Characterisation of Zea mays L. plastidial transglutaminase: interactions with thylakoid membrane proteins.

Chloroplast transglutaminase (chlTGase) activity is considered to play a significant role in response to a light stimulus and photo-adaptation of plants, but its precise function in the chloroplast is unclear. The characterisation, at the proteomic level, of the chlTGase interaction with thylakoid proteins and demonstration of its association with photosystem II (PSII) protein complexes was accomplished with experiments using maize thylakoid protein extracts. By means of a specific antibody designed against the C-terminal sequence of the maize TGase gene product, different chlTGase forms were immunodetected in thylakoid membrane extracts from three different stages of maize chloroplast differentiation. These bands co-localised with those of lhcb 1, 2 and 3 antenna proteins. The most significant, a 58 kDa form present in mature chloroplasts, was characterised using biochemical and proteomic approaches. Sequential fractionation of thylakoid proteins from light-induced mature chloroplasts showed that the 58 kDa form was associated with the thylakoid membrane, behaving as a soluble or peripheral membrane protein. Two-dimensional gel electrophoresis discriminated, for the first time, the 58-kDa band in two different forms, probably corresponding to the two different TGase cDNAs previously cloned. Electrophoretic separation of thylakoid proteins in native gels, followed by LC-MS mass spectrometry identification of protein complexes indicated that maize chlTGase forms part of a specific PSII protein complex, which includes LHCII, ATPase and pSbS proteins. The results are discussed in relation to the interaction between these proteins and the suggested role of the enzyme in thylakoid membrane organisation and photoprotection.

Cholesterol and iron availability in yolk of laying hens feed with annatto ( Bixa orellana).

Pigmented egg yolks are more attractive. Popular culture treats annatto as a powerful anticholesterolemic agent, besides being widely used in the form of industry pigment. This work evaluated the effects of the addition of annatto (Bixa orellana L.) in the feed of hens, verifying a possible alteration of cholesterol in the yolks, content of carotenes, and iron and available iron, over time. One hundred and twenty-five hens divided in control (0% - T1) and four annatto-added treatments (0.5% - T2; 1.0% - T3; 1.5% - T4, and 2.0% - T5) were used. Eggs were collected at 23, 25, 27, 29 and 30 weeks. The animals were randomly separated into five groups of five animals each. The cholesterol was measured by the colorimetric method, vitamin A (ß and α carotene) by spectrophotometry, total iron by atomic absorption spectrophotometry, and dialysable iron by dialysis. Tukey's test was used at the 5% level for comparison of the averages. Regarding cholesterol, treatments T2 and T3 did not differ significantly. However, other treatments differed ( P ≤ 0.05) from the control, decreasing the cholesterol level as the percentage of annatto in the feed increased. In time, there was a significant increase ( P ≤ 0.05). For ß and α carotene, T5 presented statistically higher values than the others ( P ≤ 0.05). With regard to total iron, T5 had higher values than the others. Dialysable iron was also higher, probably due to the increase in carotenes. Thus, we can conclude that the use of annatto in the feed of layer hens is useful, as it provokes the reduction of cholesterol and promotes an increase in the content of iron and carotenes in eggs.

Surface effects and desorption of tetracycline supramolecular complex on bovine dentine.

The aim of this in vitro study was to evaluate the antimicrobial activity, the substantivity, and surface effects of the inclusion compound tetracycline: beta-cyclodextrin on bovine roots. The antimicrobial activity was assessed by dentine slabs which had been immersed in the inclusion complex in concentrations 8.0%, 4.0%, 2.0%, 1.0%, 0.5% and 0.25% for 5min compared to a control of tetracycline hydrochloride. Each slab was tested in a broth of overnight culture of Actinobacillus actinomycetemcomitans (Y4-FDC). The inclusion complex significantly inhibited the A. actinomycetemcomitans (p<0.01) verified at concentrations from 1.0% to 8.0%. The substantivity of tetracycline was evaluated by the measure of desorption from the slabs previously immersed in solution samples and removed at 24h intervals. The tetracycline encapsulated in beta-cyclodextrin showed a flow rate near to zero order in comparison to free tetracycline. The surface morphology determined by SEM showed a more homogeneous and integrated layer with the complex compared to the effect of free tetracycline. We concluded that the root surfaces treated with tetracycline: beta-cyclodextrin release lower concentrations of active drug over 5 days at inhibitory concentrations against A. actinomycetemcomitans with enhanced disponibility in comparison to tetracycline.

Coffee Leaf Scorch Caused by a Strain of Xylella fastidiosa from Citrus.

Citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS) are two economically important diseases in Brazil caused by the bacterium Xylella fastidiosa. Strains of the bacterium isolated from the two plant hosts are very closely related, and the two diseases share sharpshooter insect vectors. In order to determine if citrus strains of X. fastidiosa could infect coffee and induce CLS disease, plant inoculations were performed. Plants of coffee, Coffea arabica 'Mundo Novo', grafted on Coffea canephora var. robusta 'Apuatão 2258' were mechanically inoculated with triply cloned strains of X. fastidiosa isolated from diseased coffee and citrus. Three months postinoculation, 5 of the 10 plants inoculated with CLS-X. fastidiosa and 1 of the 10 plants inoculated with CVC-X. fastidiosa gave positive enzyme-linked immunosorbent assay (ELISA) and/or polymerase chain reaction (PCR). Eight months postinoculation, another six plants inoculated with CVC-X. fastidiosa gave positive PCR results. The two X. fastidiosa strains were isolated from the inoculated plants and showed the same characteristics as the original clones by microscopy, ELISA, and PCR. None of the plants inoculated with sterile periwinkle wilt (PW) medium as controls gave positive reactions in diagnostic tests, and none developed disease symptoms. Six months postinoculation, seven plants inoculated with CLS-X. fastidiosa and eight inoculated with CVC-X. fastidiosa began to develop characteristic CLS symptoms, including apical and marginal leaf scorch, defoliation, and reductions of internode length, leaf size, and plant height, terminal clusters of small chlorotic and deformed leaves, and lateral shoot dieback. We have demonstrated that X. fastidiosa from citrus plants is pathogenic for coffee plants. This has important consequences for the management of CLS disease and has implications for the origin of citrus variegated chlorosis disease.

Strains of Xylella fastidiosa rapidly distinguished by arbitrarily primed-PCR.

Genomic DNAs isolated from strains of Xylella fastidiosa that caused citrus variegated chlorosis, coffee leaf scorch, Pierce's Disease of grapevine, and plum leaf scorch were analyzed by arbitrarily primed polymerase chain reaction. Purified DNA was amplified under nonstringent conditions with single primers 21 nucleotides (nt) long. Thirty-nine amplification products were observed that were useful to distinguish among the strains and to derive a similarity matrix and construct a phenogram showing possible relationships among the strains. Strains isolated from diseased coffee and citrus in Brazil were closely related to each other (coefficient of similarity of 0. 872), but only distantly related to a strain isolated from diseased grapevine in the USA (coefficient of similarity of 0.650). Strains of Xylella fastidiosa isolated from diseased plums in the USA and Brazil clustered with strains from different hosts isolated from their respective countries of origin. Thus, there may be two quite dissimilar clusters of strains of Xylella fastidiosa, one in North America and the other in South America. Each cluster contains strains that can cause disease in plum. The methods described provide a convenient and rapid method to distinguish between strains of Xylella fastidiosa that cause diseases of coffee and citrus in the same region of Brazil. This has not been possible previously. This will potentially enable the two strains to be distinguished in alternate hosts or in insect vectors.

Supraceliac clamping in the surgical treatment of abdominal aortic aneurysm. An experimental study in dogs.

Previous reports have suggested the use of supraceliac aortic clamping in the surgical treatment of abdominal aortic aneurysm of difficult approach. The objective of the present report was to study the hepatic and renal metabolic changes of three groups of dogs submitted to temporary clamping (30 minutes) of the abdominal aorta at three different levels: below the renal arteries, infrarenal group (8 dogs); above the renal arteries, suprarenal group (9 dogs); above the celiac artery, supraceliac group (9 dogs). Blood bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen, and creatinine levels were measured before clamping and 5 minutes and 24 hours after reperfusion of the aorta. Bilirubin levels remained unchanged 5 minutes and 24 hours after reperfusion in all three groups. Alkaline phosphatase levels were significantly increased in all three groups 24 hours after reperfusion. ALT levels increased significantly in the supraceliac group and AST levels increased significantly in the infrarenal and supraceliac groups 24 hours after reperfusion of the aorta. However, despite these significant increases after reperfusion, the levels of these hepatic enzymes were still within the normal range for dogs. Urea nitrogen and creatinine levels showed that renal function did not change in any of the three groups. We conclude that supraceliac, infrarenal or suprarenal aortic clamping for 30 minutes do not promote any important changes in the hepatic or renal function of dogs.

Serum gamma-glutamyl transpeptidase in chronic extrahepatic cholestasis.

