Where to look for the genes related to diaphragmatic hernia?

Analysis of approximately 150 published observations of diaphragmatic hernia (DH) in persons with structural autosomal imbalance showed several segments where DH-related genes may be found. Occurrence of DH in several patients with deletions 15q26, 8p23, 8q22, 4p16, 1q42, and 3q22 allows to propose that these segments harbor the genes which, when deleted (or truncated) may be responsible for DH. Segments 22q11, 4q28.3q32, 1q25q31.2 and 2p23p25 are good candidates for the location of genes which cause DH in trisomic condition. The genetic mechanisms of DH in tetrasomy 12p are not clear, although more than 50 cases of DH have been reported in this syndrome. Frequent coexistence of congenital heart defects and DH in some syndromes (and rarity of this association in some others) may suggest the different pathways of the DH's origin.     

Dissociation between enhanced resistance and delayed hypersensitivity induced with subcellular preparations from Listeria monocytogenes and the adjuvant dimethyl-dioctadecyl-ammonium bromide

In this study we investigated the relation between enhanced resistance and delayed hypersensitivity (DH) induced with subcellular preparations from Listeria monocytogenes and the adjuvant dimethyldioctadecylammonium bromide (DDA). Ribosomal RNA as well as cell envelope fragments (fraction I) protected mice against lethal Listeria infection. However, only fraction I induced DH against killed Listeria. For the induction of protection with fraction I or RNA as well as for the induction of DH with fraction I, preparations had to be administered in combination with DDA. Fraction I elicited a DH response in mice immunized with viable Listeria, but RNA did not. These observations pointed to a dissociation between DH and enhanced resistance induced with RNA, and to a dissociation between fraction I and RNA with respect to their ability to induce or elicit DH. Also DH and enhanced resistance induced with fraction I could be dissociated. Intracutaneous administration of fraction I induced high levels of DH without concomitant induction of protection against lethal challenge with Listeria. On the other hand, intraperitoneal administration of fraction I fully protected mice against lethal infection, but only induced a moderate DH response. DH induced with fraction I was largely specific, whereas enhance resistance induced with this preparation was nonspecific. Finally, proteinase K-sensitive proteins were found to be essential for the induction of DH but not for the induction of protection with fraction I.     

Critical but distinct roles for the pleckstrin homology and cysteine-rich domains as positive modulators of Vav2 signaling and transformation

Vav2, like all Dbl family proteins, possesses tandem Dbl homology (DH) and pleckstrin homology (PH) domains and functions as a guanine nucleotide exchange factor for Rho family GTPases. Whereas the PH domain is a critical positive regulator of DH domain function for a majority of Dbl family proteins, the PH domains of the related Vav and Vav3 proteins are dispensable for DH domain activity. Instead, Vav proteins contain a cysteine-rich domain (CRD) critical for DH domain function. We evaluated the contribution of the PH domain and the CRD to Vav2 guanine nucleotide exchange, signaling, and transforming activity. Unexpectedly, we found that mutations of the PH domain impaired Vav2 signaling, transforming activity, and membrane association. However, these mutations do not influence exchange activity on Rac and only slightly affect exchange on RhoA and Cdc42. We also found that the CRD was critical for the exchange activity in vitro and contributed to Vav2 membrane localization. Finally, we found that phosphoinositol 3-kinase activation synergistically enhanced Vav2 transforming and signaling activity by stimulating exchange activity but not membrane association. In conclusion, the PH domain and CRD are mechanistically distinct, positive modulators of Vav2 DH domain function in vivo.     

NAD-linked, factor-dependent formaldehyde dehydrogenase or trimeric, zinc-containing, long-chain alcohol dehydrogenase from Amycolatopsis methanolica.

