OCD-like behavior is caused by dysfunction of thalamo-amygdala circuits and upregulated TrkB/ERK-MAPK signaling as a result of SPRED2 deficiency.

Obsessive-compulsive disorder (OCD) is a common neuropsychiatric disease affecting about 2% of the general population. It is characterized by persistent intrusive thoughts and repetitive ritualized behaviors. While gene variations, malfunction of cortico-striato-thalamo-cortical (CSTC) circuits, and dysregulated synaptic transmission have been implicated in the pathogenesis of OCD, the underlying mechanisms remain largely unknown. Here we show that OCD-like behavior in mice is caused by deficiency of SPRED2, a protein expressed in various brain regions and a potent inhibitor of Ras/ERK-MAPK signaling. Excessive self-grooming, reflecting OCD-like behavior in rodents, resulted in facial skin lesions in SPRED2 knockout (KO) mice. This was alleviated by treatment with the selective serotonin reuptake inhibitor fluoxetine. In addition to the previously suggested involvement of cortico-striatal circuits, electrophysiological measurements revealed altered transmission at thalamo-amygdala synapses and morphological differences in lateral amygdala neurons of SPRED2 KO mice. Changes in synaptic function were accompanied by dysregulated expression of various pre- and postsynaptic proteins in the amygdala. This was a result of altered gene transcription and triggered upstream by upregulated tropomyosin receptor kinase B (TrkB)/ERK-MAPK signaling in the amygdala of SPRED2 KO mice. Pathway overactivation was mediated by increased activity of TrkB, Ras, and ERK as a specific result of SPRED2 deficiency and not elicited by elevated brain-derived neurotrophic factor levels. Using the MEK inhibitor selumetinib, we suppressed TrkB/ERK-MAPK pathway activity in vivo and reduced OCD-like grooming in SPRED2 KO mice. Altogether, this study identifies SPRED2 as a promising new regulator, TrkB/ERK-MAPK signaling as a novel mediating mechanism, and thalamo-amygdala synapses as critical circuitry involved in the pathogenesis of OCD.

Patrones de microscopia confocal para el cáncer cutáneo no melanoma y aplicaciones clínicas / Confocal Microscopy Patterns in Nonmelanoma Skin Cancer and Clinical Applications

Actualmente la microscopia confocal reflectante es la técnica diagnóstica no invasiva más prometedora para el estudio de estructuras cutáneas situadas entre la capa córnea y la dermis reticular superficial, obteniendo imágenes paralelas a la superficie cutánea en tiempo real y con una resolución microscópica similar a la observada en la histología convencional. Numerosos estudios han señalado las principales características confocales que se observan en distintas enfermedades cutáneas, tanto tumorales como inflamatorias, demostrando una buena correlación con ciertos patrones dermatoscópicos, así como con el examen histológico. Además, se han descrito algoritmos diagnósticos y patrones confocales que han demostrado unas altas tasas de sensibilidad y especificidad para el diagnóstico de tumores cutáneos de tipo melanoma y no melanoma. Las posibles aplicaciones presentes y futuras de esta tecnología son muy amplias, no solo como herramienta diagnóstica no invasiva, sino también para la evaluación de distintos procesos dinámicos como aquellos que ocurren tras la exposición de la piel a la radiación ultravioleta, o la respuesta de los tumores a terapias no invasivas como la terapia fotodinámica. Explicamos con detalle los hallazgos confocales característicos de los principales tumores cutáneos de tipo no melanoma y discutimos las posibles aplicaciones de esta novedosa técnica diagnóstica en la práctica diaria de la consulta dermatológica

