Embryonic motoneuron-skeletal muscle co-culture in a defined system.

This paper describes a significant biotechnological advancement by creating a minimalist serum-free defined system to co-culture rat mammalian nerve and muscle cells in order to form functional neuromuscular junctions. To date, all the known in vitro nerve and muscle co-culture models use serum containing media; and while functional neuromuscular junctions (NMJ) are described, they failed to detail or quantify the minimum factors needed to recreate the NMJ in vitro. In this work, we demonstrate the development of a defined motoneuron and muscle co-culture system resulting in the formation of NMJs including: 1) a new culture technique, 2) a novel serum-free medium formulation and 3) a synthetic self-assembled monolayer (SAM) substrate N-1 [3-(trimethoxysilyl) propyl] diethylenetriamine (DETA). We characterized the culture by morphology, immunocytochemistry, electrophysiology and videography. This model system provides a better understanding of the minimal growth factor and substrate interactions necessary for NMJ formation and provides a basic system that can be utilized for nerve-muscle tissue engineering, regenerative medicine and development of limb prosthetics.

Mutations in the gene encoding epsilon-sarcoglycan cause myoclonus-dystonia syndrome.

The dystonias are a common clinically and genetically heterogeneous group of movement disorders. More than ten loci for inherited forms of dystonia have been mapped, but only three mutated genes have been identified so far. These are DYT1, encoding torsin A and mutant in the early-onset generalized form, GCH1 (formerly known as DYT5), encoding GTP-cyclohydrolase I and mutant in dominant dopa-responsive dystonia, and TH, encoding tyrosine hydroxylase and mutant in the recessive form of the disease. Myoclonus-dystonia syndrome (MDS; DYT11) is an autosomal dominant disorder characterized by bilateral, alcohol-sensitive myoclonic jerks involving mainly the arms and axial muscles. Dystonia, usually torticollis and/or writer's cramp, occurs in most but not all affected patients and may occasionally be the only symptom of the disease. In addition, patients often show prominent psychiatric abnormalities, including panic attacks and obsessive-compulsive behavior. In most MDS families, the disease is linked to a locus on chromosome 7q21 (refs. 11-13). Using a positional cloning approach, we have identified five different heterozygous loss-of-function mutations in the gene for epsilon-sarcoglycan (SGCE), which we mapped to a refined critical region of about 3.2 Mb. SGCE is expressed in all brain regions examined. Pedigree analysis shows a marked difference in penetrance depending on the parental origin of the disease allele. This is indicative of a maternal imprinting mechanism, which has been demonstrated in the mouse epsilon-sarcoglycan gene.

The DYT1 GAG deletion is infrequent in sporadic and familial writer' s cramp.

A 3-base pair (GAG) deletion in the DYT1 gene has recently been found to be responsible for most cases of early-onset primary generalized dystonia. In some cases, this mutation has been associated with writer's cramp. To determine the frequency of this mutation in a larger series of patients, we examined 44 index patients with sporadic or familial (seven patients) writer's cramp for the presence of the DYT1 GAG deletion, including eight patients with segmental dystonia involving at least one upper limb. We found the mutation in none of these index patients, which confirms that isolated writer's cramp is only in rare cases a phenotypic manifestation of this mutation, even if a positive family history of writer's cramp is present.

[Training of local health managers: process of identification of strategic actions]. / Formação de gestores locais de saúde: processos para identificar estratégias de atuação.

In Brazil, in recent years, as a result of the increasing participation of county authorities in health care, a need for tools which would contribute to the better preparation of local administrators, complementary to the activities of a more academic nature, has been recognized. One of the possible alternatives is the exploitation of experiences, regarded as successful, in local health care planning and administration, by using them as material for "case studies" in activities with selected groups of health care administrators thus, stimulating the identification of those elements which contributed to the favorable results attained, and their interactions, in the quest for analogies which would facilitate the identification of new perspectives for their own situations. In this article an experience of a "case studies" development, in response to a demand from UNICEF, based on 8 counties in from the North and Northeast, which were successful in using a "focus approach" in their organization of data and their utilisation in a seminar with 21 local managers from both regions, is presented. During the seminar the local health managers attained greater knowledge of the strategies implemented and identified feasible intervention alternatives. The methodological proposition of teaching on the basis of case studies, using a conceptual strategy of grouping experiences according to specific dimensions enabled local health managers to learn from their practical experiences.

Isolation of an RNA-directed RNA polymerase-specific cDNA clone from tomato.

