A novel class A extended-spectrum beta-lactamase (BES-1) in Serratia marcescens isolated in Brazil.

Serratia marcescens Rio-5, one of 18 extended-spectrum beta-lactamase (ESBL)-producing strains isolated in several hospitals in Rio de Janeiro (Brazil) in 1996 and 1997, exhibited a high level of resistance to aztreonam (MIC, 512 microgram/ml) and a distinctly higher level of resistance to cefotaxime (MIC, 64 microgram/ml) than to ceftazidime (MIC, 8 microgram/ml). The strain produced a plasmid-encoded ESBL with a pI of 7.5 whose bla gene was not related to those of other plasmid-mediated Ambler class A ESBLs. Cloning and sequencing revealed a bla gene encoding a novel class A beta-lactamase in functional group 2be, designated BES-1 (Brazil extended-spectrum beta-lactamase). This enzyme had 51% identity with chromosomal class A penicillinase of Yersinia enterocolitica Y56, which was the most closely related enzyme and 47 to 48% identity with CTX-M-type beta-lactamases, which were the most closely related ESBLs. In common with CTX-M enzymes, BES-1 exhibited high cefotaxime-hydrolyzing activity (k(cat), 425 s(-1)). However, BES-1 differed from CTX-M enzymes by its significant ceftazidime-hydrolyzing activity (k(cat), 25 s(-1)), high affinity for aztreonam (K(i), 1 microM), and lower susceptibility to tazobactam (50% inhibitory concentration [IC(50)], 0.820 microM) than to clavulanate (IC(50), 0.045 microM). Likewise, certain characteristic structural features of CTX-M enzymes, such as Phe-160, Ser-237, and Arg-276, were observed for BES-1, which, in addition, harbored different residues (Ala-104, Ser-171, Arg-220, Gly-240) and six additional residues at the end of the sequence. BES-1, therefore, may be an interesting model for further investigations of the structure-function relationships of class A ESBLs.

IL-1 receptor antagonist in saliva; characterization in normal saliva and reduced concentration in Sjögren's syndrome (SS).

The characterization of a salivary factor cross-reacting with IL-1 receptor antagonist (IL-1Ra) is described. The apparent molecular weights of two species were 23 kD, consistent with the secreted peptide (sIL-1Ra), and 20 kD, consistent with the intracellular peptide (icIL-1Ra). It had an inhibitory activity on IL-1-stimulated fibroblasts, which is characteristic of IL-1Ra. Its source was the oral mucosa and not the salivary glands. Saliva from patients with SS contained significantly less IL-1Ra than saliva from controls. The decrease was marked in patients with early dental loss but whose xerostomia was still partial. In SS, the salivary IL-1/IL-1Ra imbalance may promote inflammatory lesions in the mouth and impede mucosal cell differentiation.

Atypical enolase isoenzyme in serum: a macroenolase formed from the alpha gamma-hybrid form.

We report an abnormal pattern for enolase (EC 4.2.1.11) isoenzymes in the serum of a patient with squamous cell lung carcinoma. The alpha alpha-isoenzyme was present but the alpha gamma form was not detected, and near the point of application on the electrophoretogram was an abnormal band. We determined that the abnormal fraction corresponded to a macroenolase, composed of the alpha gamma-isoenzyme complexed with IgG. From a practical point of view, the presence of such a macroenolase, containing gamma-subunits, results in falsely increased results for neuron-specific enolase (NSE) in procedures that determine only the NSE concentration without consideration of the enolase isoenzymes.

[Composite carcinoma of the prostate combining a small cell carcinoma and an adenocarcinoma. Apropos of a case]. / Carcinome composite de la prostate associant carcinome à petites cellules et adénocarcinome. A propos d'un cas.