Studies in the literature have shown the inhibitory action of bilirubin on serum gamma-glutamyl transpeptidase (GGT) activity. In spite of this, GGT is a sensitive diagnostic indicator, comparable to alkaline phosphatase for the diagnosis of chronic extrahepatic cholestasis (CEHC). To evaluate the relationship of bilirubin, alkaline phosphatase (AP) activity and GGT activity in CEHC we studied 30 patients with CEHC divided into two groups: A, 14 male and female subjects with serum bilirubin levels of 2 to 10 mg/dl, and B, 16 male and female subjects with serum bilirubin levels of 10.1 to 44 mg/dl. Serum GGT activity was measured by diluting serum samples 1:5 and 1:10. GGT values were equivalent in the two groups, there was no correlation between bilirubin and GGT (r = +0.179, P less than 0.01), and there was a significant correlation between GGT and AP (r = +0.627, P less than 0.01). The present results show that, if GGT inhibition by bilirubin did occur, this inhibition was not sufficient to prevent the increase in serum GGT levels and that the sensitivity of GGT as a marker for CEHC is similar to that of alkaline phosphatase.

Inhibitory effect of different anticoagulants on gamma-glutamyl transpeptidase activity.

Increased gamma-glutamyl transpeptidase (GGT) levels have been observed in patients with several types of liver diseases. However, since GGT activity can be inhibited or increased by several substances, the interpretation of these increased levels should be made with caution. The present study was designed to determine plasma GGT activity in blood samples obtained from 24 adult volunteers (13 males and 11 females aged 20 to 40 years) with the use of three different anticoagulants, i.e., ethylenediaminetetraacetic acid (EDTA), calcium fluoride and calcium citrate, and to compare it with GGT activity in serum obtained from the same blood samples. Serum GGT activity was significantly higher than plasma GGT activity (P less than 0.001) and significantly higher in men than in women (P less than 0.05), though plasma GGT activity was similar for both sexes.

Serum transaminase levels in the acute phase of chronic extrahepatic cholestasis.

Serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) levels are normal or discretely increased in rats with chronic extrahepatic cholestasis (CEHC). During the acute phase (first 72 h after biliary obstruction), however, serum transaminase values are quite elevated due to a mechanism not yet fully elucidated. Thus, this is a good experimental model, not involving hepatocellular necrosis, for the study of serum ALT and AST levels during the acute phase of CEHC. Male Wistar rats (250-350 g) were divided into two groups: group A (N = 60) was submitted to sham operation for bile duct ligation (BDL), and group B (N = 60) was submitted to BDL. Thirty and 120 min after BDL there was a 1.5-fold increase in both serum ALT and AST levels compared to sham-operated rats (P less than 0.05). Serum ALT levels were higher than AST levels as early as 30 min after BDL and the highest serum values for both transaminases were observed at 360 min which was also the last value measured. Serum AST levels increased 120 min after BDL, with no further significant increases thereafter.

Serum transaminase levels in the acute phase of chronic extrahepatic cholestasis

Serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) levels are normal or discretely increased in rats with chronic extrahepatic cholestasis (CEHC). During the acute phase (first 72 h after biliary obstruction), however, serum transminase values are quite elevated due to a mechanism not yet fully elucidated. Thus, this is a good experimental model, not involving hepatocellular necrosis, for the study of serum ALT and AST levels during the acute phase of CEHC. Male Wistar rats (250-350 g) were divided into two groups: group A(N = 60) was submitted to sham operation for bile duct ligation (BDL), and group B (N = 60) was submitted to BDL. Thirty and 120 min after BDL there was a 1.5-fold increase in both serum ALT and AST levels compared to sham-operated rats (P<0.05). Serum ALT levels were higher than AST levels as early as 30 min after BDL and the highest serum values for both transaminases were observed at 360 min which was also the last value measured. Serum AST levels increased 120 min after BDL, with no further significant increase thereafter

Serum gama-glutamyl transpeptidase in chronic extrahepatic cholestasis

Studies in the literature have shown the inhibitory action action of bilirubin on serum gama-glutamyl transpeptidase (CGT) activity. In spite of this, CGT is a sensitive diagnostic indicator, comparable to alkaline phosphatase for the diagnosis of chronic extrahepatic cholestais (CEHC). To evaluate the relationship of bilirubin, alkaline phosphatase (AP) activity and GGT activity in CEHC we studied 30 patients with CEHC divided into two groups: A, 14 male and female subjects with bilirubin levels of 10.1 to 44 mg/dl. Serum GGT activity was meaured by diluting serum samples 1:5 and 1:10. GGT values were equivalent in the two groups, there was no correlation between bilirubin and GGT (r = +0.179, P > 0.01) and there was a significant correlation between GGT and AP(r = +0.627, P < 0.01). The present results show that, if GGT inhibition by bilirubin did occur, this inhibitions was not sufficient to prevent the increase in serum GGT levels and that the sensitivity of GGT as a marker for CEHC is simular to that of alkaline phosphatase

Inhibitory effect of different anticoagulants on gamma-glutamyl transpeptidase activity

