Genotoxic effects of the cyanobacterial pentapeptide nodularin in HepG2 cells.

The cyanobacterial pentapeptide nodularin (NOD), mainly produced by genus Nodularia, is a potent inhibitor of protein phosphatases PP1 and PP2A, and causes animal mortality. The few studies available indicate that NOD is a potential non-genotoxic carcinogen. In the present study we evaluated NOD (0.01, 0.1 and 1 µg/ml) genotoxic activity in human hepatoma (HepG2) cells with the comet, γH2AX and cytokinesis block micronucleus cytome assays. In addition, induction of oxidative stress was studied. Moreover changes in the expression of selected genes from the P53 pathway, involved in the response to DNA damage (P53, GADD45α, CDKN1A, MDM2), apoptosis (BAX, BCL2) and oxidative stress (GPX1, GSR, GCLC, CAT, SOD1) were determined using qPCR. Non-cytotoxic concentrations induced time and dose dependant increase in reactive oxygen species (ROS) production and substantially increased the formation of oxidative DNA damage. In addition, elevated formation of micronuclei was detected. For the first time it has been shown that NOD deregulated the mRNA level of DNA damage (CDKN1A, GADD45α) and oxidative stress (GPX1, GSR, GCLC, CAT and SOD1) responsive genes and anti-apoptotic gene BCL2. Our results provide new evidence that NOD genotoxic effects are mediated through ROS production, already at low environmentally relevant concentrations.

LAMP assay and rapid sample preparation method for on-site detection of flavescence dorée phytoplasma in grapevine.

In Europe the most devastating phytoplasma associated with grapevine yellows (GY) diseases is a quarantine pest, flavescence dorée (FDp), from the 16SrV taxonomic group. The on-site detection of FDp with an affordable device would contribute to faster and more efficient decisions on the control measures for FDp. Therefore, a real-time isothermal LAMP assay for detection of FDp was validated according to the EPPO standards and MIQE guidelines. The LAMP assay was shown to be specific and extremely sensitive, because it detected FDp in all leaf samples that were determined to be FDp infected using quantitative real-time PCR. The whole procedure of sample preparation and testing was designed and optimized for on-site detection and can be completed in one hour. The homogenization procedure of the grapevine samples (leaf vein, flower or berry) was optimized to allow direct testing of crude homogenates with the LAMP assay, without the need for DNA extraction, and was shown to be extremely sensitive.

[The patient in the psychiatric emergency ambulance: diagnoses, reasons and comparison of layperson vs. physician viewpoints]. / Der Patient in der psychiatrischen Notambulanz: Erstdiagnosen, Vorstellungsgründe und Vergleich der Laien- vs. Arztsicht.

A survey in specialties other than psychiatry showed that "emergency room"-patients have factors other than the presenting disease that determine the usage of urgent medical evaluation. In the following prospective study 104 outpatients presenting at daytime in a university psychiatric emergency care unit were included over 6 months. Apart from social and epidemiological data, illnesses according to ICD-10, reason for presentation from the patient's point of view and in this regard the physician's evaluation were included. The most prevalent diagnoses were depression, adjustment disorders and anxiety disorders, comprising together 75 %. Organic disorders or addictive disorders were less frequent; psychoses were found in 8 %. Concerning the presentation as an emergency, 70 % of patients reported a subjective clinical deterioration but only 44 % were regarded as an urgent need in the responsible physician's point of view (Cohen's kappa 0.39). Our findings show that patients presenting as "psychiatric emergency cases" without appointment mainly suffer from depression, adjustment disorders and panic disorders. Furthermore, the layperson's point of view of clinical deterioration justifying an emergency presentation differs from physician's evaluation. The most likely cause for this disagreement between physicians and patients in the assessment to utilise a medical emergency care service in psychiatry might be dysfunctional or, respectively, negative-biased cognitions accompanying depressive syndromes.

Cost of a 5-year lung cancer survivor: symptomatic tumour identification vs proactive computed tomography screening.