NAD-linked, factor-dependent formaldehyde dehydrogenase (FD-FA1DH) of the Gram-positive methylotrophic bacterium, Amycolatopsis methanolica, was purified to homogeneity. It is a trimeric enzyme with identical subunits (molecular mass 40 kDa) containing 6 atoms Zn/enzyme molecule. The factor is a heat-stable, low-molecular-mass compound, which showed retention on an Aminex HPX-87H column. Inactivation of the factor occurred during manipulation, but activity could be restored by incubation with dithiothreitol. The identity of the factor is still unknown. It could not be replaced by thiol compounds or cofactors known to be involved in metabolism of C1 compounds. Of the aldehydes tested, only formaldehyde was a substrate. However, the enzyme showed also activity with higher aliphatic alcohols and the presence of the factor was not required for this reaction. Methanol was not a substrate, but high concentrations of it could replace the factor in the conversion of formaldehyde. Presumably, a hemiacetal of formaldehyde is the genuine substrate, which, in the case of methanol, acts as a factor leading to methylformate as the product. This view is supported by the fact that formate could only be detected in the reaction mixture after acidification. Inhibition studies revealed that the enzyme contains a reactive thiol group, being protected by the binding of NAD against attack by heavy-metal ions and aldehydes. Studies on the effect of the order of addition of coenzyme and substrate suggested that optimal catalysis required NAD as the first binding component. Substrate specificity and the induction pattern clearly indicate a role of the enzyme in formaldehyde oxidation. However, since FD-FA1DH was also found in A. methanolica grown on n-butanol, but not on ethanol, it may have a role in the oxidation of higher aliphatic alcohols as well. FD-FA1DH and the factor from A. methanolica are very similar to a combination already described for Rhodococcus erythropolis [Eggeling, L. & Sahm, H. (1985) Eur. J. Biochem. 150, 129-134]. NAD-linked, glutathione-dependent formaldehyde dehydrogenase (GD-FA1DH) resembles FD-FA1DH in many respects. Since glutathione has so far not been detected in Gram-positive bacteria, FD-FA1DH could be the counterpart of this enzyme in Gram-positive bacteria. Alignment of the N-terminal sequence (31 residues) of FD-FA1DH with that of GD-FA1DH from rat liver indeed showed similarity (30% identical positions). However, comparable similarity was found with class I alcohol dehydrogenase from this organism and with cytosolic alcohol dehydrogenase from Saccharomyces cerevisiae, isozyme 1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Different regulation of the Trio Dbl-Homology domains by their associated PH domains

Guanine nucleotide exchange factors for Rho-GTPases (Rho-GEFs) invariably share a catalytic Dbl-Homology (DH) domain associated with a Pleckstrin Homology (PH) domain, whose function in Rho-GEF activation is not well understood. Trio is the first member of an emerging family of Dbl proteins containing two Rho-GEF domains (GEFD1 and GEFD2). TrioGEFD1 activates the GTPases RhoG and Rac1, while TrioGEFD2 acts on RhoA. In this study, we have investigated the roles of the two PH domains of Trio in Rho-GEF activity. We show that TrioPH1 is required for GEFD1-mediated induction of actin cytoskeleton remodeling and JNK activation. TrioPH1 is involved both in the catalytic activity and in the subcellular localization of its associated DH domain, by acting as a cytoskeletal targeting signal. Moreover, TrioPH1 in association with DH2 activates the JNK pathway, by an unknown mechanism independent of DH2 catalytic activity. TrioPH2 does not behave as a targeting module in intact cells. TrioPH2 inhibits DH2-dependent stress fiber formation, which correlates with the TrioPH2-mediated inhibition of DH2 GEF activity. In addition, expression in the neuron-like PC12 cell line of the intact Trio protein deleted of each PH domain shows that only TrioPH1 is required for Trio-induced neurite outgrowth. Taken together, these data demonstrate that the two PH domains play a different role in the control of Trio Rho-GEF function.     

海藻酸钠/壳聚糖微胶囊固定化大肠杆菌的研究

本文以大肠杆菌ＤＨ5α为模型体系 ,探索了大肠杆菌ＤＨ5α用海藻酸钠 壳聚糖 (ＡＣＡ)微胶囊培养的可行性 ,并观察了微囊化大肠杆菌ＤＨ5α细胞生长与物料渗透性能 ,通过将ＡＣＡ微胶囊移植到实验组小鼠体内 ,考察了ＡＣＡ微胶囊作为口服药物载体的可能性。1　材料和方法1.1　材料壳聚糖 ,本实验室改性所得 ;海藻酸钠 ,ＫｅｌｃｏＤｉｖｏｆＭｅｒ ｃｋＣｏ .Ｉｎｃ .ＵＳＡ ;其它试剂均为国产分析纯。大肠杆菌ＤＨ5α ,长春生物制品所 ;ＬＢ培养基 ,华美生物制品公司提供。昆明系小白鼠 18～ 2 0ｇ ,解放军大连高等医学专科学校实验动物中…     

Conformational aspects and hyperpotent agonists of diapause hormone for termination of pupal diapause in the corn earworm

