Specific roles for Group V secretory PLA2 in retinal iron-induced oxidative stress. Implications for age-related macular degeneration.

Iron accumulation and oxidative stress are hallmarks of retinas from patients with age-related macular degeneration (AMD). We have previously demonstrated that iron-overloaded retinas are a good in vitro model for the study of retinal degeneration during iron-induced oxidative stress. In this model we have previously characterized the role of cytosolic phospholipase A2 (cPLA2) and calcium-independent isoform (iPLA2). The aim of the present study was to analyze the implications of Group V secretory PLA2 (sPLA2), another member of PLA2 family, in cyclooxygenase (COX)-2 and nuclear factor kappa B (NF-κB) regulation. We found that sPLA2 is localized in cytosolic fraction in an iron concentration-dependent manner. By immunoprecipitation (IP) assays we also demonstrated an increased association between Group V sPLA2 and COX-2 in retinas exposed to iron overload. However, COX-2 activity in IP assays was observed to decrease in spite of the increased protein levels observed. p65 (RelA) NF-κB levels were increased in nuclear fractions from retinas exposed to iron. In the presence of ATK (cPLA2 inhibitor) and YM 26734 (sPLA2 inhibitor), the nuclear localization of both p65 and p50 NF-κB subunits was restored to control levels in retinas exposed to iron-induced oxidative stress. Membrane repair mechanisms were also analyzed by studying the participation of acyltransferases in phospholipid remodeling during retinal oxidation stress. Acidic phospholipids, such as phosphatidylinositol (PI) and phosphatidylserine (PS), were observed to show an inhibited acylation profile in retinas exposed to iron while phosphatidylethanolamine (PE) showed the opposite. The use of PLA2 inhibitors demonstrated that PS is actively deacylated during iron-induced oxidative stress. Results from the present study suggest that Group V sPLA2 has multiple intracellular targets during iron-induced retinal degeneration and that the specific role of sPLA2 could be related to inflammatory responses by its participation in NF-κB and COX-2 regulation.

Fulfilling Koch's postulates confirms the monopartite nature of tomato leaf deformation virus: a begomovirus native to the New World.

The monopartite nature of the begomovirus tomato leaf deformation virus (ToLDeV) reported in Peru is demonstrated here. The DNA molecule cloned from an infected plant was shown to be fully infectious in tomatoes inducing leaf curling and stunted growth similar to that observed in field-infected plants. The viral DNA was reisolated from systemically infected tissues of inoculated plants, thus fulfilling Koch's postulates. ToLDeV was demonstrated, therefore, as the causal agent of the disease syndrome widespread in tomato crops in Peru. This virus was shown to be present throughout the major tomato-growing regions of this country, both in tomatoes and wild plants. Analyses of the sequences of 51 ToLDeV isolates revealed a significant genetic diversity with three major genetic types co-circulating in the population. A geographical segregation was observed which should be taken into account for virus control. Constraints to genetic divergence found for the C4 gene of ToLDeV isolates suggest a relevant function for this protein. The results obtained confirm ToLDeV as a monopartite begomovirus native to the New World, which is a significant finding for this region.

Non-alcoholic steatohepatitis: what can we learn from animal models?

Non-alcoholic fatty liver disease (NAFLD) is one of the most frequent causes of abnormal liver function and correlates with central adiposity, obesity, insulin resistance, the metabolic syndrome and type 2 diabetes mellitus. The pathological spectrum of NAFLD ranges from fatty liver to non-alcoholic steatohepatitis (NASH), advanced fibrosis, cirrhosis, and even hepatocellular carcinoma. Though NAFLD and NASH are becoming a major public health problem, ethical constraints on obtaining human liver tissue limit the interpretability of the data and the ability to delineate cause and effect from complex, interactive disease pathogenic pathways. Animal models of NASH can provide critical information leading to identify potential drug targets and to understand their molecular mechanisms, and are platforms for compound screening in drug development and for the assessment of novel therapeutic strategies. This review is aimed to offer an updated overview of the nutritional, genetic and pharmacologic animal models of NASH. Though the information derived from these models has clear relevance for the comprehension of the molecular basis of human disease, most of them fail to reproduce the full spectrum of liver pathology and the metabolic context that characterizes human NASH. Consequently, it is necessary to establish animal models that can best mimic the actual etiology, progression, and pathogenesis of the disease, and prove effectiveness for examining and selecting compounds with potential therapeutic benefit in NASH.