BACKGROUND: Our objective was to analyse the cost effectiveness of computed tomography (CT) screening for lung cancer in terms of the cost per long-term survivor, which has not been evaluated to date. METHODS: Estimations were computed based on data from the Surveillance, Epidemiology, and End Results registries covering years 1999-2003. The design framework of our model allowed for the incorporation of multiple values taken from the epidemiological and clinical literature to be utilised for cost inputs, scope of patients screened, diagnostic staging, and survival percentages applied separately to two cohorts: age 40-79 and 60-79 years. This enabled the analysis of over 1400 scenarios, each containing a unique set of input values, for which the estimated cost per 5-year survivor (CP5YS) was compared between the symptom-detected and proactive screening approaches. RESULTS: Estimated CP5YS were higher for the symptom-detected approach in all 729 scenarios analysed for the cohort ages 60-79 years, ranging from approximately $5800 to $116,700 increased cost per 5-year survivor (CP5YS). For the cohort ages 40-79 years, 75% of the 729 scenarios analysed showed increased CP5YS for the symptom-detected approach ranging from $5700 to $110,000 increased CP5YS. Total costs and total 5-year survivors were higher for the proactive screening method for all scenarios analysed across both cohorts with increases ranging from 50-256% and 98-309%, respectively. CONCLUSION: The predicted increase in long-term survival with CT screening and the potential for better utilisation of health-care dollars in terms of CP5YS, particularly when screening patients over the age of 60 years, are critically important considerations in directing effective future lung cancer management strategy.

[Picrotoxin in the treatment of Menière's disease]. / Picrotoxin in der Therapie des Morbus Menière.

OBJECTIVE: In medical long-term treatment of Menière's disease picrotoxin suppositories are an uncommon method of prophylaxis. In spite of its empirical benefit in clinical use and its lack of side effects, there are few clinical studies about the therapeutical effect of picrotoxin. In this study we evaluate the effectiveness of picrotoxin compared to the therapy with betahistine in Menière's disease. MATERIAL AND METHODS: In a prospective clinical trial we examined 41 patients, 18 of them were treated with betahistine 3x12 mg, 23 had a therapy with picrotoxin-suppositories at 0.001 g three times a week. Frequency and intensity of the vertigo attacks were evaluated before and under treatment. Mean follow up time was 12 months. RESULTS: In both groups a reduction of the attacks' frequency and intensity could be noticed, which was statistically significant for all the two groups. Thus, in the course of the treatment we observed a significant stronger effectiveness of picrotoxin, regarding the frequency and intensity of vertigo attacks. Discussing our own results we review the state of the art in medical long-term treatment in Menière's disease. CONCLUSION: Because of its clinical benefit and the lack of side effects Picrotoxin is a reasonable alternative in medical long- term treatment of Menière's disease, which should have an important role in the treatment cascade.

Low-frequency distortion product otoacoustic emission test compared to ECoG in diagnosing endolymphatic hydrops.

Previous studies assumed that specific audiometric measures like low-frequency biasing were noninvasive and inexpensive techniques for diagnosing endolymphatic hydrops (EH). The aim of this study was to compare the results of low-frequency DPOAE (LF-DPOAE) with those of transtympanic electrocochleography (ECoG) in patients with Menière's disease (MD). The prospective study included 50 patients, 22 to 72 years old, who were diagnosed with Menière's disease according to the criteria laid down by the American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS) in 1995. LF-DPOAE and ECoG were performed on the same day after standard audiometry. Enlarged amplitude of summation potential to action potential ratio was used as the diagnostic criterion for EH. The results were compared to those of LF-DPOAE. In this audiometric examination we used the modulation index (MI) to detect EH if MI < 0.5. ECoG yielded a result in 46 of the 50 patients examined. An enlarged SP/AP ratio was found in 23 patients (50%). The results of 33 patients could be compared, i.e. assessable results in both tests. The two tests yielded the same results in 13 patients and different ones in 20 patients. ROC analysis and Mann-Whitney statistics showed no significant correlation between the two tests. With ECoG as the gold standard for verifying EH, we found that LF-DPOAE is not yet a suitable method for diagnosing EH.