Diapause hormone (DH) is a peptide well known to induce embryonic diapause in the commercial silkmoth Bombyx mori. More recently, this same neuropeptide was reported to break diapause in pupae of the agriculturally important Heliothis/Helicoverpa complex. In this study we examine the efficacy and potency of a select group of structural analogs of the native hormone in Helicoverpa zea and report the structures of several analogs that are considerably more potent than DH in breaking diapause. Among the most potent analogs (PK-Etz, PK-2Abf, 901) were those with structural components that enhance resistance to peptidases that degrade and inactivate the native peptide in vivo, which may account, at least in part, for the observed increase in potency for these analogs. Analog 901 was previously demonstrated to both enhance biostablility and bioavailability properties in adult heliothines and thus may be a potential candidate for topical application as a diapause-terminating agent. The significant activity observed for two restricted conformation analogs is consistent with an active conformation for diapause hormone that features a transPro within a type I beta-turn in the C-terminal region. DH is also known to successfully break diapause only within a fairly narrow temperature range. While DH is effective at 21 degrees C, it is not effective at 18 degrees C. Likewise, the analogs were effective at 21 degrees C but not at 18 degrees C. By contrast, 20-hydroxyecdysone, a steroid hormone that is also capable of breaking diapause is effective at both temperatures, thus suggesting that DH and the ecdysteroids act through different mechanisms to terminate diapause.     

Topoisomerase I associates specifically with simian virus 40 large-T-antigen double hexamer-origin complexes

Topoisomerase I (topo I) is required for releasing torsional stress during simian virus 40 (SV40) DNA replication. Recently, it has been demonstrated that topo I participates in initiation of replication as well as in elongation. Although T antigen and topo I can bind to one another in vitro, there is no direct evidence that topo I is a component of the replication initiation complex. We demonstrate in this report that topo I associates with T-antigen double hexamers bound to SV40 origin DNA (T(DH)) but not to single hexamers. This association has the same nucleotide and DNA requirements as those for the formation of double hexamers on DNA. Interestingly, topo I prefers to bind to fully formed T(DH) complexes over other oligomerized forms of T antigen associated with the origin. High ratios of topo I to origin DNA destabilize T(DH). The partial unwinding of a small-circular-DNA substrate is dependent on the presence of both T antigen and topo I but is inhibited at high topo I concentrations. Competition experiments with a topo I-binding fragment of T antigen indicate that an interaction between T antigen and topo I occurs during the unwinding reaction. We propose that topo I is recruited to the initiation complex after the assembly of T(DH) and before unwinding to facilitate DNA replication.     

Distribution and activity of a Dippu DH31-like peptide in the large milkweed bug Oncopeltus fasciatus

The milkweed bug, Oncopeltus fasciatus, is a plant feeding hemipteran. While there has been much research done on the neurohormonal control of the post-feeding diuresis in the blood-feeding hemipteran, Rhodnius prolixus, little is known about the control of the post-feeding diuresis in O. fasciatus. One of the neurohormones that may play a role in this rapid diuresis belongs to the calcitonin-like diuretic hormone (DH31) family of insect peptides. In this study we demonstrate the presence of DH31-like immunoreactivity in the central nervous system (CNS) and gut of O. fasciatus 5th instars. As well, DH31-like material was quantified and partially purified from the CNS of 5th instar O. fasciatus using reversed-phase liquid chromatography (RPLC) and monitored with an enzyme-linked immunosorbent assay (ELISA). When tested on O. fasciatus 5th instar Malpighian tubules, DH31-like peptides significantly increased the rate of secretion over saline controls. The results suggest that there is a DH31-like peptide(s) present in the CNS of O. fasciatus and that this peptide may play a role in the control of Malpighian tubule secretion.     

A DNase I hypersensitive site flanks an origin of DNA replication and amplification in Sciara

In chromosomes of metazoa, the assembly of the genome into chromatin makes an important but poorly understood contribution to determining where DNA replication will initiate. We addressed this issue by studying the developmental progression of the location of the DNA replication origin (ORI) and alterations in chromatin structure in one of the best-mapped ORIs in metazoa, that found in DNA puff II/9A of the fly Sciara coprophila. We found that DNA synthesis for both normal chromosomal endoduplication and DNA amplification initiates within the same 5.5 kb EcoRI fragment. We showed that irrespective of the mode of ORI function--replication or amplification--chromatin over the 1 kb major ORI is never remodeled into a conventional DNase I hypersensitive site (DH site). Instead, we found that the major site of alterations to chromatin structure at this locus is a large (approximately 400 bp) DH site located 600 bp away from the major ORI, at a position where the frequency of replication initiation events falls dramatically. We describe a tight positive correlation between ORI activity, strength of this DH site, and the intranuclear titer of protein factor(s) that bind the DH site in a sequence-specific manner. We propose that the Sciara replicator in locus II/9A is composed of sequences that reside within the ORI per se as well as sequences encompassed by the DH site.     