Reflectance confocal microscopy is currently the most promising noninvasive diagnostic tool for studying cutaneous structures between the stratum corneum and the superficial reticular dermis. This tool gives real-time images parallel to the skin surface; the microscopic resolution is similar to that of conventional histology. Numerous studies have identified the main confocal features of various inflammatory skin diseases and tumors, demonstrating the good correlation of these features with certain dermatoscopic patterns and histologic findings. Confocal patterns and diagnostic algorithms have been shown to have high sensitivity and specificity in melanoma and nonmelanoma skin cancer. Possible present and future applications of this noninvasive technology are wide ranging and reach beyond its use in noninvasive diagnosis. This tool can also be used, for example, to evaluate dynamic skin processes that occur after UV exposure or to assess tumor response to noninvasive treatments such as photodynamic therapy. We explain the characteristic confocal features found in the main nonmelanoma skin tumors and discuss possible applications for this novel diagnostic technique in routine dermatology practice

Confocal microscopy patterns in nonmelanoma skin cancer and clinical applications.

Reflectance confocal microscopy is currently the most promising noninvasive diagnostic tool for studying cutaneous structures between the stratum corneum and the superficial reticular dermis. This tool gives real-time images parallel to the skin surface; the microscopic resolution is similar to that of conventional histology. Numerous studies have identified the main confocal features of various inflammatory skin diseases and tumors, demonstrating the good correlation of these features with certain dermatoscopic patterns and histologic findings. Confocal patterns and diagnostic algorithms have been shown to have high sensitivity and specificity in melanoma and nonmelanoma skin cancer. Possible present and future applications of this noninvasive technology are wide ranging and reach beyond its use in noninvasive diagnosis. This tool can also be used, for example, to evaluate dynamic skin processes that occur after UV exposure or to assess tumor response to noninvasive treatments such as photodynamic therapy. We explain the characteristic confocal features found in the main nonmelanoma skin tumors and discuss possible applications for this novel diagnostic technique in routine dermatology practice.

Anti-tumor effects of peptide analogs targeting neuropeptide hormone receptors on mouse pheochromocytoma cells.

Pheochromocytoma is a rare but potentially lethal chromaffin cell tumor with currently no effective treatment. Peptide hormone receptors are frequently overexpressed on endocrine tumor cells and can be specifically targeted by various anti-tumor peptide analogs. The present study carried out on mouse pheochromocytoma cells (MPCs) and a more aggressive mouse tumor tissue-derived (MTT) cell line revealed that these cells are characterized by pronounced expression of the somatostatin receptor 2 (sst2), growth hormone-releasing hormone (GHRH) receptor and the luteinizing hormone-releasing hormone (LHRH) receptor. We further demonstrated significant anti-tumor effects mediated by cytotoxic somatostatin analogs, AN-162 and AN-238, by LHRH antagonist, Cetrorelix, by the cytotoxic LHRH analog, AN-152, and by recently developed GHRH antagonist, MIA-602, on MPC and for AN-152 and MIA-602 on MTT cells. Studies of novel anti-tumor compounds on these mouse cell lines serve as an important basis for mouse models of metastatic pheochromocytoma, which we are currently establishing.

Identification of the biosynthetic gene cluster for 3-methylarginine, a toxin produced by Pseudomonas syringae pv. syringae 22d/93.

The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (K(m), 7 mM; k(cat), 85 min(-1)). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.

Long-term changes in pharyngeal airway dimensions following activator-headgear and fixed appliance treatment.