A 3600-bp RNA-directed RNA polymerase (RdRP)-specific cDNA comprising an open reading frame (ORF) of 1114 amino acids was isolated from tomato. The putative protein encoded by this ORF does not share homology with any characterized proteins. Antibodies that were raised against synthetic peptides whose sequences have been deduced from the ORF were shown to specifically detect the 127-kD tomato RdRP protein. The immunoresponse to the antibodies correlated with the enzymatic activity profile of the RdRP after chromatography on Q-, poly(A)-, and poly(U)-Sepharose, hydroxyapatite, and Sephadex G-200 columns. DNA gel blot analysis revealed a single copy of the RdRP gene in tomato. RdRP homologs from petunia, Arabidopsis, tobacco, and wheat were identified by using polymerase chain reaction. A sequence comparison indicated that sequences homologous to RdRP are also present in the yeast Schizosaccharomyces pombe and in the nematode Caenorhabditis elegans. The previously described induction of RdRP activity upon viroid infection is shown to be correlated with an increased steady state level of the corresponding mRNA. The possible involvement of this heretofore functionally elusive plant RNA polymerase in homology-dependent gene silencing is discussed.

A single nucleotide substitution converts potato spindle tuber viroid (PSTVd) from a noninfectious to an infectious RNA for nicotiana tabacum.

Mechanical inoculation of Nicotiana tabacum with the PSTVd isolate KF 440-2 from the host plant tomato resulted in the de novo emergence, replication, and accumulation of a new "tobacco variant," designated PSTVd NT. It produces no symptoms in tobacco but, like PSTV KF 440-2, severe ones in tomato. The sequence analysis of PSTVd NT revealed a single nucleotide substitution from C-->U at position 259. Autonomous viroid replication was also induced in tobacco by genomic integration of oligomeric cDNA copies of PSTVd KF 440-2. Although these cDNAs contained the original tomato-specific C259, the circular PSTVd RNA subsequently accumulating in tobacco also exhibited the C259-->U259 substitution. In the secondary structure of PSTVd, nucleotide 259 is part of an internal loop analogous to loop E of eukaryotic 5S rRNA and presumed to be the only bulged extrahelical nucleotide of this loop. The C259 in PSTVd KF 440-2 and in practically all other isolates and the U259 in PSTVd NT of the loop E-like structure might be involved in protein binding and in viroid processing. The new variant PSTVd NT is genetically stable in both tobacco and tomato.

Molecular analysis of the gene family of the signal recognition particle (SRP) RNA of tomato.

The sequence variants of the signal recognition particle (SRP) RNA gene family from four tomato cultivars have been isolated and characterized which indicated the existence of SRP RNA pseudogenes. Sequence analysis revealed two conserved sequence motifs in the upstream region, a TATA-like box and an upstream sequence element (USE), 'TCCCACATCG', both located at a conserved distance to the transcription start point. These elements are identical to the DNA-dependent RNA polymerase III (pol III)-specific promoters of U-rich small nuclear RNA (UsnRNA) genes of plants. Moreover, T-rich stretches are found at the 3' end of the coding regions of the SRP RNA genes which could act as typical pol III termination signals. These findings and recent results from site-directed mutation analysis of the SRP RNA genes from Arabidopsis thaliana indicate that, in contrast to mammalian systems, plant pol III SRP RNA genes are most probably regulated by external promoter elements. According to the identical promoter organization between plant U3-, U6snRNA, MRP-like RNA and SRP RNA genes, one can group these genes into the 'pol III(EXT)USE' subclass of externally regulated USE-dependent pol III genes.

Characterization of the signal recognition particle (SRP) RNA population of tomato (Lycopersicon esculentum).

Molecular cloning of 30 cDNAs and subsequent characterization of the corresponding SRP RNA from four cultivars of tomato (Lycopersicon esculentum) revealed altogether 14 sequence variants, which could be ordered into six groups. The expression of five representatives from these groups was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) in different cultivars and different tissues. Although one cultivar-specific SRP RNA variant could be detected in the leaf SRP RNA population, identical SRP RNA populations seem to be present in the four different cultivars as well as in different tissues, such as leaves, flowers, fruits, stems and roots. Sequence comparison revealed that several variants might have evolved by recombination of two different SRP RNA sequences. On the basis of five SRP RNA variants, the current secondary structure model was refined and a new conserved structural element was detected. Comparative sequence analysis of domain II from all known SRP RNA homologues reveals a remarkable conservation of this element. As demonstrated previously, the corresponding area overlaps with a region that interact with the SRPp68/p72 heterodimer and/or with ribosomes. Based on structural and functional considerations, we propose that the domain IV structure together with the highly conserved area of domain II constitutes the essential core of the SRP RNA.

RNA-directed de novo methylation of genomic sequences in plants.

One monomeric and three oligomeric potato spindle tuber viroid (PSTVd) cDNA units were introduced into the tobacco genome via the Agrobacterium-mediated leaf-disc transformation. Southern analysis of the integrates revealed that only their PSTVd-specific sequences become fully methylated, whereas the flanking T-DNA and the genomic plant DNA remain unaltered. Viroid cDNA methylation could only be observed after autonomous viroid RNA-RNA replication had taken place in these plants. These findings demonstrate that a mechanism of de novo methylation of genes might exist that can be induced and targeted in a sequence-specific manner by their own mRNA.