Progressive loss of knee extension after injury. Cyclops syndrome due to a lesion of the anterior cruciate ligament.

Cyclops syndrome has been defined as a loss of knee extension due to impingement of a pedunculated proliferative tissue mass after anterior cruciate ligament reconstruction. We describe four patients who were operated on for progressive loss of knee extension after minor knee injury. During the arthroscopic procedure, the consistent finding was a fibrous pedunculated nodule adhering to the anterolateral aspect of the original anterior cruciate ligament, obstructing extension by impingement in the anterior aspect of the knee. At least part of the anterior cruciate ligament was intact in all cases. These patients were compared with seven patients who developed cyclops syndrome after anterior cruciate ligament reconstruction. Clinical and arthroscopic findings were the same in both groups. An arthroscopic excision of the nodule, performed an average of 12 weeks after knee trauma or after reconstruction, gave very good results. Histologic examination of the excised nodules from both groups showed fibroelastic connective tissue proliferation, thromboangiitis, and areas of necrotic bone and foreign body giant cell granuloma. On the basis of our observations, we conclude that formation of a fibrous pedunculated nodule may occur after an anterior cruciate ligament injury as well as after surgical reconstruction of the anterior cruciate ligament.

Chondroblastoma of the patella associated with an aneurysmal bone cyst.

Chondroblastoma is a rare, benign tumor of bone, accounting for about 1% of all bone tumor cases. It tends to affect the epiphyseal ends of long bones, most often in males during the first and second decades of life. It has well-characterized radiographic and histologic features but despite its histologically benign appearance a few cases of metastases have been reported. Local recurrences after curettage and bone grafting occur in 11% to 25% of cases. The features of a patellar chondroblastoma are the same as for other locations. In reviewing the literature we found an unusually high male-to-female ratio. It is interesting that the usual treatment of the patellar chondroblastoma has been patellectomy, whereas curettage and bone grafting has predominated in the other locations. We present a computer tomography and magnetic resonance imaging study of a case of chondroblastoma of the patella associated with an aneurysmal bone cyst. To our knowledge, it is the seventh case reported and the second with computer tomography and magnetic resonance imaging studies. We also review and discuss in detail all the cases of patellar chondroblastoma that we found in the literature.

Genomic organization and alternative splicing of the human and mouse RPTPrho genes.

BACKGROUND: Receptor protein tyrosine phosphatase rho (RPTPrho, gene symbol PTPRT) is a member of the type IIB RPTP family. These transmembrane molecules have been linked to signal transduction, cell adhesion and neurite extension. The extracellular segment contains MAM, Ig-like and fibronectin type III domains, and the intracellular segment contains two phosphatase domains. The human RPTPrho gene is located on chromosome 20q12-13.1, and the mouse gene is located on a syntenic region of chromosome 2. RPTPrho expression is restricted to the central nervous system. RESULTS: The cloning of the mouse cDNA, identification of alternatively spliced exons, detection of an 8 kb 3'-UTR, and the genomic organization of human and mouse RPTPrho genes are described. The two genes are comprised of at least 33 exons. Both RPTPrho genes span over 1 Mbp and are the largest RPTP genes characterized. Exons encoding the extracellular segment through the intracellular juxtamembrane 'wedge' region are widely spaced, with introns ranging from 9.7 to 303.7 kb. In contrast, exons encoding the two phosphatase domains are more tightly clustered, with 15 exons spanning approximately 60 kb, and introns ranging in size from 0.6 kb to 13.1 kb. Phase 0 introns predominate in the intracellular, and phase 1 in the extracellular segment. CONCLUSIONS: We report the first genomic characterization of a RPTP type IIB gene. Alternatively spliced variants may result in different RPTPrho isoforms. Our findings suggest that RPTPrho extracellular and intracellular segments originated as separate modular proteins that fused into a single transmembrane molecule during a later evolutionary period.

Modulation of GABA(A) receptor subunit mRNA levels in olivocerebellar neurons of purkinje cell degeneration and weaver mutant mice.