The aim of this study was to evaluate changes in the pharyngeal airway in growing children and adolescents and to compare these with a group of children who received activator-headgear Class II treatment. The sample consisted of 64 children (32 males and 32 females), 32 had a combined activator-headgear appliance for at least 9 months (study group) followed by fixed appliance therapy in most patients, while the other half received only minor orthodontic treatment (control group). Lateral cephalograms before treatment (T1, mean age 10.4 years), at the end of active treatment (T2, mean age 14.5 years), and at the long-term follow-up (T3, mean age 22.1 years) were traced and digitized. To reveal the influence of somatic growth, body height measurements were also taken into consideration. A two-sample t-test was applied in order to determine differences between the groups. At T1, the study group had a smaller pharynx length (P = 0.030) and a greater ANB angle (P < 0.001) than the controls. The pharyngeal area and the smallest distance between the tongue and the posterior pharyngeal wall also tended to be smaller in the study group. During treatment (T1-T2), significant growth differences between the two groups were present: the study group had a greater reduction in ANB (P < 0.001) and showed a greater increase in pharyngeal area (P = 0.007), pharyngeal length (P < 0.001) and the smallest distance between the tongue and the posterior pharyngeal wall (P = 0.038). At T2, the values for the study group were similar to those of the control group and remained stable throughout the post-treatment interval (T2-T3). Activator-headgear therapy has the potential to increase pharyngeal airway dimensions, such as the smallest distance between the tongue and the posterior pharyngeal wall or the pharyngeal area. Importantly, this increase seems to be maintained long term, up to 22 years on average in the present study. This benefit may result in a reduced risk of developing long-term impaired respiratory function.

Stimulation of cortisol during mental task performance in a provocative virtual environment.

Fully immersive and stereoscopic Virtual Environments (VE) represent a powerful multimedia tool for laboratory-based simulations of distinct scenarios including scenarios for evaluating stressful situations resembling reality. Thus far, cortisol secretion as a neuroendocrine parameter of stress has not been evaluated within a Virtual Reality (VR)-based paradigm. In this study 94 healthy volunteers were subjected to a provocative VR-paradigm and a cognitive stress task. Provocative in this context means the VE was designed to provoke physiological reactions (cortisol secretion) within the respective users by purpose. It was tested (a) if a fully dynamic VE as opposed to a static VE can be regarded as a stressor and (b) if such a fully dynamic VE can modify an additional response to a cognitive stressor presented within the VE additionally. Furthermore, possible gender-related impacts on cortisol responses were assessed. A significant cortisol increase was observed only after the combined application of the fully dynamic VE and the cognitive stressor, but not after application of the dynamic VE or the cognitive stressor alone. Cortisol reactivity was greater for men than for women. We conclude that a fully dynamic VE does not affect cortisol secretion per se, but increases cortisol responses to a dual task paradigm that includes performance of a stressful mental task. This provides the basis for the application of VR-based technologies in neuroscientific research, including the assessment of the human Hypothalamus-Pituitary-Adrenal (HPA) axis regulation.

Prevalences of hyperhomocysteinemia, unfavorable cholesterol profile and hypertension in European populations.

BACKGROUND: Hyperhomocysteinemia (HHCY) is a risk factor for cardiovascular diseases (CVD). HHCY may interact with hypertension (HTEN) and an unfavorable cholesterol profile (UNFAVCHOL) to alter the risk of CVD. OBJECTIVES: To estimate the prevalences of HHCY (1) isolated and (2) in combination with UNFAVCHOL and/or HTEN in different age categories. To provide information that may improve the screening and treatment of subjects at risk of CVD. DESIGN: Cross-sectional data on 12,541 men and 12,948 women aged 20 + y were used from nine European studies. RESULTS: The prevalence of isolated HHCY was 8.5% in subjects aged 20-40 y, 4.7% in subjects aged 40-60 y and 5.9% in subjects aged over 60 y. When combining all age groups, 5.3% had isolated HHCY and an additional 5.6% had HHCY in combination with HTEN and/or UNFAVCHOL. The combinations of risk factors increased with age and, except for HHCY&UNFAVCHOL, were more prevalent than predicted by chance. Of the young subjects (20-40 y), 24% suffered from one or more of the investigated CVD risk factors. This figure was 75.1% in the old subjects (60+ years). CONCLUSIONS: A substantial number of subjects in selected European populations have HHCY (10.9%). In half of these cases, subjects suffer also from other CVD risk factors like UNFAVCHOL and HTEN. Older people in particular tend to have more than one risk factor. Healthcare professionals should be aware of this when screening and treating older people not only for the conventional CVD risk factors like UNFAVCHOL and HTEN but also HHCY, as this can easily be reduced through increased intake of folic acid via supplement or foods fortified with folic acid.