Hepatitis C virus, oxidative stress and steatosis: current status and perspectives.

Reactive oxygen and nitrogen species (ROS/RNS), whether produced endogenously as a consequence of normal cell functions or derived from external sources, pose a constant threat to cells living in an aerobic environment. When the production of ROS/RNS overrides the antioxidant capability of the target cells, oxidative damage may occur as a consequence of the interaction with DNA, protein, and lipids. Hepatitis C virus (HCV) is a major cause of viral hepatitis. Although the molecular mechanisms of HCV pathogenesis remain unclear, oxidative stress is emerging as a key step and a major initiator in the development and the progression of liver damage, and the evaluation of oxidative stress may be useful for a better understanding of the pathogenesis of hepatitis C. Liver steatosis is one of the most important histopathological features in patients with chronic hepatitis C. Both viral and host factors contribute to the development of steatosis, and putative defects caused by ROS/RNS may be involved through abnormalities in lipid metabolism. This review is aimed to offer an updated overview of the relationship between oxidative stress and HCV infection, focusing on the significance of ROS/RNS in the pathogenesis of liver disease. The potential role played by oxidative stress in the pathogenic mechanisms of HCV-related steatosis is also discussed.

Potential of flavonoids as anti-inflammatory agents: modulation of pro-inflammatory gene expression and signal transduction pathways.

Flavonoids are a large class of naturally occurring compounds widely present in fruits, vegetables, and beverages derived from plants. Reports have suggested that these compounds might be useful for the prevention of a number of diseases, partly due to their anti-inflammatory properties. It has been demonstrated that flavonoids are able to inhibit expression of isoforms of inducible nitric oxide synthase, ciclooxygenase and lipooxygenase, which are responsible for the production of a great amount of nitric oxide, prostanoids and leukotrienes, as well as other mediators of the inflammatory process such as cytokines, chemokines or adhesion molecules. Modulation of the cascade of molecular events leading to the over-expression of those mediators include inhibition of transcription factors such as nuclear factor kappa B, activator protein 1, signal transducers and activators of transcription, CCAAT/enhancer binding protein and others. Effects on the binding capacity of transcription factors may be regulated through the inhibition of protein kinases involved in signal transduction, such as mitogen activated protein kinases. Although the numerous studies published with in vitro approaches allow identifying molecular mechanisms of flavonoid effects, the limited bioavailability of these molecules makes necessary validation in humans. Whatever the case, the data available make clear the potential utility of dietary flavonoids or new flavonoid-based agents for the possible treatment of inflammatory diseases. The present review summarizes recent research data focusing on the modulation of the expression of different inflammatory mediators by flavonoids and the effects on cell signaling pathways responsible for their anti-inflammatory activity.

Anti-inflammatory properties of dietary flavonoids.

Flavonoids are a group of natural substances that are located in sources of vegetal origin. More than 4,000 varieties of flavonoids have been identified. All of them are phenyl-benzopyrones of low molecular weight with a basic structure formed by two benzene rings united through a heterocyclic pyrane or pyrone. Besides their relevance in plants, flavonoids are important for human health. Their antioxidant capacity confers a therapeutic potential in cardiovascular diseases, gastric or duodenal ulcers, cancer or hepatic pathologies. Also important are their antiviral and anti-allergic actions, as well as their anti-thrombotic and anti-inflammatory properties. Prostaglandins and nitric oxide biosynthesis is involved in inflammation, and isoforms of inducible nitric oxide synthase (iNOS) and of cyclooxygenase (COX-2) are responsible for the production of a great amount of these mediators. It has been demonstrated that flavonoids are able to inhibit both enzymes, as well as other mediators of the inflammatory process such as reactive C protein or adhesion molecules. Modulation of the cascade of molecular events leading to the overexpression of those mediators include inhibition of transcription factors such as nuclear factor kappa B and AP-1, through the inhibition of protein kinases involved in signal transduction. Increased antioxidant defenses through activation of the NF-E2 related factor 2 (Nrf2) also contribute to the anti-inflammatory capacity of flavonoids.