Increased gama-glutamyl transpeptidase (GGT) levels have been observed in pacients with several types of liver diseases. However, since GGT activity can be inhibited or incrased by several substances, the interpretation of these increased levels should be made with caution. The present study was designed to determine plasma GGT activity in blood samples obtained from 24 adult volunters (13 males and 11 females aged 20 to 40 years) with the use of three different anticoagulants, i.e., ethylenediaminetetraacetic acid (EDTA), calcium fluoride and calcium citrate, and to compare it with GGT activity in serum obtanied from the same blood samples. Serum GGT activity wa significantly higher than plasma GGT activity (P < 0.001) and singnificantly higher in men than in women (P < 0.05), though plasma GGT activity was similar for both sexes

Effect of hepatic artery ligation on liver metabolism in rats

The rats an experimental model of orthotopic liver transplant that does not need arterial revascularization of the transplanted liver. the objective of the present study was to investigate the effect of hepatic artery ligation on hepatic metabolism in rats. After 2, 3, or 24 h hepatic artery ligation, no significant differences in serum levels of alanine aminotransferase were observed. Bile flow decreased significantly 3 h after arterial ligation (P < 0.05) and returned to normal after 24 h (P < 0.05)

Heparin-induced increase in plasma and serum gamma-glutamyl transpeptidase activity.

gamma-Glutamyl transpeptidase (GGT) activity may be a sensitive index of hepatobiliary dysfunction. GGT activity, however, has been shown to be affected by several factors in different clinical situations. The present study was designed to determine the effect of heparin on serum and plasma GGT activity. Blood samples were collected from 15 normal male Wistar rats weighing 200-250 g for the determination of GGT activity in plasma prepared with heparin (group P(H), N = 10), in serum (group S, N = 15) and in serum with heparin added (group S + H, N = 10). GGT activity was 45.1 +/- 9.5 U/l (mean +/- SD) for serum compared with 161.2 +/- 46.1 U/l for serum plus heparin and 93.3 +/- 30.9 U/l for plasma prepared with heparin. The mean for each group was significantly different from the means for the other groups. These data demonstrate that GGT activity measurements should be made on serum in the absence of heparin, which produces elevated results.

Effect of hepatic artery ligation on liver metabolism in rats.

The rat is an experimental model of orthotopic liver transplant that does not need arterial revascularization of the transplanted liver. The objective of the present study was to investigate the effect of hepatic artery ligation on hepatic metabolism in rats. After 2, 3 or 24 h of hepatic artery ligation, no significant differences in serum levels of alanine aminotransferase were observed. Bile flow decreased significantly 3 h after arterial ligation (P less than 0.05) and returned to normal after 24 h (P less than 0.05).

Heparin-induced increase in plasma and serum gama-glutamyl transpeptidase activity

Gama-Glutamyl transpeptidase (GGT) activity may be a sensitive index of hepatobiliary dysfunction. GGT activity, however, has been shown to be affected by several factors in different clinical situations. The present study was designed to determine the effect of heparin on serum and plasma GGT activity. Blood samples were collected from 15 normal male Wistar rats weighing 200-250 g for the determination of GGT activity in plasma prepared with heparin (group P(H),N = 10), in serum (group S,N = 15) and in serum with heparin added (group S + H,N = 10). GGT activity was 45.1 + or - 9.5 U/l (mean + or - SD) for serum compared with 161.2 + or - 46.1 U/l for serum plus heparin and 93.3 + or - 30.9 U/l for plasma prepared with heparin. The mean for each group was significantly different from the means for the other groups. These data demonstrate that GGT activity measurements should be made on serum in the absence of heparin, which produces elevated results

Epidemiological aspects of acute viral hepatitis in Säo Paulo, Brazil

A necessidade de maior número de dados sobre prevalência das hepatites por vírus em nosso meio levou-nos a estudar a freqüência das hepatites por vírus na Unidade de Fígado de Säo Paulo. Foram estudados 154 pacientes atendidos consecutivamente de novembro de 1980 a novembro de 1984. O emprego de marcadores para hepatite A (anti-VHA-IgM), hepatite B (AgHBs, anti-HBc e anti HBs) e a ausência dos mesmos para hepatite näo-A, näo-B (NANB), permitiu verificar a freqüência das mesmas que foi respectivamente, de 52,6%, 27,3% e 20,1%. A hepatite A caracterizou-se pela maior freqüência em jovens, contacto prévio com doente ou ingestäo de alimento suspeito. Na hepatite B os dados epidemiológicos preponderantes foram transmissäo parenteral näo-transfusional, contacto prévio e alta incidência em profissionais de saúde. Na hepatite NANB predominaram os casos pós-transfusionais (34,5%). Ausência de antecedentes epidemiológicos foi observada em 30-40% dos pacientes dos três grupos