Metabolism of propafenone and verapamil by cryopreserved human, rat, mouse and dog hepatocytes: comparison with metabolism in vivo.

In the present study we examined the metabolism of [(14)C]propafenone (P) and [(14)C]verapamil (V) using cryopreserved human, dog (Beagle), rat (Sprague-Dawley) and mouse (NMRI) hepatocytes. The percentage ratios of the metabolites were identified after extraction by HPLC with UV and radioactivity detection. Phase-II metabolites were cleaved using beta-glucuronidase. Metabolism of the drugs by cryopreserved hepatocytes was compared with that in the respective species in vivo. All phase-I and -II metabolites known from in vivo experiments: 5-hydroxy-P (5-OH-P); 4'-hydroxy-P (4'-OH-P); N-despropyl-P (NdesP) and the respective glucuronides, were identified after incubation with cryopreserved hepatocytes. Interspecies differences were observed concerning the preferential position of propafenone hydroxylation: 5-OH-P made up 91, 51, 16 and 3% of the total metabolites after incubation with cryopreserved human ( n=4), dog ( n=3), rat ( n=3) and mouse ( n=4) hepatocytes respectively. These results are consistent with interspecies differences known from in vivo experiments. The metabolism of V is more complex than that of P. Nevertheless, all phase-I metabolites known from in vivo experiments and the expected glucuronides were identified after incubation with cryopreserved hepatocytes from all four species. As expected from the results of in vivo experiments, there were no major interspecies differences with respect to phase-I metabolites although the conjugation of verapamil phase-I metabolites by cryopreserved canine hepatocytes was much weaker than for the other species. In conclusion, phase-I and phase-II metabolism of P and V was evaluated using hepatocytes in vitro. All of the relevant interspecies differences known from in vivo experiments were identified after short-term incubation with cryopreserved hepatocytes in suspension.

Complete nucleotide sequence and analysis of pPSR1 (72,601 bp), a pPT23A-family plasmid from Pseudomonas syringae pv. syringae A2.

Plasmid pPSR1 is a conjugative plasmid originally isolated from Pseudomonas syringae pv. syringae A2, and is a member of the recently described pPT23A plasmid family. We have determined the complete sequence of pPSR1 and found the plasmid to be 72,601 bp in length, encoding 55 ORFs. Putative functions were assigned to 49 ORFs; of these, 24 (49.0%) are involved in plasmid replication, maintenance or conjugation, 17 (34.7%) have roles in virulence or ecological fitness, and eight (16.3%) encode transposase functions as part of mobile elements. pPSR1 carries the effector gene orf34, the mutagenic DNA repair operon rulAB which confers tolerance to ultraviolet radiation, and two genes for methyl-accepting chemotaxis proteins, one of which was located within the novel transposon Tn 5395. The streptomycin resistance transposon Tn 5393a, which carries a strA-strB determinant, was found inserted immediately downstream of the pPSR1 repA gene. Functional analysis of the replication region of pPSR1 indicated that the repA gene and flanking upstream and downstream sequences are required for autonomous replication in P. syringae. Hybridization analyses of the distribution of 11 of the pPSR1 ORFs indicated that many of the ecologically important ORFs were confined to the pathovar P. syringae pv. syringae -either to strains from the local population from which pPSR1 was originally isolated, or strains from a worldwide collection. Conjugative transfer genes and a gene encoding a transcriptional regulator were more widely distributed among several P. syringae pathovars. The sequence analysis of pPSR1 suggests that pPT23A-family plasmids evolve by accumulating genes that are important for host-pathogen interactions or growth on plant hosts, which are incorporated onto a conserved backbone encoding conjugation and stability determinants.

Thermoregulated expression of virulence factors in plant-associated bacteria.

