Proteome analysis of differentially displayed proteins as a tool for the investigation of symbiosis.

Two-dimensional gel electrophoresis was used to identify differentially displayed proteins expressed during the symbiotic interaction between the bacterium Sinorhizobium meliloti strain 1021 and the legume Melilotus alba (white sweetclover). Our aim was to characterize novel symbiosis proteins and to determine how the two symbiotic partners alter their respective metabolisms as part of the interaction, by identifying gene products that are differentially present between the symbiotic and non-symbiotic states. Proteome maps from control M. alba roots, wild-type nodules, cultured S. meliloti, and S. meliloti bacteroids were generated and compared. Over 250 proteins were induced or up-regulated in the nodule, compared with the root, and over 350 proteins were down-regulated in the bacteroid form of the rhizobia, compared with cultured cells. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprint analysis, in conjunction with data base searching, were used to assign putative identity to nearly 100 nodule, bacterial, and bacteroid proteins. These included the previously identified nodule proteins leghemoglobin and NifH as well as proteins involved in carbon and nitrogen metabolism in S. meliloti. Bacteroid cells showed down-regulation of several proteins involved in nitrogen acquisition, including glutamine synthetase, urease, a urea-amide binding protein, and a PII isoform, indicating that the bacteroids were nitrogen proficient. The down-regulation of several enzymes involved in polyhydroxybutyrate synthesis and a cell division protein was also observed. This work shows that proteome analysis will be a useful strategy to link sequence information and functional genomics.

Elevated levels of synthesis of over 20 proteins results after mutation of the Rhizobium leguminosarum exopolysaccharide synthesis gene pssA.

The protein expression profiles of Rhizobium leguminosarum strains in response to specific genetic perturbations in exopolysaccharide (EPS) biosynthesis genes were examined using two-dimensional gel electrophoresis. Lesions in either pssA, pssD, or pssE of R. leguminosarum bv. viciae VF39 or in pssA of R. leguminosarum bv. trifolii ANU794 not only abolished the capacity of these strains to synthesize EPS but also had a pleiotropic effect on protein synthesis levels. A minimum of 22 protein differences were observed for the two pssA mutant strains. The differences identified in the pssD and pssE mutants of strain VF39 were a distinct subset of the same protein synthesis changes that occurred in the pssA mutant. The pssD and pssE mutant strains shared identical alterations in the proteins synthesized, suggesting that they share a common function in the biosynthesis of EPS. In contrast, a pssC mutant that produces 38% of the EPS level of the parental strain showed no differences in its protein synthesis patterns, suggesting that the absence of EPS itself was contributing to the changes in protein synthesis and that there may be a complex interconnection of the EPS biosynthetic pathway with other metabolic pathways. Genetic complementation of pssA can restore wild-type protein synthesis levels, indicating that many of the observed differences in protein synthesis are also a specific response to a dysfunctional PssA. The relevance of these proteins, which are grouped as members of the pssA mutant stimulon, remains unclear, as the majority lacked a homologue in the current sequence databases and therefore possibly represent a novel functional network(s). These findings have illustrated the potential of proteomics to reveal unexpected higher-order processes of protein function and regulation that arise from mutation. In addition, it is evident that enzymatic pathways and regulatory networks are more interconnected and more sensitive to structural changes in the cell than is often appreciated. In these cases, linking the observed phenotype directly to the mutated gene can be misleading, as the phenotype could be attributable to downstream effects of the mutation.

Proteome analysis of the model microsymbiont Sinorhizobium meliloti: isolation and characterisation of novel proteins.

Sinorhizobium meliloti is an agriculturally and ecologically important microbe due to its capacity to establish nitrogen-fixing symbiosis with plant legumes. Two-dimensional gel electrophoresis of total cellular protein was used to establish a proteome reference map for the model microsymbiont Sinorhizobium meliloti strain 1021. The extent of changes in the gene expression of cells grown in a defined medium at different growth phases was established. After examination of over 2000 resolved protein spots, a minimum of 52 reproducible changes in protein expression levels were detected when early exponential phase cells were compared to late exponential phase cells. In contrast, induction of nodulation gene expression by the addition of the flavonoid luteolin to cells did not result in detectable changes in protein expression at either early or late exponential phase. N-terminal microsequencing of eighteen unknown constitutive proteins plus four proteins, induced or up-regulated in late exponential phase cells, allowed the identification of proteins not previously described in rhizobia. These included an amide-binding protein, a putative hydrolase of the glyoxalase II protein family, a nucleoside diphosphate kinase, and a 5'-nucleotidase. N-terminal microsequencing was also valuable in revealing N-terminal post-translational processing and assigning a subcellular location to the analysed protein. Proteome analysis will provide a powerful analytical tool to complement the sequencing of the genome of strain 1021.