Anti-inflammatory properties of dietary flavonoids / Propiedades antiinflamatorias de los flavonoides de la dieta

Flavonoids are a group of natural substances that are located in sources of vegetal origin. More than 4,000 varieties of flavonoids have been identified. All of them are phenyl-benzopyrones of low molecular weight with a basic structure formed by two benzene rings united through a heterocyclic pyrane or pyrone. Besides their relevance in plants, flavonoids are important for human health. Their antioxidant capacity confers a therapeutic potential in cardiovascular diseases, gastric or duodenal ulcers, cancer or hepatic pathologies. Also important are their antiviral and anti-allergic actions, as well as their anti-thrombotic and anti-inflammatory properties. Prostaglandins and nitric oxide biosynthesis is involved in inflammation, and isoforms of inducible nitric oxide synthase (iNOS) and of cyclooxygenase (COX-2) are responsible for the production of a great amount of these mediators. It has been demonstrated that flavonoids are able to inhibit both enzymes, as well as other mediators of the inflammatory process such as reactive C protein or adhesion molecules. Modulation of the cascade of molecular events leading to the overexpression of those mediators include inhibition of transcription factors such as nuclear factor kappa B and AP-1, through the inhibition of protein kinases involved in signal transduction. Increased antioxidant defenses through activation of the NF-E2 related factor 2 (Nrf2) also contribute to the anti-inflammatory capacity of flavonoids

Los flavonoides son un grupo de las sustancias naturales que se encuentran en fuentes de origen vegetal, existiendo más de 4.000 variedades. Todos son fenilbenzopironas de peso molecular bajo con una estructura básica formada por dos anillos heterocíclicos de benceno unidos a través de un pirano o de una pirona. Además de su función en las plantas, los flavonoides son importantes para la salud humana. Su capacidad antioxidante confiere un potencial terapéutico en enfermedades cardiovasculares, úlceras gástricas o duodenales, cáncer o patologías hepáticas. También son importantes sus acciones antivirales y antialérgicas, así como sus características antitrombóticas y antiinflamatorias. La síntesis de prostaglandinas y de óxido nítrico está implicada en la inflamación, e isoformas de la óxido nítrico sintetasa (iNOS) y de la ciclooxigenasa (COX-2) son responsables de la producción de una gran cantidad de estos mediadores. Se ha demostrado que los flavonoides pueden inhibir ambas enzimas, así como otros mediadores del proceso inflamatorio tales como la proteína C reactiva o diversas moléculas de adhesión. La modulación de la cascada de los acontecimientos moleculares que conducen al aumento en la expresión de estos mediadores incluye la inhibición de factores de transcripción tales como el factor nuclear kappaB y el factor AP-1, a través de la inhibición de diferentes proteína quinasas. Otros factores, tales como el incremento de las defensas antioxidantes a través del factor Nrf2 pueden también contribuir a las propiedades antiinflamatorias de los flavonoides

Diagnostic imaging in sheep hepatic fascioliasis: ultrasound, computer tomography and magnetic resonance findings.

Radiological features and biochemical changes were investigated during the parenchymal and ductal phases of chronic Fasciola hepatica infection in sheep. The activities of plasma aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and gamma-glutamyl transpeptidase (GGT), plasma levels of IgG anti- F. hepatica and serial ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI) findings were studied in eight sheep infected with 150 F. hepatica metacercariae. Experimental fluke infection provoked an increase in plasma level of IgG directed against F. hepatica and in plasma LDH and AST activities from 4 weeks after infection. Enzyme activities did not significantly differ from the baseline after 15 and 12 weeks for LDH and AST, respectively. GGT activity increased from 9 weeks postinfection and still remained significantly elevated at 18 weeks. In the parenchymal phase, both CT and MRI showed nodular lesions in five animals and MRI could also detect early tracks in the subcapsular area in three sheep. Ultrasound findings were nonspecific in this phase. Ductal dilatation was shown by ultrasound, CT and MRI in almost all animals, although MRI was inferior to CT in depicting a mild ductal dilatation. Moving echogenic forms in the dilated bile ducts were observed by ultrasound from 9 weeks postinfection in seven of the eight sheep. Moving worms were only demonstrated in four sheep at CT and in a single sheep at MRI. This study shows that radiological findings may be useful in studies of fluke-induced liver damage.