Anxiolytic-like effects of substance P administration into the dorsal, but not ventral, hippocampus and its influence on serotonin

Substance P (SP) is known to be involved in processes related to learning and memory, fear, anxiety and stress. SP and NK1 receptors are localized in the hippocampus, a brain structure involved in learning and memory as well as emotional processes. As there is evidence for differential functions of the ventral (VH) and dorsal (DH) hippocampus in a variety of behaviors, we here evaluated the effects of injections of SP into the VH and DH in rats submitted to the elevated plus-maze (EPM) and open field (OF) tests. The results obtained showed that infusions of 100 and 1000 ng of SP into the DH, but not VH, increased open arm activity in the EPM and in the central zone of the OF, indicative of anxiolytic-like action. These effects were observed in the absence of significant changes in general motor activity. In an additional experiment to examine whether these effects of SP are mediated by local serotoninergic mechanisms, extracellular concentrations of this monoamine were assessed by use of in vivo microdialysis. Infusions of SP into the DH did not influence the extracellular concentration of serotonin. These data indicate that neurokinins in the DH, but not VH, are involved in mechanisms associated with anxiety and that the mediation of SP in anxiety-related behaviors is independent of local serotonergic mechanisms.     

Characterization of circulating and cutaneous IgA immune complexes in patients with dermatitis herpetiformis

Dermatitis herpetiformis (DH) is a chronic, blistering skin disease characterized in part by deposits of IgA at the dermal-epidermal junction. Eighty-five percent of DH patients have granular IgA deposits and have an associated gluten-sensitive enteropathy (GSE). In contrast, 15% of DH patients have a linear pattern of IgA deposits and no associated intestinal abnormality. Although circulating IgA antibodies against skin are not present in these patients, 40% of DH patients do have IgA-containing circulating immune complexes (IgA-CIC). The role and origin of the cutaneous IgA and the IgA-CIC in patients with DH are unknown; however, the association of GSE with the granular IgA deposits suggests that a mucosal immune response may be important in the pathogenesis of DH. We have characterized the IgA subclass composition of the cutaneous IgA deposits in patients with DH, and have isolated and characterized the IgA-CIC from these patients. Twenty-nine of 29 patients with DH and granular IgA deposits were found to have only IgA1 deposits. Ten of 11 patients with linear IgA deposits also had only IgA1 deposits; one of 11 had IgA2 deposits. Isolated IgA-CIC from the sera of eight patients with DH and granular IgA deposits were found to contain both IgA1 (58% +/- 5, mean percent of total IgA +/- SEM) and IgA2 (42% +/- 5), as were IgA-CIC from two patients with ordinary GSE without cutaneous IgA deposits. The IgA subclass composition of the isolated immune complexes was significantly different from the serum IgA1 and IgA2 composition (serum IgA1 = 76% +/- 6; IgA2 = 24% +/- 5, p less than 0.025, Student's t-test), and suggests that the IgA-CIC may arise from gut-associated lymphoid tissue (GALT). Sequential anti-IgA1 absorption of serum which contained IgA-CIC did not remove all the IgA-CIC, suggesting that the complexes circulate as separate IgA1 and IgA2 complexes. The finding of IgA1 alone in the skin of patients with DH suggests that the cutaneous IgA may not arise from GALT, or that IgA1, possibly arising in GALT, is preferentially bound to DH skin. Because IgA-containing CIC which contain both IgA1 and IgA2 were found in the serum of patients with DH and with ordinary GSE, it seems unlikely that IgA-containing CIC are responsible for the cutaneous IgA deposits seen in DH.     