In olivocerebellar circuits, changes in the subunit composition of GABA(A) receptors occur at a time of extensive synaptic remodeling. In the deep cerebellar nuclei, GABA(A) receptor alpha1, beta2, and gamma2 subunit mRNA expression increases throughout neonatal development, whereas in the inferior olivary complex, the perinatal combination of alpha3, alpha5, beta3, and gamma2 mRNAs switches to the adult combination of alpha2, alpha4, beta3 and gamma1 during postnatal week 2. In situ hybridization was used to examine changes in subunit expression in the olivocerebellar nuclei of Purkinje cell degeneration and weaver mutant mice. In Purkinje cell degeneration, subunit transcripts decreased below control levels in olivary neurons; however, alpha1, beta2, and gamma2 transcript levels were slightly increased in the medial nucleus of the deep cerebellar nuclei. In weaver olivary neurons, although the switch from early- to late-onset subunit mRNAs occurred as in normal mice, transcript levels were differentially modulated by the mutation. Our studies indicate that major alterations in synaptic connectivity do not prevent developmentally programmed switches in GABA(A) receptor gene expression but can modulate the timing and level of transcript expression in afferent and efferent neurons.

Cyclops syndrome occurring after partial rupture of the anterior cruciate ligament not treated by surgical reconstruction.

Cyclops syndrome is one of the specific causes of loss of extension of the knee following anterior cruciate ligament (ACL) reconstruction. The syndrome is manifested by progressive loss of extension associated with pain and audible clunk at terminal extension caused by a pedunculated nodule of fibrovascular proliferative tissue usually arising from the graft. The entity has been described recently and has been reported exclusively as a complication of ACL reconstructions. We report the case of a patient with symptoms and arthroscopic and histological findings compatible with cyclops syndrome that developed after a partial ACL rupture that was not treated by surgical reconstruction. A different etiology and classical histological and immunohistological microscopic analysis of the nodule presented in this report may further clarify the pathogenesis of the cyclops syndrome.

Harmaline-induced changes in gamma aminobutyric acid(A) receptor subunit mRNA expression in murine olivocerebellar nuclei.

Increased CNS activity in the form of electrically or chemically induced seizures is known to alter the properties of GABA(A) receptors. The tremorgen, harmaline, causes a bursting pattern of activity in inferior olivary neurons, the effects of which are transmitted throughout the olivocerebellar circuit to other regions of the CNS. In situ hybridization was used to determine the effect of this increased activity on gamma aminobutyric acid(A) (GABA(A)) receptor subunit gene expression in the cerebellar Purkinje cell layer, deep cerebellar nuclei and inferior olivary complex of adult mice. In Purkinje cells, the expression of alpha(1), beta(2), and gamma(2) mRNAs was increased only slightly (<5%) by harmaline administration, while in deep cerebellar neurons, beta(2) transcript levels were initially elevated (26%), but dropped to control levels immediately thereafter. The expression of alpha(2), alpha(4), beta(3) and gamma(1) mRNAs in olivary neurons was affected differentially by harmaline administration. The alpha(4) transcript was increased, reaching >60% above control levels at 6 h post-injection. A smaller increase was observed for alpha(2) mRNA, while beta(3) and gamma(1) transcripts dropped below control levels during the same period. The expression of corticotropin-releasing factor mRNA was also elevated in the olivary complex. These data indicate that while Purkinje cells and deep cerebellar neurons are only minimally affected, harmaline induced changes in cellular properties may result in increased numbers of alpha(4)-containing, diazepam-insensitive, GABA(A) receptors in olivary neurons.

Physician practice management companies: should physicians be scared?

Physician practice management companies (PPMCs) manage nonclinical aspects of physician care and control physician groups by buying practice assets. Until recently, PPMCs were a favorite of Wall Street. Suddenly, in early 1998, the collapse of the MedPartners-PhyCor merger led to the rapid fall of most PPMC stock, thereby increasing wariness of physicians to sell to or invest in PPMCs. This article explores not only the broken promises made by and false assumptions about PPMCs, but also suggests criteria that physicians should use and questions would-be PPMC members should ask before joining. Criteria include: demonstrated expertise, a company philosophy that promotes professional autonomy, financial stability, freedom from litigation, and satisfied physicians already in the PPMC. The authors recommend that physicians seek out relatively small, single-specialty PPMCs, which hold the best promise of generating profits and permitting professional control over clinical decisions.