[Spontaneous extradural spinal hematoma. Case report]. / Hematoma extradural intrarraquidiano espontâneo. Relato de caso.

We report a case of a 55-year-old woman, that has a mild hypertension, in use of a betablocker drugs, who had a sudden spinal cord compression syndrome with intense cervical pain, tetraplegia and sensitivity level in C4. Extradural spinal hematoma of the cervical spine was diagnosed by magnetic resonance. The patient underwent a laminectomy and aspiration of the hematoma. The patient is on physiotherapy and presents progressive motor and sensitivity improvement.

[Spontaneous epidural hematoma. Report of two cases]. / Hematoma epidural espontâneo. Relato de dois casos.

Spontaneous epidural hematomas are rarely described in literature. They are associated with infectious diseases of the skull, coagulation disorders, vascular malformations of the dura-mater and metastasis to the skull. The authors report two cases of spontaneous epidural hematoma of different etiologies, and study parameters of hemostasis.

Determination of plasmid-encoded functions in Rhizobium leguminosarum biovar trifolii using proteome analysis of plasmid-cured derivatives.

We have used proteome analysis of derivatives of R. leguminosarum biovar trifolii strain ANU843, cured of indigenous plasmids by a direct selection system, to investigate plasmid-encoded functions. Under the conditions used, the plasmid-encoded gene products contributed to only a small proportion of the 2000 proteins visualised in the two-dimensional (2-D) protein map of strain ANU843. The level of synthesis of thirty-nine proteins was affected after curing of either plasmid a, c or e. The differences observed upon plasmid curing included: protein loss, up/down-regulation of specific proteins and novel synthesis of some proteins. This suggests that a complex interplay between the cured plasmid and the remaining replicons is occurring. Twenty-two proteins appeared to be absent in the cured strains and these presumably are encoded by plasmid genes. Of these, a small heat shock protein, a cold shock protein, a hypothetical YTFG-29.7 kDa protein, and the alpha and beta subunits of the electron transfer flavoprotein were identified by N-terminal microsequencing and predicted to be encoded by plasmid e. Four of the sequenced proteins putatively encoded on plasmid e and two encoded on plasmid c were novel. In addition, curing of plasmid e and c consistently decreased the levels of 3-isopropylmalate dehydratase and malate dehydrogenase, respectively, suggesting that levels of these proteins may be influenced by plasmid-encoded functions. A protein with homology to 4-oxalocrotonate tautomerase, which is involved in the biodegradation of phenolic compounds, was found to be newly synthesised in the strain cured of plasmid e. Proteome analysis provides a sensitive tool to examine the functional organisation of the Rhizobium genome and the global gene interactions which occur between the different replicons.

[Intraspinal enterogenous cyst. Case report]. / Cisto enterógeno intrarraquiano. Relato de caso.

The authors report the case of a 7 years old boy with cervical pain and tetraparesis progressing by two months. He presented an intraspinal cystic lesion from C4 to C6. Exeresis of the lesion was undergone by anterior way through a corpectomy of C5-C6 followed by reconstruction with bone graft of the iliac crest and metallic plaque. Anatomic diagnosis of the lesion was enterogenous cyst. Aspects of clinical presentation, embryology and treatment of these cysts are discussed.

New Rhizobium leguminosarum flavonoid-induced proteins revealed by proteome analysis of differentially displayed proteins.

Proteome analysis was used to establish the first two-dimensional protein map of Rhizobium. R. leguminosarum bv. trifolii strain ANU843 was grown in defined medium in the presence and absence of the flavonoid 7,4'-dihydroxyflavone. Over 1,700 constitutive proteins were resolved, representing about 30% of the estimated genomic output. Proteome analysis of flavonoid-treated cells was done to reveal differentially displayed proteins. The results showed that while the global expression pattern of proteins was largely unaltered by the treatment, four inducible proteins were observed. The four inducible proteins and 20 constitutively expressed proteins were subjected to sequence analysis to provide internal standards for the construction of a two-dimensional Rhizobium protein data base. The identity of 12 proteins, including NodE and NodB, was established. NodE was present throughout the growth of the cells but was diminished in amount in stationary phase cells whereas NodB was not detected in the later stages of growth. Two of the induced proteins sequenced did not match any known nodulation gene product, with one of these being present in mid-late log and stationary phase cells and possessing four consecutive His residues at the N-terminal sequencing was successful with 100 to 200 fmol of protein. Proteome analysis provides a sensitive new tool to examine plant-microbe interactions.