High Genetic Stability of the Begomovirus Tomato yellow leaf curl Sardinia virus in Southern Spain Over an 8-Year Period.

ABSTRACT The evolution of the plant single-stranded DNA virus Tomato yellow leaf curl Sardinia virus (TYLCSV) (genus Begomovirus, family Geminiviridae) has been monitored for 8 years after its appearance in southern Spain. Variation within three genomic regions of 166 TYLCSV isolates collected from three locations was assessed by single-strand conformation polymorphism (SSCP) analysis. According to SSCP, the intergenic region (IR) was the most variable. Low genetic diversity was found within the population and geographical or temporal differences were not evident. Nucleotide sequences of specific genomic regions of haplotypes identified by SSCP indicated close relationships among them. Therefore, the Spanish TYLCSV population appears to represent a single, undifferentiated population. The analysis of IR sequences for a subsample of 76 randomly chosen isolates confirmed the limited genetic diversity revealed by the SSCP analysis. A tendency to a lineal increase in diversity over time was observed in Málaga and Almería subpopulations; however, no accumulation of mutations in single isolates was evident. Negative selection to variation seems to operate to conserve certain regions of the genome. Thus, the low genetic diversity found in the studied TYLCSV population might be the result of a founder effect with subsequent selection against less fit variants arising by mutation.

Effects of melatonin on fuel utilization in exercised rats: role of nitric oxide and growth hormone.

We have previously reported that melatonin modifies carbohydrate and lipid utilization in exercised rats, maintaining glycemia and reducing plasma and liver lactate and plasma beta-hydroxybutyrate. This study was undertaken to determine whether effects on fuel metabolism were related to changes in nitric oxide (NO) production or growth hormone (GH) secretion. Male Wistar rats received melatonin i.p. at a dose of 0.5 mg/kg body weight 30 min before being exercised to exhaustion on a treadmill at a speed of 24 m/min and a 12% slope. Melatonin ameliorated the decrease in plasma glucose and the increase in plasma urea, free fatty acid, beta-hydroxybutyrate, and nitrite induced by exercise. Melatonin-treated exercised rats had significantly elevated liver glycogen content and hepatic tissue showed a lowered expression of both inducible and constitutive NO synthase (iNOS and cNOS). Administration of the NO inhibitor NG-nitro-L-arginine (L-NAME) to exercised rats caused a significant reduction in plasma nitrite, but liver glycogen and biochemical parameters in blood did not significantly differ from untreated exercised animals, indicating the absence of a direct association between melatonin effects on fuel metabolism and NO levels. Although results of treatment with pyridostigmine, a cholinergic agonist drug that stimulates GH release, partially differed from that of melatonin, modulation of GH secretion could play a role in the metabolic actions of the hormone because effects of melatonin on exercised rats were almost completely blocked by simultaneous administration of L-NAME.

Natural recombination between Tomato yellow leaf curl virus-is and Tomato leaf curl virus.

The complete genome sequences (2791 and 2793 nt) of isolates of Tomato yellow leaf curl virus-Is (TYLCV-Is) from Spain (SP72/97) and Portugal (Port2/95) were determined. These isolates are closely related to TYLCV-Is isolates reported in Japan (Japan-A and Japan-S) and Israel (Israel/Mild). Comparison of all sequenced isolates of TYLCV-Is showed that part of the genome comprising the intergenic region and the 5'-end of the rep gene of the Iran and Israel isolates was not closely related to that of other isolates. Phylogenetic analyses suggest that the Israel and Iran isolates may have chimeric genomes that have arisen by recombination between TYLCV-Is-like and tomato leaf curl virus (ToLCV)-like ancestors. The TYLCV-Is donors of the Iran and the Israel genomes were closely related to each other and to other known TYLCV-Is isolates. However, the ToLCV donors differed from each other, although both were related to ToLCV isolates from India (Bangalore-2 and Bangalore-4).

Morphologic and biochemical changes caused by experimentally induced dicroceliosis in hamsters (Mesocricetus auratus).

