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.

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.

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.

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.

Biosynthesis and regulation of coronatine, a non-host-specific phytotoxin produced by Pseudomonas syringae.

Many P. syringae pathovars are known to produce low-molecular-weight, diffusible toxins in infected host plants. These phytotoxins reproduce some of the symptoms of the relevant bacterial disease and are effective at very low concentrations. Phytotoxins generally enhance the virulence of the P. syringae pathovar which produces them, but are not required for pathogenesis. Genes encoding phytotoxin production have been identified and cloned from several P. syringae pathovars. With the exception of coronatine, toxin biosynthetic gene clusters are generally chromosomally encoded. In several pathovars, the toxin biosynthetic gene cluster also contains a resistance gene which functions to protect the producing strain from the biocidal effects of the toxin. In the case of phaseolotoxin, a resistance gene (argK) has been utilized to engineer phaseolotoxin-resistant tobacco plants. Although P. syringae phytotoxins can induce very similar effects in plants (chlorosis and necrosis), their biosynthesis and mode of action can be quite different. Knowledge of the biosynthetic pathways to these toxins and the cloning of the structural genes for their biosynthesis has relevance to the development of new bioactive compounds with altered specificity. For example, polyketides constitute a huge family of structurally diverse natural products including antibiotics, chemotherapeutic compounds, and antiparasitics. Most of the research on polyketide synthesis in bacteria has focused on compounds synthesized by Streptomyces or other actinomycetes. It is also important to note that it is now possible to utilize a genetic rather than synthetic approach to biosynthesize novel polyketides with altered biological properties (Hutchinson and Fujii, 1995; Kao et al., 1994; Donadio et al., 1993; Katz and Donadio, 1993). Most of the reprogramming or engineering of novel polyketides has been done using actinomycete PKSs, but much of this technology could also be applied to polyketides synthesized by Pseudomonas when sufficient sequence information is available. It is important to note that Pseudomonas produces a variety of antimicrobial compounds from the polyketide pathway, including mupirocin (pseudomonic acid) (Feline et al., 1977), pyoluteorin (Cuppels et al., 1986), and 2-4 diacetylphloroglucinol (Phl) (Bangera and Thomashow, 1996). Pseudomonic acid is valued for its pharmaceutical properties as an antibiotic (Aldridge, 1992), whereas pyoluteorin and Phl have antifungal properties (Howell and Stipanovic, 1980; Keel et al., 1992). A thorough understanding of the biosynthetic pathway to polyketide phytotoxins such as coronatine may ultimately lead to the development of novel compounds with altered biological properties. Thus, specific genes in the biosynthetic pathways of P. syringae phytotoxins could be deployed in other systems to develop new compounds with a wide range of activities.

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.

Use of translational fusions to the maltose-binding protein to produce and purify proteins in Pseudomonas syringae and assess their activity in vivo.

A simple approach is described for the production and purification of proteins in Pseudomonas syringae. The strategy involves the use of the tac promoter, the maltose-binding protein, and the broad-host-range vector, pRK415. This approach was used to partially purify two proteins involved in coronatine biosynthesis from P. syringae. The activity of the fusions was demonstrated in vivo in complementation experiments using the appropriate mutants.

Resistance to ultraviolet light in Pseudomonas syringae: sequence and functional analysis of the plasmid-encoded rulAB genes.

The indigenous plasmids, pPSR1 and pPSR5, were each shown to confer resistance to ultraviolet light (UV) in Pseudomonas syringae (Ps) pv. syringae FF5. The UV-resistance (UVR) determinant was subcloned from a cosmid library of pPSR1, and sequence analysis revealed the presence of two ORFs, designated rulAB which are homologous to the Escherichia coli umuDC mutagenic DNA repair systems and other plasmid-encoded UVR operons. Amino acid (aa) alignments indicated that RulAB are most closely related to the RumAB proteins from plasmid R391, sharing 40.5% and 48.6% aa identity with RumA and RumB, respectively. UV sensitivity assays with the cloned rulAB genes indicated that the expression of UVR in Ps required a functional recA gene.

Characterization of the genes controlling the biosynthesis of the polyketide phytotoxin coronatine including conjugation between coronafacic and coronamic acid.

Pseudomonas syringae pv. glycinea PG4180 produces a chlorosis-inducing phytotoxin, coronatine (COR), which consists of a polyketide component, coronafacic acid (CFA), which is coupled via amide bond formation to coronamic acid (CMA), an ethylcyelopropyl amino acid (aa) derived from isoleucine. P. syringae pv. syringae strains PS51 and PS61, which do not synthesize coronafacoyl compounds (conjugates between CFA and aa), acquired the ability to produce CFA and COR when transformed with p4180A, a 90-kb indigenous plasmid in PG4180. Tn5 mutagenesis indicated that the COR biosynthetic genes in PG4180 are clustered within a 30-kb region on p4180A. The phenotype of selected COR-defective mutants was determined by supplying them with CFA and CMA and by complementation studies with cloned DNA from the COR biosynthetic cluster. Using this approach, the regions encoding CFA and CMA synthesis and coupling activity were localized to the 24-, 12.5- and 2.3-kb regions of the cluster, respectively. Mutants in a 6-kb region required the addition of both CFA and CMA for COR synthesis, which may indicate a regulatory role for this part of the cluster. PS51 and PS61 transconjugants containing cloned DNA from the coupling region produced COR when supplied with CFA and CMA, indicating that coupling activity was cloned and expressed in bacteria lacking the COR biosynthetic cluster.

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.

Expression and analysis of coronafacate ligase, a thermoregulated gene required for production of the phytotoxin coronatine in Pseudomonas syringae.

Coronafacic acid, the polyketide component of the phytotoxin coronatine, is activated and coupled to coronamic acid via amide bond formation, a biosynthetic step presumably catalyzed by the coronafacate ligase (cfl) gene product. In the present study, cfl was fused to the carboxy terminus of malE, which encodes the maltose-binding protein (MBP), and overexpressed in Escherichia coli. Immunoblot analysis indicated that Cfl contained an ATP-binding region, a motif conserved in enzymes which activate their substrates by adenylation. MBP-Cfl was overproduced and purified from Pseudomonas syringae and the protein fusion was used to generate antisera. Anti-MBP-Cfl antibodies and a transcriptional fusion of the cfl promoter to a promoterless glucuronidase gene were used to follow the temporal expression of coronafacate ligase. The results indicated that transcription of cfl is temperature-sensitive. Furthermore, a nonpolar mutation in cfl suggested that the gene may have a role in coronafacic acid biosynthesis.

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.

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.

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.