On the interaction of alpha-crystallin with unfolded proteins.

alpha-Crystallin, a major protein component of the lens, has chaperone-like properties whereby it prevents destabilised proteins from precipitating out of solution. It does so by forming a soluble high-molecular-weight (HMW) complex. A spectroscopic investigation of the HMW complex formed between a variety of unfolded proteins and bovine alpha-crystallin is presented in this paper. As monitored by fluorescence spectroscopy, a large amount of the hydrophobic probe, 8-anilino-1-naphthalene sulfonate (ANS) binds to the HMW complex implying that the complexed proteins (alcohol dehydrogenase (ADH), gamma-crystallin and rhodanese) are bound in an unfolded, possibly molten-globule state. The interaction between the anionic surfactant, sodium dodecyl sulfate (SDS) and ADH at high temperatures gives rise to a similar large increase in ANS fluorescence to that for the complex between alpha-crystallin and ADH. SDS, like alpha-crystallin, therefore complexes to proteins in their unfolded state leaving a large hydrophobic surface exposed to solvent. Unlike other chaperones (e.g., GroEL, DnaK and SecB), alpha-crystallin does not interact with unfolded, hydrophobic but stable proteins (e.g., reduced and carboxymethylated alpha-lactalbumin and alpha-casein). It is concluded that alpha-crystallin will only complex with proteins that are about to precipitate out of solution, i.e., ones that are severely compromised. 1H-NMR spectroscopy of the HMW complex formed between alpha-crystallin and gamma-crystallin indicates that the short C-terminal extension of alpha B-crystallin, but not that of alpha A-crystallin, has lost its flexibility in the complex implying that the former is involved in interactions with the unfolded gamma-crystallin molecule, possibly electrostatically via its two C-terminal lysine residues.

[Hepatic glycogenosis: the clinical, biochemical and enzymatic aspects in a group of pediatric patients]. / Glucogenosis hepática: aspectos clínicos, bioquímicos y enzimáticos en un grupo de pacientes pediátricos.

Nine children with clinical diagnosis of glycogenoses were studied, types were confirmed through determination of levels and structure of glycogen, stimulation with glucagon and enzymatic defect analyses. Eight patients suffered glycogenoses type III and one, type VI. The major age group un type III was 1 to 2 years old (62.5%), the type VI was diagnosed in a preschool boy. Mean clinical features were: hepatomegaly, doll-like facies and short height. Major biochemical alterations were: transaminases elevation in both types, hypertriglyceridemia, hyperglycemia, metabolic acidosis and hyperuricemia only in glycogenoses III. One III type patient presented cardiovascular alterations. All patients showed increased concentrations of erythrocyte glycogen, with normal structure in type VI and abnormal in 75% of type III. Tree fourths of type III patients had a positive response to glucagon stimulation. No one presented glucose 6 phosphatase deficiency.

[Giardia lamblia: comparison of two diagnostic methods and evaluation of response to treatment with metronidazole]. / Giardia lamblia: comparación de dos métodos de diagnóstico y evaluación de la respuesta al tratamiento con metronidazol.

During the period from March through November 1989, 70 children who were attended at the Pediatric Department at Central Hospital in Valencia, were enrolled in the study, it was thought that Giardia lamblia infection might be present. Giardia L. were identified using two different diagnostic procedures: from stool samples and duodenal aspirates for cysts or trophozoites examination. These children were treated with Metronidazole three dosage of 15, 30 and 50 mg/kg per day for a ten day period. Our study showed predominant giardiasis in children with ages ranging from 2 to 6 years old (60%) with a relationship between female and male sex 1.05:1. In this series, 72.8% of patients presented normal nutrition, and 55.7% of them were from the suburban area. The most frequent symptoms were abdominal pain, diarrhea, vomiting, abdominal distention, constipation and flatulence. The infants prevalent symptom was diarrhea (83.3%) and the older children and school children prevalent symptom was abdominal pain with 78.5 and 100% respectively. In this study, stool examination was positive in 97.1% of the children and duodenal aspirate was positive in all 70 children (100%); the first procedure showed predominant Giardia cysts (88.2%) and the second one showed predominant trophozoites (47.1%). All 70 patients (100%) were cured with Metronidazole to different dosage. Side effects were seen with only the maxim dose, such as nausea 40%, headache 10% and appearance of yeast into 50% of duodenal aspirate.