BACKGROUND AND PURPOSE: The aim of the study reported here was to investigate the pathomorphologic changes caused by experimentally induced dicroceliosis and their correlation with hepatobiliary function. METHODS: Studies were carried out at days 80 and 120 after oral inoculation of hamsters with 40 metacercariae of Dicrocoelium dendriticum. RESULTS: The parasite-induced pathologic changes were assessed by presence of fluke eggs in feces, increased plasma alanine transaminase and aspartate transaminase activities and morphologic alterations. Dicroceliosis was characterized by bile ductular proliferation and enlargement of the bile duct surface area caused by hyperplastic cholangitis in septal bile ducts. The liver from infected animals contained portal tracts infiltrated with small to moderate numbers of lymphocytes, macrophages, and eosinophils. Simultaneously, there was an increase in portal tract collagen that extended to the interlobular septa and caused pressure atrophy of the hepatic parenchyma. The concentration of thiobarbituric acid-reactive substances and the ratio of oxidized to reduced glutathione, measured as markers of oxidative stress, were significantly increased. CONCLUSIONS: The presence of oxidative alterations could be related to the morphologic evidence of chronic inflammatory response as well as to liver cellular injury indicated by cellular swelling, and increased presence of peroxisomes and lysosomes.

Oxidative stress and changes in liver antioxidant enzymes induced by experimental dicroceliosis in hamsters.

The purpose of our study was to assess the effects of experimental dicroceliosis on the antioxidant defense capability of the liver in hamsters. Studies were carried out at 80 and 120 days after infection with an oral dose of 40 metacercariae of Dicrocoelium dendriticum. The parasitic pathology was ascertained by the presence of fluke eggs in feces, increased serum ALT and AST activities, and histological findings. The concentration of thiobarbituric acid-reactive substances (TBARS) and the ratio of oxidized to reduced glutathione (GSSG/GSH), measured as markers of oxidative stress, were significantly increased [TBARS: +40% and +84% at 80 and 120 days postinfection (p.i.), respectively; GSSG/GSH: +200% and +117%]. Dicroceliosis increased Se-dependent glutathione peroxidase (GPx) activity in both cytosol (+24% and +46%) and mitochondria (+73% and +41%). Superoxide dismutase activity was significantly reduced in cytosol (-19% and -38%) and mitochondria (-20% and -39%). No significant change was found in the activity of Se-independent GPx or catalase. The ratio of glutathione peroxidase/glutathione reductase at 80 and 120 days p.i. was increased by 25% and 63%, respectively. Gamma-glutamyl cysteinyl synthetase activity was increased by 27% and 20%, respectively. Our data indicate that although dicroceliosis courses with activation of antioxidant enzymes and glutathione synthesis, inefficient scavenging of reactive oxygen species takes place, resulting in oxidative liver damage.

Displacement of Tomato Yellow Leaf Curl Virus (TYLCV)-Sr by TYLCV-Is in Tomato Epidemics in Spain.

ABSTRACT A progressive displacement of tomato yellow leaf curl virus (TYLCV)-Sr by TYLCV-Is was observed in tomato epidemics in southern Spain based on incidence data of both virus species obtained during surveys conducted between 1996 and 1998. Ecological factors that might be involved in such a displacement, such as competition of TYLCV-Sr and TYLCV-Is in tomato, transmission by local biotypes (B and Q) of Bemisia tabaci, and presence in weeds and alternate crops, have been analyzed. No selective advantage is observed for TYLCV-Sr or TYLCV-Is in tomato plants either infected via Agrobacterium tumefaciens or via B. tabaci. However, TYLCV-Is is more efficiently vectored by local biotypes of B. tabaci; and common bean, a bridge crop between tomato crops, is a host for TYLCV-Is but not TYLCV-Sr. Therefore, common bean acts as a reservoir for TYLCV-Is. These two factors are probably responsible for the displacement of TYLCV-Sr by TYLCV-Is as the causative agent of epidemics in tomato in southern Spain.

Enhanced bile formation induced by experimental dicrocoeliosis in the hamster.