Pathogenic bacteria with habitats inside and outside a given host react to changes in environmental parameters by synthesizing gene products specifically needed during pathogenic or saprophytic growth. Temperature effects have been investigated in detail for pathogens of warm-blooded hosts, and major principles governing the temperature-sensing mechanism have been uncovered. Generally, transcription of virulence genes in these pathogens is induced at higher temperatures (37-41 degrees C), which are typical for body cavities and host tissues. However, effects of temperature on virulence determinants in plant pathogenic bacteria have not been focused on in detail. Interestingly, almost all virulence genes of plant pathogenic bacteria studied with respect to temperature exhibit increased transcription at temperatures well below the respective growth optima. This includes virulence determinants such as those directing bacteria-to-plant gene transfer, plant cell-wall-degrading enzymes, phytotoxins, ice nucleation activity, exopolysaccharide production, and the type III protein secretion machinery. Although many of the studied phytopathogens cause "cold-weather" diseases, the ecological rationale for this phenomenon remains to be studied in detail. This mini-review summarizes our current knowledge on thermoregulation of cellular processes taking place in bacterial phytopathogens in response to temperature changes. Since the temperature range of interest is different from that relevant to pathogens of mammals, one envisions novel principles of thermo-sensing in bacteria interacting with plants.

Characterization and mutational analysis of three allelic lsc genes encoding levansucrase in Pseudomonas syringae.

In the plant pathogen Pseudomonas syringae pv. glycinea PG4180 and other bacterial species, synthesis of the exopolysaccharide levan is catalyzed by the extracellular enzyme levansucrase. The results of Southern blotting and PCR analysis indicated the presence of three levansucrase-encoding genes in strain PG4180: lscA, lscB, and lscC. In this study, lscB and lscC were cloned from a genomic library of strain PG4180. Sequence analysis of the two lsc genes showed that they were virtually identical to each other and highly similar to the previously characterized lscA gene. lscA and lscC had a chromosomal location, whereas lscB resided on an indigenous plasmid of PG4180. Mutants with impaired expression of individual lsc genes and double mutants were generated by marker exchange mutagenesis. Determination of levansucrase activities in these mutants revealed that the lscB gene product was secreted but not that of lscA or lscC. Our results indicated that lscB and lscC but not lscA contributed to periplasmic levan synthesis of PG4180. The lscB lscC double mutant was completely defective in levan formation and could be complemented by either lscB or lscC. Our data suggested a compartment-specific localization of two lsc gene products, with LscB being the secreted, extracellular enzyme and LscC being the predominantly periplasmic levansucrase. Results of Western blot analyses indicated that lscA was not expressed and that lscA was not associated with levansucrase activities in any particular protein fraction. LscA could be detected in PG4180 only when transcribed from the vector-borne P(lac) promoter. PCR screening in various P. syringae strains with primers derived from the three characterized lsc genes demonstrated the presence of multiple Lsc isoenzymes in other P. syringae pathovars.

Interactions of Pseudomonas syringae pv. glycinea with host and nonhost plants in relation to temperature and phytotoxin synthesis.

Pseudomonas syringae pv. glycinea PG4180 causes bacterial blight of soybean and produces the phytotoxin coronatine (COR) in a temperature-dependent manner. COR consists of a polyketide, coronafacic acid (CFA), and an amino acid derivative, coronamic acid, and is produced optimally at 18 degrees C whereas no detectable synthesis occurs at 28 degrees C. We investigated the impact of temperature on PG4180 during compatible and incompatible interactions with soybean and tobacco plants, respectively. After spray inoculation, PG4180 caused typical bacterial blight symptoms on soybean plants when the bacteria were grown at 18 degrees C prior to inoculation but not when derived from cultures grown at 28 degrees C. The disease outcome was quantified by determination of bacterial populations in planta. The temperature effect was not observed when PG4180 was artificially infiltrated into soybean leaves, indicating that the pre-inoculation temperature and phytotoxin synthesis were important for bacterial invasion via natural plant openings. In the incompatible interaction, PG4180 elicited the hypersensitive response (HR) on tobacco plants regardless of the bacterial pre-inoculation temperature. However, the HR was significantly delayed when tobacco plants were treated with cells of the CFA-overproducing derivative, PG4180.N9, which were derived from cultures grown at 18 degrees C, compared with parallels incubated at 28 degrees C. CFA biosynthesis by PG4180.N9 was optimal at 18 degrees C and negligible at 28 degrees C. The impact of CFA synthesis on the HR was studied with different growth media, mutants, and transconjugants of PG4180, indicating that the amount of synthesized CFA but not that of COR influenced the outcome of the HR. Feeding experiments with purified coronafacoyl compounds suggested that the observed delay of the HR was mediated by CFA, shedding further light on CFA's putative role as a molecular mimic of the plant signaling molecule, jasmonic acid.