Identification and characterization of RPTP rho, a novel RPTP mu/kappa-like receptor protein tyrosine phosphatase whose expression is restricted to the central nervous system.

We describe the cloning, chromosomal localization and characterization of RPTPrho, a new member of the RPTPmu/kappa phosphatase subfamily. Receptor tyrosine phosphatases in this subfamily are comprised of a MAM domain near the N-terminal, an immunoglobulin-like domain, four fibronectin type III repeats, a single transmembrane domain, and a large juxtamembrane segment followed by two intracellular phosphatase domains. An alternatively spliced mini-exon was identified in the extracellular segment of RPTPrho, between the fourth fibronectin type III repeat and the transmembrane domain. The RPTPrho gene was mapped to human chromosome 20 and mouse chromosome 2. Northern blot analysis demonstrated that RPTPrho expression was restricted to the central nervous system, and in situ hybridization studies showed that the RPTPrho transcript was distributed throughout the murine brain and spinal cord. Exceptionally high levels of the transcript were present in the cortex and olfactory bulbs during perinatal development, but were down-regulated during postnatal week two. The motifs found in the extracellular segment of type II receptor protein tyrosine phosphatases are commonly found in neural cell adhesion molecules, suggesting that RPTPrho may be involved in both signal transduction and cellular adhesion in the central nervous system.

Novel receptor protein tyrosine phosphatase (RPTPrho) and acidic fibroblast growth factor (FGF-1) transcripts delineate a rostrocaudal boundary in the granule cell layer of the murine cerebellar cortex.

We have identified a novel receptor-like protein tyrosine phosphatase (RPTPrho) transcript whose expression in the cerebellar cortex is restricted to the granule cell layer of lobules 1-6. Acidic fibroblast growth factor (FGF-1) mRNA follows a similar cerebellar expression pattern. Together, the two markers define a sharp boundary in lobule 6, slightly caudal to the primary fissure. Anterior and posterior compartments became discernible only during postnatal weeks two and six, for RPTPrho and FGF-1, respectively. A rostrocaudal boundary in lobule 6 of the murine cerebellar cortex has also been identified morphologically by the effects of the meander tail mutation. The position of the RPTPrho and FGF-1 boundary on the rostrocaudal axis of the cerebellar cortex was close to, but not coincident with, the caudal extent of the disorganized anterior lobe of meander tail and the rostral extent of Otx-2 expression. The restricted pattern of FGF-1 and RPTPrho implies that these molecules may have specific signaling roles in the tyrosine phosphorylation/dephosphorylation pathway in the anterior compartment of the adult cerebellar cortex.

The GABAA receptor alpha 6 subunit gene (Gabra6) is tightly linked to the alpha 1-gamma 2 subunit cluster on mouse chromosome 11.

We have established that the GABAA receptor alpha 6 (Gabra6) and alpha 1 (Gabra 1) subunit genes are tightly linked on mouse chromosome 11 by analysing the strain distribution patterns of RFLPs for the two genes and microsatellite markers flanking these genes in 26 BXD recombinant inbred strains. These results further demonstrate clustering of the GABAA receptor subunit genes on mouse chromosomes and the synteny for these clusters between the mouse and human genomes.

Embryonic and postnatal expression of four gamma-aminobutyric acid transporter mRNAs in the mouse brain and leptomeninges.