The purpose of this investigation was to determine the effects of experimental dicrocoeliosis on bile formation in the hamster. Studies were carried out at 120 days after infection with an oral dose of 40 metacercariae of Dicrocoelium dendriticum. A significant elevation in bile flow (+20%) and in the biliary output of glutathione (+34%), bile acid (+59%), cholesterol (+108%), phospholipids (+99%) and alkaline phosphatase (+36%) was observed in the infected animals. The bile-to-plasma [14C] mannitol ratio increased to values greater than 1 and there was a reduced contribution (-26%) of biliary tree to bile formation. Those data suggest that enhancement in choleresis had a canalicular origin. The presence of oxidative stress, evidenced by the increased oxidized/reduced glutathione ratio and TBARS concentrations, may contribute to the elevated glutathione efflux into bile. Enhancement in bile acid output was not due to qualitative or quantitative changes in bile acid metabolism, as indicated by the absence of significant modification in liver cholesterol 7alpha-hydroxylase activity and bile acid profile in bile. Increase in the ability of the canalicular membrane to export bile acids was not involved, since maximal secretion rate for exogenously administered taurocholate was decreased. When bile flow, bile acid and biliary lipid secretion was determined in colchicine-pretreated animals differences between control and infected animals were abolished, suggesting that stimulation of the transcytotic vesicle pathway plays an important role in the alteration of the biliary function caused by dicrocoeliosis.

Improvement of the print-capture polymerase chain reaction procedure for efficient amplification of DNA virus genomes from plants and insect vectors.

A rapid and simple procedure is described to amplify efficiently geminivirus DNA genomes by improving the print-capture polymerase chain reaction (PCR) procedure reported recently for RNA viruses. This method, termed print-PCR (P-PCR), allows direct amplification of DNA from infected plant or whitefly tissues printed directly on Whatman 3MM paper, without the need of any grinding, incubation, or washing steps previous to the amplification reaction. P-PCR reduces sample manipulation and avoids previous extraction of nucleic acids, thereby diminishing the possibilities of cross-contamination between samples. P-PCR has been successfully applied to whiteflies and various plant species infected by two different tomato yellow leaf curl viruses, TYLCV-Sr and TYLCV-Is, and for the amplification of the full-length genome of TYLCV-Is from infected plants.

Cholestasis and alterations of glutathione metabolism induced by tacrolimus (FK506) in the rat.

BACKGROUND: Tacrolimus (FK506) is an immunosuppressive agent used for the prevention of allograft rejection after organ transplantation. The aim of this study was to investigate the effects of chronic tacrolimus treatment on bile secretion in rats. METHODS: Tacrolimus was administered intraperitoneally at doses of 0.2, 0.5, and 0.8 mg/kg/day for 6 weeks. RESULTS: Bile flow was significantly reduced at doses of 0.5 mg/kg and 0.8 mg/kg (-25% and -32%, respectively). Bile acid secretion was not significantly modified, but bicarbonate secretion decreased at doses of 0.5 mg/kg and 0.8 mg/kg (-23% and -29%, respectively). Glutathione secretion was significantly reduced at doses of 0.5 mg/kg (-29%) and 0.8 mg/kg (-49%). Liver glutathione concentration was reduced at the higher dose (-17%). Liver gamma-glutamyl-cysteinyl synthetase activity was elevated (+22%, +10, and +15%) and gamma-glutamyl transpeptidase activity was reduced (-18%, -40%, and -25%) at all doses. Dichlorofluorescein and thiobarbituric acid-reactive substance concentrations were not significantly modified. Liver glutathione peroxidase activity increased at doses of 0.5 mg/kg (+65%) and 0.8 mg/kg (+56%). Kidney concentration of thiobarbituric acid-reactive substances was significantly increased at doses of 0.5 mg/kg (+17%) and 0.8 mg/kg (+12%). CONCLUSIONS: Our data indicate that tacrolimus at high doses induces cholestasis by inhibiting primarily biliary excretion of glutathione and, to a lesser extent, bicarbonate. The decrease in biliary glutathione secretion is not due to a lower synthesis or degradation and could be related to its increased sinusoidal efflux.

First Report of Tomato Yellow Leaf Curl Virus-Is in Spain: Coexistence of Two Different Geminiviruses in the Same Epidemic Outbreak.