A case of combined adenocarcinoma and small cell carcinoma of the prostate is described in a 58-year-old-man. Prostatic acid phosphatases and neuron specific enolase were found elevated in the serum. At autopsy the lung was free of tumor. The liver was replaced by numerous metastatic nodules and a voluminous mesenteric metastasis extended into the wall of the vessels (aorta and vena cava). Microscopic examination showed a small cell carcinoma component of the oat cell type and an adenocarcinoma component constituting 10% of the total tumor volume. By immunostaining, the small cell carcinoma component is neuron specific enolase+ and prostatic specific antigen-. The adenocarcinoma component is neuron specific enolase- and prostatic specific antigen+.

Serum neuron-specific/nonneuronal enolase ratio in the diagnosis of neuroblastomas.

Pretreatment samples from 24 children with neuroectodermal tumors (two ganglioneuromas, 22 neuroblastomas) and from 106 others with various tumors were submitted to the enzymatic determination of the serum neuron-specific enolase (NSE). The enzymatic procedure employed in this study allows the systematic determination of the NSE and of the nonneuronal enolase (NNE), thus permitting the calculation of the ratio of the two enolase components. Like results obtained with other procedures, enzymatic determined serum NSE results were raised in a high proportion of Stage IV neuroblastoma (100%) but elevated values also were found in a considerable number of the other tumors (29.2%) like Wilms' tumor, lymphomas, and soft tissue sarcomas. The use of the NSE/NNE ratio which characterizes NSE elevations originating from relative poor or rich sources of NSE, represents an additional index for improving the specificity of the NSE results in the diagnosis of neuroblastomas. With a cutoff value fixed at 7.5%, the specificity of the test is 85.9%. When this limit is fixed at 15%, the specificity reaches 95.3% whereas 81.8% of the results of Stage IV neuroblastomas are still above this value.

Enzymatic determination of serum neuron-specific enolase in small cell lung cancers. Utility of the serum neuron-specific enolase/serum nonneuronal enolase ratio.

Increasing interest is shown in the determination of the serum neuron-specific enolase for the diagnosis and the follow-up studies of small cell lung cancers. We report results obtained by an enzymatic procedure that permits the simultaneous determination of the neuron and nonneuron-specific enolase and the calculation of the ratio of these two components. The utility of this ratio which characterizes elevations of the serum neuron-specific enolase from a poor or rich source of this component was tested in 38 patients with small cell lung carcinoma and in 57 subjects suffering from other bronchogenic cancers. The control group consisted of 37 blood donors and 56 patients with respiratory disease. For the diagnosis, the sensitivity and the specificity of the enzymatically determined neuron-specific enolase compared well with published results obtained by radioimmunoassay and enzymoimmunoassay. The use of the ratio clearly increases the specificity of the test, since only 5.3 percent of false positive results are found when bronchogenic tumors other than small cell carcinoma are studied. The sensitivity was 76 and 100 percent in diagnosis of limited and extensive forms, respectively. The use of this ratio in the follow-up of the patients and for the determinations in hemolyzed samples is set out.

Preparation and purification of gamma gamma enolase (neuron-specific enolase) using high performance anion exchange chromatography.

A simple and rapid method, using only two chromatographic steps, is described for the purification and preparation of gamma gamma enolase isoenzymes from human and beef brain extracts. In the first step, a crude gamma gamma enolase was obtained by chromatography on Q-Sepharose Fast Flow column. The crude fraction was then purified by high performance anion exchange chromatography on a Mono-Q column. gamma gamma enolase obtained in this manner was shown to be homogeneous by two dimensional polyacrylamide gel electrophoresis and by high performance gel permeation chromatography. The yield of gamma gamma enolase by this method was 7-8 mg of pure enzyme per 100 g of brain.

Interference by platelets in the electrophoretic determination of enolase isoenzymes in plasma.

Serum and plasma gave different electrophoretic patterns when enolase isoenzymes were evaluated by electrophoresis on cellulose acetate. Plasma isoenzyme bands were more intense, and there was an additional one (band P) that was not present in serum. We show that, under the conditions of electrophoresis, some of the residual platelets in the plasma are ruptured, releasing intracellular enolases and consequently leading to intensification of the isoenzyme bands. The band P originated from the remaining unruptured platelets. Thus plasma samples must be platelet-free for determination of enolase isoenzyme to be reliable.