[Hepatic abscess in children: analysis of 20 cases]. / Abscesos hepáticos en niños: análisis de 20 casos.

Between March 1986 and March 1989, 20 children were diagnosed as having liver abscesses at the Pediatric Department of the Central Hospital in Valencia. 10 were amebic and 10 pyogenic abscesses. 85% occurred in children and 15% in infants. 15 of them (75%) were located in the right lobe, 80% of amebic abscesses had positive serological tests. An etiologic agent was found in 50% of the pyogenic abscesses (3: E. coli, 2: S. Aureus). 80 to 90% of patients presented abnormal prothrombin time. Sedimentation rate was very high in both groups, even at the discharge time. Hepatomegaly, fever, anemia and abdominal pain were the main clinical features. The macroscopic aspect of the aspirate was not helpful in the diagnosis. 60% of amebic liver abscesses needed only medical treatment but 80% of pyogenic abscesses deserved medical treatment and needle aspiration.

[Tumor form of neurocysticercosis: surgical removal of a cysticercus measuring 70X77 mm and treatment with praziquantel]. / Forma tumoral da neurocisticercose: exérese de cisticerco de 70X77 mm e tratamento com praziquantel: relato de caso.

The authors report a case of a female patient, age 22, who presented episodes of focal seizures, right hemiparesis and dysphasia of five months duration. The neurologic exam revealed bilateral papilledema and computarized tomography of the head showed a cystic lesion (70X77 mm) in the left fronto-temporo-parietal area, midline shift and similar lesions scattered in the brain parenchyma. She was submitted to a left craniotomy with the diagnosis of cerebral cysticercosis and the major cyst (Cysticercus racemosus) and a small cortical cyst (10X10 mm) were removed to aliviate the increased intracraneal pressure and for histopathological examination. Thirteen days after surgery she was discharged without neurological deficits, except mild papilledema. One month later she was treated with praziquantel (50 mg/kg/day) during 21 days. The CSF examination was normal before the treatment started and showed a transient eosinophilic cellular reaction with positivation of the complement fixation test for cysticercosis while on treatment. The CT-Scan performed before the medical treatment revealed ventricular dilatation and several low density areas (10 to 40 mm) scattered in the cerebral parenchyma, including the operative site. CT-Scan performed three months later showed small low density areas only in the left cerebral hemisphere, including the operative site, calcification in the parenchyma, and slight dilatation of the left lateral ventricle. The clinical evolution was uneventful and the neurologic examination was normal three months later.

Fistula liquorica pela cicatriz umbilical como complicacao de derivacao ventriculo-peritoneal. Relato de 2 casos. / Cerebraspinal fluid fistula through umbilical cicatrix as a complication of ventriculoperitoneal shunt. Report of 2 cases

Os autores relatam 2 casos de criancas hidrocefalicas tratadas com derivacao ventriculo-peritoneal, que apresentaram fistula liquorica pela cicatriz umbilical, que surgiram, 14 e 10 meses apos a instalacao do sistema de derivacao. Em 1 caso houve extrusao do cateter pela cicatriz umbilical. As condutas terapeuticas pertinentes sao discutidas em comparacao com a literatura

Aderencia do cateter atrial a veia jugular interna em derivacao ventriculo-atrial. / Adherence of the atrial catheter to internal jugular vein in ventriculoatrial shunt. A case report

O caso relatado refe-se a uma crianca hidrocefalica tratada com derivacao ventriculo-atrial e que, 6 anos e 6 meses apos revisao para alongamento do cateter atrial, apresentou firme aderencia deste a veia jugular interna, no ponto da conexao para o alongamento do cateter (proximo a juncao com a subclavia). Para a retirada do mesmo, foi necessaria a disseccao completa e a ligadura da veia jugular interna. A revisao da literatura mostrou 1 caso relatado com fixacao do cateter na veia cava, sem que houvesse conexao no local