Characterization of plasmids encoding the phytotoxin coronatine in Pseudomonas syringae.

Coronatine (COR) is a nonhost-specific phytotoxin that substantially contributes to the virulence of several pathovars (pvs.) of Pseudomonas syringae. The COR gene cluster in P. syringae is generally plasmid-encoded in pvs. atropurpurea, glycinea, morsprunorum, and tomato but chromosomally encoded in pv. maculicola. In the present study, we investigated whether the COR plasmids in four pathovars shared other traits including self-transmissibility, conserved oriV/par loci, and insertion sequences (ISs) known to reside on other plasmids in P. syringae. Three COR plasmids were shown to be self-transmissible, and all COR plasmids shared a related oriV/par region. Two COR plasmids hybridized to IS801, an IS element widely distributed in P. syringae. Further analysis of p4180A, a 90-kb COR plasmid in P. syringae pv. glycinea, indicated that multiple copies of IS801 were present on this plasmid, and all copies mapped outside the COR gene cluster. Sequence analysis of the region adjacent to the COR gene cluster in p4180A indicated the presence of additional IS elements including IS870, IS51, and IS1240. The IS elements borne on p4180A may have contributed to horizontal transfer of the COR gene cluster and the evolution of the COR biosynthetic pathway.

The transcriptional activator CorR is involved in biosynthesis of the phytotoxin coronatine and binds to the cmaABT promoter region in a temperature-dependent manner.

A modified two-component regulatory system consisting of the histidine protein kinase CorS and two highly homologous response regulators, CorR and CorP, controls biosynthesis of the polyketide phytotoxin coronatine (COR) by Pseudomonas syringae pv. glycinea PG4180 in a temperature-dependent manner. COR synthesis is maximal at 18 degrees C but does not occur at 28 degrees C. Fusions of CorR and CorP to the maltose-binding protein (MBP) were overproduced in Escherichia coli and P. syringae PG4180, and tested for functionality by complementation of corR and corP mutants of PG4180, respectively. The cmaABT promoter region was defined by deletion mapping, and the DNA-binding capability of CorR and CorP was examined by gel retardation assays. When overproduced in P. syringae at 18 degrees C and purified, MBP-CorR was shown to bind specifically to a 218-bp DNA fragment corresponding to positions -841 to -623 bp upstream of the transcriptional start site of the cmaABT operon. In contrast, MBP-CorP and MBP itself, when overproduced in P. syringae and E. coli at 18 degrees C and 28 degrees C, respectively, did not bind to the 218-bp fragment or to any other DNA fragment analyzed. The CorP protein lacks typical DNA-binding motifs, suggesting that it might modulate the function of CorR. However, addition of purified MBP-CorP did not alter the DNA-binding activity of MBP-CorR. On the other hand, this activity was completely abolished when MBPCorR was overproduced at 28 degrees C or in a corS mutant, indicating that the binding of CorR depended on the growth temperature at which it was produced and was controlled by CorS. In addition, overproduction of MBP-CorR in a corP mutant of PG4180 also yielded inactive protein, underlining the importance of CorP for CorR activation. We propose that CorR is activated by CorS at low temperature and that CorP is required for this activation before CorR can bind to DNA.

