees, S wave in Lead V6 of greater than 7 mm, R/S voltage ratio in Lead V6 of less than 2, and negative T wave in Lead V6. In addition, preoperative cardiac catheterization data showed that the patients exhibiting postoperative azotemia had more severe pulmonary stenosis, a smaller pulmonary-to-systemic flow ratio (Qp/Qs), and a larger left ventricular cavity than nonazotemic patients. The incidence of postoperative low cardiac output state (LOS) was significantly higher in the azotemic patients. These findings suggest that a combination of the severe form of TOF and a large left ventricle increase susceptibility to LOS and postoperative renal dysfunction. The cause and the clinical significance of the large left ventricular cavity in adults with TOF are discussed.

Primary repair of interrupted aortic arch and severe aortic stenosis in neonates.

Two infants, aged 36 days old (Case 1) and 18 days old (Case 2) with interrupted aortic arch types B and A, respectively, and with severe aortic stenosis, were successfully operated on by use of pulsatile cardiopulmonary bypass. The great arteries were normally related in Case 1 and were transposed in Case 2. Repair involved the following procedure: ligation of the patent ductus arteriosus, restoration of aortic continuity with an 8 mm polytetrafluoroethylene graft, placement of an internal patch to tunnel all left ventricular blood from the left ventricle through the ventricular septal defect into the pulmonary artery in Case 1 and patch closure of the ventricular septal defect in Case 2, transection of the main pulmonary artery, anastomosis between the proximal pulmonary artery and the ascending aorta, and interposition of a valved conduit between the right ventricle and the distal pulmonary artery. The operative field could be approached easily through a median sternotomy. Postoperative cardiac catheterization revealed satisfactory anatomical and hemodynamic results in both cases.

Partial purification and properties of citrate synthase from sea urchin eggs.

Citrate synthase [EC 4.1.3.7] was purified from sea urchin eggs about 14-fold with a 23% yield, based on the activity of the crude extract. The molecular weight of the enzyme was about 100,000 as determined by gel filtration. The optimum pH was about 7.8 in 100 mM Tris-HCl. The apparent Km values for acetyl-CoA and for oxaloacetate were 33 and 3.2 muM, respectively. Monovalent and divalent cations inhibited the enzyme. Iodoacetamide, pCMB, EDTA, NaF, and dithiothreitol did not affect the enzyme activity. Oxaloacetate protected the enzyme against heat denaturation. Among nucleotides, ATP was the most potent inhibitor of the enzyme. The inhibition by ATP was competitive with respect to acetyl-CoA and mixed with respect to oxaloacetate.

Purification and properties of mitochondrial malate dehydrogenase from unfertilized eggs of the sea urchin, Anthocidaris crassispina.

Purification and characterization of mitochondrial malate dehydrogenase [EC 1.1.1.37] from unfertilized eggs of the sea urchin, Anthocidaris crassispina, are described. The purification method consisted of dextran sulfate fractionation, Blue Dextran Sepharose chromatography, Phenyl-Sepharose hydrophobic chromatography and DEAE-cellulose chromatography. The enzyme was purified 771-fold with a 7% yield from the crude extract. The purified enzyme appeared homogeneous on polyacrylamide gel electrophoresis under both native and denatured conditions. After incubation at 45 degrees C for 50 min, the enzyme lost about 90% of its activity. In the presence of NADH, however, the enzyme was protected against the heat denaturation. The native enzyme had a molecular weight of about 65,000 and probably consisted of two identical subunits. In the reduction of oxaloacetate with NADH, a broad optimum pH ranging from 8.2 to 9.4 was found with 50 mM Tris-HCl and glycine-NaOH buffers. Sodium phosphate buffer apparently activated the enzyme. The apparent Km values for oxaloacetate and NADH were 19 microM and 30 microM, respectively. The optimum pH for malate oxidation with NAD+ was 10.2 in 50 mM NaHCO3-Na2CO3 buffer. The apparent Km values for malate and NAD+ were 7.0 mM and 0.6 mM, respectively. Zinc ion, sulfite ion, p-chloromercuriphenylsulfonate and adenine nucleotides strongly inhibited the enzyme.

Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding the Candida utilis urate oxidase (uricase).

A urate oxidase (uricase) gene was cloned from Candida utilis with an oligonucleotide probe based on the amino acid sequence of cyanogen bromide-cleaved uricase. The uricase gene contains 909 base pairs and encodes a protein with a predicted mass of 34,193 Da. Candida uricase was similar (49% match in amino acid sequence) to the uricase from Aspergillus flavus. The uricase from Candida utilis has four cysteines and one of them, Cys168, participates in the enzyme activity. This enzyme was expressed to a level of about 20% of total cellular protein in an Escherichia coli cell as a soluble and functional form.