Determination of serum neuron-specific enolase by differential immunocapture.

A new method for the determination of serum neuron-specific enolase is presented. It consists of two steps: first, an immunocapture of gamma-subunit containing isoenzymes by absorption on immobilized anti-gamma antibodies; second, bioluminescence assay of enolase activities in untreated control samples and in the supernates of antibody treated samples. Total and alpha alpha activities are obtained, from which the neuron-specific enolase activity (alpha gamma + gamma gamma) can then be calculated by difference. As compared to the procedures currently in use, the immunocapture method is very rapid (30 min) and is more suitable for small series of determinations as needed in clinical chemistry applications. Reference interval values for serum found by this method agree with published data. When tested with samples from patients suffering from neuroblastoma or small cell lung cancer, it confirms the specific elevations in neuron-specific enolase activity previously described for these cancers, using other analytical approaches.

Rapid electrophoretic determination of neuron-specific enolase isoenzymes in serum.

This assay procedure for each of the two neuron-specific enolases (alpha gamma and gamma gamma) and the non-neuronal enolase (alpha alpha) in serum involves two steps: electrophoretic separation of the three isoenzymes--alpha alpha, alpha gamma, and gamma gamma--on cellulose acetate, and bioluminescence measurement of total enolase activity. From these data, the activity concentrations (U/L) of the three isoenzymes in serum are calculated. Both measurement steps are based on the enzymatic activity of enolase and thus differ from the immunological methods currently in use, which require the availability of specific antibodies. The method is rapid (approximately 30 min for both steps) and requires only 10 microL of serum for the complete analysis. Studies of normal children and adults, and of patients suffering from neuroblastoma and small-cell lung cancer, show that it is suitable for clinical use. Furthermore, the fact that both neuron-specific isoenzymes of enolase can be systematically separated is an advantage over immunological techniques in determining isoenzyme patterns for pathological samples.

Isolation of free and membrane-bound polysomes and mRNA highly active in translation and reverse transcription from small discrete regions of rat brain.

A procedure is described for the preparation of total polysomes, membrane-bound and free polysomes and polysomal mRNA from as little as 5 mg or less of brain tissue. These preparations were highly active when tested for translation and reverse transcription in vitro. Using this method, polysomes and mRNA from rat cerebral cortex, cerebellum, hippocampus and hypothalamus were compared. The results showed that membrane-bound polysomes were more active than free polysomes in protein synthesis. The activities of polysomes and mRNA for protein and cDNA synthesis were dependent on the specific brain structures from which they were obtained. Polysomes from cerebellum and hypothalamus incorporated amino acids more actively than those from cerebral cortex or hippocampus, when tested in a reticulocyte lysate system. Cerebellar mRNA also showed the highest activity for cDNA syntehsis as compared to mRNAs from the other three tissues.

An ultramicro bioluminescence assay of enolase: application to human cerebrospinal fluid.

A highly sensitive method based on bioluminescence is described for the assay of enolase which can measure as little as 0.4 X 10(-6) IU of activity. This corresponds to an amount of enzyme present in 1-2 microliters of normal human cerebrospinal fluid and is therefore easily applicable to clinical samples of CSF which can only be obtained in very small amounts. The reproducibility of the method is very high within a broad range of enzyme concentrations and the assay is linear from 0.4 X 10(-6) IU up to at least 50 X 10(-6) IU of enzyme. This would permit application of the method to biological samples containing low as well as high enolase activities and especially for monitoring changes in enolase concentrations in the CSF and in the serum, as a function of pathological lesions in the central nervous system and other tissues.

Structure and biological activity of polysomes stained with Coomassie blue.