Hyperhomocysteinemia in high-aged subjects: relation of B-vitamins, folic acid, renal function and the methylenetetrahydrofolate reductase mutation.

Moderate hyperhomocysteinemia is an atherogenic risk factor and plays an important role in geriatrics. Here, we have investigated the role of hyperhomocysteinemia in two elderly groups: 104 longeval subjects of 85-102 years, 100 seniors aged 65-75 years, and 75 controls of 19-60 years. Elevated homocysteine levels were found in 58% of longeval subjects in comparison with 32% in seniors. The homocysteine level in serum correlated positively with age as well as serum creatinine, and inversely with serum folate, but there was no correlation with serum B-vitamins. The frequency of vitamin B deficiency in serum of longeval subjects compared to seniors was as follows: vitamin B6 43% vs. 22%, vitamin B12 20% vs. 8%, and folic acid 1% in both groups. Increased serum creatinine levels (> 1.1 mg/dl) were found in 63% of the longeval subjects and in 32% of seniors. The 677-missense mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, responsible for moderate homocysteine elevation, was found in 35, 37 and 27% of alleles in longeval persons, senior subjects and younger controls, respectively, showing no significant difference in frequency distributions of the MTHFR gene mutation. It can be concluded that hyperhomocysteinemia is very common with increased age. Its importance as an atherogenic risk factor with advanced age has to be clarified.

Thermoregulated expression and characterization of an NAD(P)H-dependent 2-cyclohexen-1-one reductase in the plant pathogenic bacterium Pseudomonas syringae pv. glycinea.

The phytopathogenic bacterium Pseudomonas syringae pv. glycinea PG4180.N9 causes bacterial blight of soybeans and preferably infects its host plant during periods of cold, humid weather conditions. To identify proteins differentially expressed at low temperatures, total cellular protein fractions derived from PG4180.N9 grown at 18 and 28 degreesC were separated by two-dimensional gel electrophoresis. Of several proteins which appeared to be preferentially present at 18 degreesC, a 40-kDa protein with an isoelectric point of approximately 5 revealed significant N-terminal sequence homology to morphinone reductase (MR) of Pseudomonas putida M10. The respective P. syringae gene was isolated from a genomic cosmid library of PG4180, and its nucleotide sequence was determined. It was designated ncr for NAD(P)H-dependent 2-cyclohexen-1-one reductase. Comparison of the 1,083-bp open reading frame with database entries revealed 48% identity and 52% similarity to the MR-encoding morB gene of P. putida M10. The ncr gene was overexpressed in Escherichia coli, and its gene product was used to generate polyclonal antisera. Purified recombinant Ncr protein was enzymatically characterized with NAD(P)H and various morphinone analogs as substrates. So far, only 2-cyclohexen-1-one and 3-penten-2-one were found to be substrates for Ncr. By high-pressure liquid chromatography analysis, flavin mononucleotide could be identified as the noncovalently bound prosthetic group of this enzyme. The distribution of the ncr gene in different Pseudomonas species and various strains of P. syringae was analyzed by PCR and Southern blot hybridization. The results indicated that the ncr gene is widespread among P. syringae pv. glycinea strains but not in other pathovars of P. syringae or in any of the other Pseudomonas strains tested.

Comparison of Ethylene Production by Pseudomonas syringae and Ralstonia solanacearum.