Epidemics of tomato yellow leaf curl have occurred annually in greenhouse- and field-grown tomato (Lycopersicon esculentum Mill.) crops in southern Spain since 1992 (2). The nucleotide sequences of two tomato yellow leaf curl virus (TYLCV) isolates from this region, TYLCV-M (GenBank accession no. Z25751) and TYLCV-Alm (L27708), have been determined and these isolates are closely related to isolates reported from Italy (X61153 and Z28390), suggesting the existence of a geographical cluster of closely related TYLCV isolates in the Western Mediterranean Basin (2). In June 1997, new and unusually severe symptoms of stunting, yellowing, and curling of leaflet margins, with a marked reduction in leaf size, were observed in some plants of a greenhouse-grown tomato crop in Almeria (southeastern Spain). Tomato plants showing milder symptoms similar to those previously described for TYLCV infection in that region (2) were also present in the same greenhouse. Total nucleic acids extracts from plants exhibiting both types of symptoms were analyzed by dot blot hybridization with a probe prepared by random priming on a 1,674-bp SalI fragment of the pSP95 clone of TYLCV-M (3). A strong reaction was obtained with the samples that showed mild symptoms, whereas a weak reaction was observed with the severely affected plants. Specific pairs of primers were prepared to amplify the complete pre-coat (V1) (MA10: 5'-ATGTGGGATCCTTTATTAAATG-3'; MA11: 5'-TCAGGGCTTCTGTACATTC-3') and C2 (MA12: 5'-TAAAGACTCTTAAAAAATGACC-3'; MA13: 5'-AATGCAATCTTCGTCACC-3') genes based on TYLCV-M sequence. With polymerase chain reaction (PCR), the expected fragments were amplified from extracts of both types of plants. The PCR products were submitted to single-strand conformation polymorphism (SSCP) analysis. Clearly distinguishable SSCP patterns were obtained: one for the plants with mild symptoms, identical to that of known TYLCV-M infected plants, and another for the plants with more severe symptoms. Further analyses done on tomato samples collected from the same area showed that both SSCP patterns were present simultaneously in several severely affected plants. The nucleotide sequences of the V1 and C2 PCR products from two samples differing in their SSCP pattern were obtained by direct sequencing, and compared with available TYLCV sequences. The sequences corresponding to the sample with mild symptoms were 100% identical to those previously reported for TYLCV-M. In contrast, the sequences from the sample that showed severe symptoms (GenBank accesion no. AF022219 for V1, and AF022220 for C2) were only 80 and 76% identical to TYLCV-M V1 and C2 genes, respectively, but were 99% identical to the sequence reported for an isolate of TYLCV-Is from Israel (X15656). Epidemics in tomato caused by TYLCV-Is have been recently reported from Portugal (1). Our results demonstrate that the unusually severe symptoms observed are associated with an isolate of TYLCV-Is that coexists in the field with the milder TYLCV previously reported from this area. This is the first report of the occurence of TYLCV-Is in Spain. References: (1) D. Louro et al. Plant Dis. 80:1079, 1996. (2) E. Noris et al. Arch. Virol. 135:165, 1994.

Effects of experimental dicrocoeliosis on oxidative drug metabolism in hamster liver.

The purpose of this investigation was to determine the effects of experimental dicrocoeliosis on the hepatic oxidative drug-metabolizing system in hamsters. Studies were carried out 80 and 120 days after infestation with an oral dose of 40 metacercariae of Dicrocoelium dendriticum. The parasitic pathology was ascertained by detection of the fluke eggs in faeces, increased serum alanine aminotransferase and aspartate aminotransferase activities, and postmortem and histological findings. Cytochrome P-450 concentration, aniline hydroxylase activity and ethoxycoumarin O-deethylase activity were significantly decreased in both groups of infected animals. Aminopyrine N-demethylase activity and erythromycin N-demethylase activity were only reduced 120 days after infection. Effects on drug metabolizing enzymes were unrelated to changes in the physical state of the microsomal membrane, as assessed by measurement of fluorescence polarization. The results of this study indicate that the capacity of the liver for handling drugs and xenobiotics may be impaired as a consequence of dicrocoeliosis.