[Myocardial infarct and cerebral vascular disorders associated with high temperature and carbon monoxide in a metropolitan area of southeastern Brazil]. / Infarto do miocárdio e acidente vascular cerebral associados à alta temperatura e monóxido de carbono em área metropolitana do sudeste do Brasil.

Many studies have been published about the relationship between life style and genetic risk factors and stroke and heart attack, but there have been few about the relationship between atmospheric pollution, specifically with carbon monoxide, and temperature and stroke and heart attack. With a view to filling this gap the relationship between values of carbon monoxide concentration in the air, maximum and average by day, and maximum temperature by day and the number of new cases of CVA and IM admitted as emergencies at the biggest hospital complex of S. Paulo city was analysed. If was concluded, by the use of multivariable regression analysis, that 2.1% and 4.9% of heart attack admissions were due to carbon monoxide air pollution and high temperature, respectively. 2.8% of stroke admissions were due high temperature. Stroke and carbon monoxide were not associated in this population.

Malabsorption of carbohydrate foods by urban blacks.

Prevalences of non-infective bowel diseases are very low in South African urban blacks compared with the white population. In seeking elucidation, using breath hydrogen measurements in series of black and white subjects, small-bowel transit time was determined, and the malabsorption of maize, wheat, and rice investigated. Median transit times in both ethnic groups were similar. Rice was fully, but wheat incompletely absorbed by both groups. Maize, the staple food of blacks, was incompletely absorbed by them, although completely absorbed by the white subjects. Carbohydrate consumption is high in the black population (60-65% of total energy intake). It is probable that in blacks, despite their now eating a low-fibre diet, an expected increase in large-bowel diseases has been inhibited in part by the protective mechanism of fermentation of malabsorbed maize and wheat.

Limitations of faecal chymotrypsin as a screening test for chronic pancreatitis.

Faecal chymotrypsin was measured in patients with chronic pancreatitis and in healthy black urban and rural control subjects. In the patients, significantly lower values of faecal chymotrypsin were obtained (mean (SD) 2.4 1.79 U/g stool) whereas in urban control subjects, values were within the normal range (mean (SD) 13.2 (11.9)). In rural black control subjects, however, the faecal chymotrypsin value was significantly lower (mean (SD) 7.1 (5.1)) than in urban black control subjects. It is suggested that faecal pH may influence faecal chymotrypsin values. The mean faecal pH in rural black subjects (pH 6.14) was significantly lower than that in urban control subjects (pH 6.77) and in patients with chronic pancreatitis (pH 6.61). Moreover, mean faecal chymotrypsin is high (20.0 U/g stool) at a pH greater than 7. Between pH 6 and 7 the mean value drops to 8.6 U/g stool and below pH 6 mean faecal chymotrypsin is in the abnormal range (4.4 U/g stool). Hence, low values for faecal chymotrypsin may be due to lower faecal pH (less than 6) in healthy control subjects. For diagnostic purposes, the faecal pH value should be determined if a low faecal chymotrypsin value is obtained.

Analgesics and gastric ulcers. A case-control study in an urbanised black population.

The patterns of analgesic ingestion in urbanised black patients with gastric ulcer disease were assessed. A strong statistical association was found between the quantity ingested and the disease. There was no difference in the type of analgesic and smoking and alcohol habits between gastric ulcer patients and controls in this study.

The prolonged course of gastrointestinal tuberculosis.

Review of biopsy specimens showed that a patient incorrectly diagnosed 7 years ago as having Crohn's disease actually had histologically proven gastrointestinal tuberculosis of 7 years' duration. It is significant that gastrointestinal tuberculosis and Crohn's disease can mimic each other not only in their clinical, radiologic, and histologic manifestations but also temporally. We discuss the interrelationship between AIDS, tuberculosis, and inflammatory bowel disease.

Mutagenicity of antiepileptic drugs. II. Phenytoin, primidone and phenobarbital.

The antiepileptic drugs, phenytoin, primidone and phenobarbital, were tested in Chinese hamster ovary (CHO) cells for their ability to induce sister-chromatid exchanges (SCEs) and structural chromosomal aberrations (CAs), with and without metabolic activation. SCEs and CAs were analyzed in the same cell population. The results are negative.

[Bronchopulmonary infiltrates in chronic inflammatory bowel disease]. / Bronchopulmonale Infiltrate bei chronisch entzündlicher Darmerkrankung.

High temperatures, night sweat, chest pain, cough and dyspnoea suddenly occurred in a 54-year-old patient. The serious disease was accompanied by variable pulmonary infiltrations. Chemical pathology showed maximally increased sedimentation rates, slight leucocytosis and anaemia. Complete serology was negative. The occurrence of large intestinal ileus required laparatomy and after commencement of treatment with steroids the overall state improved, pulmonary symptoms disappeared, and radiographically demonstrable infiltration were clearly regressing. Histology revealed presence of acute ulcerative colitis. Lung infiltrates probably represented extraintestinal manifestation of the chronic inflammatory bowel disease. In contrast to experience from the literature lung infiltrations in this case preceded clinical manifestations of the underlying disease.