ABSTRACT Strains of Pseudomonas syringae pv. pisi and Ralstonia solanacearum produced ethylene at rates 20- and 200-fold lower, respectively, than strains of P. syringae pvs. cannabina, glycinea, phaseolicola, and sesami. In the current study, we investigated which ethylene biosynthetic pathways were used by P. syringae pv. pisi and R. solanacearum. Neither the activity of an ethylene-forming enzyme nor a corresponding efe gene homolog could be detected in R. solanacearum, suggesting synthesis of ethylene via 2-keto-4-methyl-thiobutyric acid. In contrast, 2-oxoglutarate-dependent ethylene formation was observed with P. syringae pv. pisi, and Southern blot hybridization revealed the presence of an efe homolog in this pathovar. The efe genes from P. syringae pvs. cannabina, glycinea, phaseolicola, pisi, and sesami were sequenced. Nucleotide sequence comparisons indicated that the efe gene in pv. pisi was not as highly conserved as it was in other P. syringae pathovars. The pv. pisi efe homolog showed numerous nucleotide substitutions and a deletion of 13 amino acids at the C-terminus of the predicted gene product. These sequence alterations might account for the lower rate of ethylene production by this pathovar. All ethylene-producing P. syringae pathovars were virulent on bush bean plants. The overlapping host range of these pathovars suggests that horizontal transfer of the efe gene may have occurred among bacteria inhabiting the same host.

Cloning, nucleotide sequence, and expression in Escherichia coli of levansucrase genes from the plant pathogens Pseudomonas syringae pv. glycinea and P. syringae pv. phaseolicola.

Plant-pathogenic bacteria produce various extracellular polysaccharides (EPSs) which may function as virulence factors in diseases caused by these bacteria. The EPS levan is synthesized by the extracellular enzyme levansucrase in Pseudomonas syringae, Erwinia amylovora, and other bacterial species. The lsc genes encoding levansucrase from P. syringae pv. glycinea PG4180 and P. syringae pv. phaseolicola NCPPB 1321 were cloned, and their nucleotide sequences were determined. Heterologous expression of the lsc gene in Escherichia coli was found in four and two genomic library clones of strains PG4180 and NCPPB 1321, respectively. A 3. 0-kb PstI fragment common to all six clones conferred levan synthesis on E. coli when further subcloned. Nucleotide sequence analysis revealed a 1,248-bp open reading frame (ORF) derived from PG4180 and a 1,296-bp ORF derived from NCPPB 1321, which were both designated lsc. Both ORFs showed high homology to the E. amylovora and Zymomonas mobilis lsc genes at the nucleic acid and deduced amino acid sequence levels. Levansucrase was not secreted into the supernatant but was located in the periplasmic fraction of E. coli harboring the lsc gene. Expression of lsc was found to be dependent on the vector-based Plac promoter, indicating that the native promoter of lsc was not functional in E. coli. Insertion of an antibiotic resistance cassette in the lsc gene abolished levan synthesis in E. coli. A PCR screening with primers derived from lsc of P. syringae pv. glycinea PG4180 allowed the detection of this gene in a number of related bacteria.

Analysis of genes involved in biosynthesis of coronafacic acid, the polyketide component of the phytotoxin coronatine.

Coronafacic acid (CFA) is the polyketide component of coronatine (COR), a phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae. The genes involved in CFA biosynthesis are encoded by a single transcript which encompasses 19 kb of the COR gene cluster. In the present study, the nucleotide sequence was determined for a 4-kb region located at the 3' end of the CFA biosynthetic gene cluster. Three open reading frames were identified and designated cfa8, cfa9, and tnp1; the predicted translation products of these genes showed relatedness to oxidoreductases, thioesterases, and transposases, respectively. The translational products of cfa8 and cfa9 were overproduced in Escherichia coli BL21; however, tnp1 was not translated in these experiments. Mutagenesis and complementation analysis indicated that cfa8 is required for the production of CFA and COR. Analysis of a cfa9 mutant indicated that this gene is dispensable for CFA and COR production but may increase the release of enzyme-bound products from the COR pathway; tnp1, however, had no obvious function in CFA or COR biosynthesis. A genetic strategy was used to produce CFA in a P. syringae strain which lacks the COR gene cluster; this approach will be useful in future studies designed to investigate biosynthetic products of the CFA gene cluster.