Dermatitis herpetiformis sera or goat anti-transglutaminase-3 transferred to human skin-grafted mice mimics dermatitis herpetiformis immunopathology

Dermatitis herpetiformis (DH) is characterized by deposition of IgA in the papillary dermis. However, indirect immunofluorescence is routinely negative, raising the question of the mechanism of formation of these immune deposits. Sárdy et al. (2002. J. Exp. Med. 195: 747-757) reported that transglutaminase-3 (TG3) colocalizes with the IgA. We sought to create such deposits using passive transfer of Ab to SCID mice bearing human skin grafts. IgG fraction of goat anti-TG3 or control IgG were administered i.p. to 20 mice. Separately, sera from seven DH patients and seven controls were injected intradermally. Biopsies were removed and processed for routine histology as well as direct immunofluorescence. All mice that received goat anti-TG3 produced papillary dermal immune deposits, and these deposits reacted with both rabbit anti-TG3 and DH patient sera. Three DH sera high in IgA anti-TG3 also produced deposits of granular IgA and TG3. We hypothesize that the IgA class anti-TG3 Abs are directly responsible for the immune deposits and that the TG3 is from human epidermis, as this is its only source in our model. These deposits seem to form over weeks in a process similar to an Ouchterlony immunodiffusion precipitate. This process of deposition explains the negative indirect immunofluorescence results with DH serum.     

Influence of cytokine inhibitors on concentration and activity of MMP-1 and MMP-3 in disc herniation

Introduction  

Spontaneous resorption of disc herniation (DH) after sciatica is well documented. The matrix metalloproteinases (MMP)-1 and MMP-3 are enzymes potentially involved in this process. Glucocorticoid injections are commonly used for treatment, and other anti-inflammatory molecules like tumor necrosis factor (TNF) inhibitors are under clinical investigation. However, little is known about the effect of these molecules on DH resorption.     

The role and proposed mechanism by which oestradiol 17β-hydroxysteroid dehydrogenase regulates breast tumour oestrogen concentrations

Synthesis of the biologically active oestrogen, oestradiol, within breast tumours makes an important contribution to the high concentrations of oestrogens which are present in malignant breast tissues. In breast tumours, oestrone is preferentially converted to oestradiol by the Type I oestradiol 17β-hydroxysteroid dehydrogenase (E2DH). Several growth factors, such as insulin-like growth factor Type I, and cytokines, such as Tumour Necrosis Factor (TNF), have been shown to stimulate E2DH activity in MCF-7 breast cancer cells. As little is known about the regulation of Type I E2DH expression and activity in other breast cancer cell lines, the expression and activity of this enzyme was examined in other oestrogen receptor positive and also oestrogen receptor negative breast cancer cell lines. As it is possible that E2DH activity may be limited by co-factor availability, the effects of exogenous co-factors on enzyme activity in these cell lines was also investigated. For T47D and BT20 breast cancer cells, the addition of exogenous co-factors was found to enhance enzyme activity. TNF, in addition to stimulating E2DH activity in MCF-7 cells, also increased activity in T47D and MDA-MB-231 cells, although to a lesser extent than in MCF-7 cells. An investigation of signalling pathways involved in the regulation of E2DH activity revealed that stimulation of both the protein kinase C (PKC) and PKA pathways may be involved in regulation of E2DH activity. As several growth factors and cytokines have now been found to be involved in regulating E2DH activity, the role that macrophages and lymphocytes have in supplying these factors and the mechanism by which these factors may stimulate tumour growth, is also reviewed.     

Critical role of the pleckstrin homology domain in Dbs signaling and growth regulation

Dbl family proteins act as guanine nucleotide exchange factors and positive regulators of Rho GTPase function by stimulating formation of the active, GTP-bound state. All Dbl family Rho guanine nucleotide exchange factors possess an invariant tandem domain structure consisting of a Dbl homology (DH) catalytic domain followed by a pleckstrin homology (PH) regulatory domain. We determined previously that the PH domain of Dbs was critical for the intrinsic catalytic activity of the DH domain in vitro and for Dbs transformation in vivo. In this study, we evaluated the role of phosphoinositide binding to the PH domain in regulating the DH domain function of Dbs in vitro and in vivo. We determined that mutation of basic amino acids located within the beta1-beta2 and beta3-beta4 loops of the PH domain resulted in impaired phospholipid binding in vitro, yet full guanine nucleotide exchange activity in vitro was retained for RhoA and Cdc42. Surprisingly, these mutants were compromised in their ability to activate Rho GTPases in vivo and to cause transformation of NIH 3T3 cells. However, Dbs subcellular localization was impaired by these PH domain mutations, supporting a role for phospholipid interactions in facilitating membrane association. Despite the importance of phospholipid binding for Dbs function in vivo, we found that Dbs signaling and transforming activity was not stimulated by phosphatidylinositol 3-kinase activation. We suggest that the PH domain of Dbs facilitates two distinct roles in the regulation of DH domain function, one critical for GTPase association and activation in vitro and one critical for phosphoinositide binding and GTPase interaction in vivo, that together promote Dbs association with membranes.     