The distribution of gamma-aminobutyric acid (GABA) transporter mRNAs (mGATs) was studied in mouse brain during embryonic and postnatal development using in situ hybridization with radiolabeled oligonucleotide probes. Mouse GATs 1 and 4 were present in the ventricular and subventricular zones of the lateral ventricle from gestational day 13. During postnatal development, mGAT1 mRNA was distributed diffusely throughout the brain and spinal cord, with the highest expression present in the olfactory bulbs, hippocampus, and cerebellar cortex. The mGAT4 message was densely distributed throughout the central nervous system during postnatal week 1; however, the hybridization signal in the cerebral cortex and hippocampus decreased during postnatal weeks 2 and 3, and in adults, mGAT4 labeling was restricted largely to the olfactory bulbs, midbrain, deep cerebellar nuclei, medulla, and spinal cord. Mouse GAT2 mRNA was expressed only in proliferating and migrating cerebellar granule cells, whereas mGAT3 mRNA was absent from the brain and spinal cord throughout development. Each of the four mGATs was present to some degree in the leptomeninges. The expression of mGATs 2 and 3 was almost entirely restricted to the pia-arachnoid, whereas mGATs 1 and 4 were present only in specific regions of the membrane. Although mGATs 1 and 4 may subserve the classical purpose of terminating inhibitory GABAergic transmission through neuronal and glial uptake mechanisms, GABA transporters in the pia-arachnoid may help to regulate the amount of GABA available to proliferating and migrating neurons at the sub-pial surface during perinatal development.

Characterization of the 1B promoter of fibroblast growth factor 1 and its expression in the adult and developing mouse brain.

The present study elucidates the molecular structure of a murine fibroblast growth factor 1 (FGF-1) promoter and describes its distribution in the adult and developing mouse brain. A cDNA clone coding for FGF-1 was isolated from a mouse brain cDNA library. Nucleotide sequence analysis revealed that the clone contained, in addition to the protein coding region, an untranslated exon (FGF-1B) 34 base pairs upstream of the translation start codon ATG. The mouse cDNA clone corresponded to the sole FGF-1 transcript in the brain. An RNase protection assay was used to map the transcription start site of the 1B promoter. The sequences upstream from the major transcription initiation site lacked consensus TATA or CAAT boxes. In situ hybridization with cRNA probes specific for the 1B transcript showed the message to be restricted largely to sensory and motor nuclei in the brainstem, and to the ventral spinal cord and cerebellum. Although occasional brainstem nuclei were labeled at low levels by embryonic day 18, the majority of nuclei became detectable autoradiographically during postnatal weeks 1 and 2, and adult levels of grain density were reached during the 3rd and 4th postnatal weeks. FGF-1B mRNA was expressed in phylogenetically older brain regions, which are involved primarily in processing information from exteroceptive sensory mechanoreceptors and in motor control. The relatively late developmental expression suggests a role for FGF-1 in neuronal maturation, rather than in neurogenesis.

[Favorable effects of epsilon-aminocaproic acid on the children with nephrotic syndrome and Schonlein-Henoch syndrome treated with corticosteroids]. / Korzystny wplyw kwasu epsilon-aminokapronowego u dzieci chorych na zespól nerczycowy i zespól Schönleina-Henocha leczonych kortykosteroidami.

An effect of EACA given in the daily dose of 85-230 mg/kg for 1-1-days on the activity of certain plasma protease inhibitors in 7 children with steroid-sensitive and steroid-dependent nephrotic syndrome (age between 3.5 and 18 years), and in 6 children with Schönlein-Henoch syndrome (aged between 3.5 and 6 years). Additionally, an effect of EACA on clinical status, dynamics of improvement, proteinuria and/or erythrocyturia, and incidence of adverse reactions was studied. It was found that EACA significantly increased antithrombin III activity by approximately 68.8% proteinase alpha 1-inhibitor by 41.8% alpha 2-antiplasmin by 55% in patients with nephrotic syndrome, and increased an activity of protease alpha 1-inhibitor by 75% in patients with Schönlein-Henoch syndrome. EACA given together with corticosteroids enhanced their efficiency manifested--especially in children with Schönlein-Henoch syndrome--by a rapid diminishment of skin changes, proteinuria and erythrocyturia. A drop in blood pressure, loose stools, upper respiratory inflammation, and fever were most frequent adverse reactions. EACA given alone produced rapidly increasing edema in patients with hephrotic syndrome. It seems that EACA may be used as an adjuvant therapy in some cases of nephrotic and Schönlein-Henoch syndromse.