Requirement for C-terminal sequences in regulation of Ect2 guanine nucleotide exchange specificity and transformation

Ect2 was identified originally as a transforming protein and a member of the Dbl family of Rho guanine nucleotide exchange factors (GEFs). Like all Dbl family proteins, Ect2 contains a tandem Dbl homology (DH) and pleckstrin homology (PH) domain structure. Previous studies demonstrated that N-terminal deletion of sequences upstream of the DH domain created a constitutively activated, transforming variant of Ect2 (designated DeltaN-Ect2 DH/PH/C), indicating that the N terminus served as a negative regulator of DH domain function in vivo. The role of sequences C-terminal to the DH domain has not been established. Therefore, we assessed the consequences of mutation of C-terminal sequences on Ect2-transforming activity. Surprisingly, in contrast to observations with other Dbl family proteins, we found that mutation of the invariant tryptophan residue in the PH domain did not impair DeltaN-Ect2 DH/PH/C transforming activity. Furthermore, although the sequences C-terminal to the PH domain lack any known functional domains or motifs, deletion of these sequences (DeltaN-Ect2 DH/PH) resulted in a dramatic reduction in transforming activity. Whereas DeltaN-Ect2 caused formation of lamellipodia, DeltaN-Ect2 DH/PH enhanced actin stress fiber formation, suggesting that C-terminal sequences influenced Ect2 Rho GTPase specificity. Consistent with this possibility, we determined that DeltaN-Ect2 DH/PH activated RhoA, but not Rac1 or Cdc42, whereas DeltaN-Ect2 DH/PH/C activated all three Rho GTPases in vivo. Taken together, these observations suggest that regions of Ect2 C-terminal to the DH domain alter the profile of Rho GTPases activated in vivo and consequently may contribute to the enhanced transforming activity of DeltaN-Ect2 DH/PH/C.     

Localized amyloidosis in nasopharyngeal carcinoma diagnosed by fine needle aspiration and electron microscopy. A case report

BACKGROUND: The association of amyloidosis and tumors is well known, but only rarely has it been found in the nasopharynx. Moreover, only a single case of tumor-associated amyloidosis in the nasopharynx has been diagnosed by exfoliative cytology and reported in the English-language literature. We describe a case of localized amyloidosis in metastatic nasopharyngeal carcinoma diagnosed by fine needle aspiration and confirmed by histology and electron microscopy. CASE: Bilateral neck enlargement appeared six months before consultation in a 57-year-old man. Both masses were resected, and during exploration of the nasopharynx, several blind biopsies were taken. Smears from the fine needle aspirate showed malignant epithelial cells, consistent with metastatic carcinoma, and Congo red stain showed the presence of amyloid. Histologic examination of the resected cervical masses and nasopharyngeal biopsies confirmed the diagnosis of nasopharyngeal carcinoma, nonkeratinizing variant with amyloid, and metastases in two lymph nodes. Electron microscopic examination of a lymph node showed extracellular, ribbon-like amyloid deposits as well as masses of amorphous amyloid, apparently intracellular. CONCLUSION: The origin of this form of amyloidosis is unclear in this patient since he had no other known etiologic factors for the condition. Increased awareness of the potential association of these two conditions and the use of fine needle aspiration may reveal a higher frequency than hitherto reported.     

Changes in key enzymes of viral-RNA biosynthesis in chloroplasts from PVY and TMV infected tobacco plants

Effects of the infection with tobacco mosaic virus (TMV) and potato virus Y (PVY) on chloroplasts from susceptible tobacco plants were determined. Changes in ribonucleases (RNases), phosphomonoesterase (PME), phosphodiesterase (PDE), glucose-6-phosphate dehydrogenase (G6P DH), 6-phosphogluconate dehydrogenase (6PG DH), glucokinase (GK), and fructokinase (FK) activities in thylakoid/envelope and stroma fractions were studied. Slight increase in the activities of PME, PDE, G6P DH and 6PG DH of thylakoid/envelope fraction as well as of RNases, PME, PDE, G6P DH, 6PG DH, GK and FK of stroma fraction was found in chloroplasts isolated from leaf tissues infected with PVY. Infection with TMV produced higher increase in enzymes activities in chloroplasts; especially, PME, G6P DH and 6PG DH in fraction of thylakoid/envelope, and RNases, PME, PDE, G6P DH, 6PG DH, and GK in stroma fraction.This study was supported by grant No. 522/02/0708 of the Grant Agency of the Czech Republic.