Polysomes prestained with Coomassie blue were fractionated on sucrose density gradients giving rise to visible bands corresponding to different size classes of aggregates. Coomassie blue staining enhanced the capacities of brain and liver polysomes to synthesize proteins in vitro, including the synthesis of neuron-specific enolase. This positive action of the dye was restricted to polysomes and was not manifested when mRNAs isolated from prestained polysomes were tested in in vitro translation or reverse transcription, indicating that the action of the dye consists in stabilization of polysomal structure.

[Congenital anomaly of serum pseudocholinesterase originating in neonatal respiratory distress]. / Anomalie congénitale des pseudocholinestérases sériques à l'origine d'une détresse respiratoire néonatale.

Prolonged suxamethonium-induced apnoea was observed after obstetrical anaesthesia in a 30 year old woman with abnormal plasma cholinesterases (homozygous Ea1-Ea1). Flaccidity and apnoea in the child required controlled ventilation for 30 min. Possible mechanisms underlying prolonged apnoea after the use of suxamethonium for obstetrical anaesthesia are discussed. Atypical pseudocholinesterases were identified using quantitative dosage of enzymatic activity and inhibition of atypical pseudocholinesterases by dibucaine, fluoride, chloride, scoline and urea. This was carried out in the patient, her baby and family, thus identifying the genotype of the different family members.

Role of tissue specific factors in the translation of brain messenger ribonucleic acids in vitro.

The activity of brain ribosomal subunits was examined by measuring (a) the saturation of ribosomes with nascent polypeptides and the rates of release of completed chains; (b) interaction of the subunits with brain messenger RNA to form polysomal aggregates; (c) formation of polyphenylalanine in the presence of polyuridylic acid at high magnesium concentration and (d) inhibition by aurine tricarboxylic acid. The results showed that a portion of the subunits were defective in forming initiation complexes with brain messenger RNA, but translated polyuridylate efficiently. The subunits that did form polysomes were more competent than the heterologous systems (derived from Krebs ascites cells, reticulocytes or wheat germ) in carrying out reinitiations of brain mRNA translation. Both the homologous and the heterologous systems translated brain mRNA and synthesized the two brain specific proteins S-100 and the neuron specific enolase, indicating that each of the systems had all the necessary factors. However, homologous initiation factors, aminoacyl-tRNA synthetases and transfer RNAs were more effective, particularly at suboptimal concentrations. Our results suggest that discriminative translation of brain messenger RNA may take place based on relative proportions of required components in the reaction milieu rather than by the presence or absence of one or more special messenger RNA specific factors.

The correlation of adenosine deaminase and purine nucleoside phosphorylase activities in human lymphocytes subpopulations and in various lymphoid malignancies.

Adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) were measured in normal human and in malignant lymphoid cells. Thymocytes had high ADA activity (21.2 +/- 6.8 10(3) nM/h/mg) and low PNP activity (1.2 +/- 0.6), whereas T peripheral blood lymphocytes (PBL) had low ADA activity (1.20 +/- 0.22) and high PNP activity (2.8 +/- 1.3). Moreover cortico-thymocytes had higher ADA and lower PNP levels than medullary thymocytes. A linear correlation was observed between ADA and PNP activities in both thymocytes and T-PBL. Cells from 13 patients with T acute lymphoblastic leukemia (ALL) and 10 patients with T lymphoblastic lymphoma (LL) had very high levels of ADA (respectively 13.0 +/- 5.4 and 22.8 +/- 14) and low levels of PNP (respectively 1.9 +/- 0.8 and 2.5 +/- 1.4). However no clear relationship appeared between subgroups of these T-cell malignancies defined by their patterns of surface antigens, revealed by reactivity with monoclonal antibodies, and ADA and PNP levels, and there was no correlation between the two enzymes. In contrast, cells from 31 patients with HLA-DR+ common ALL had significantly low values of ADA as compared to cells from six patients with HLA-DR- common ALL and a linear correlation was observed between ADA and PNP in cells from children with non-T, non-B ALL. These results show that specific stages of T-cell development may be characterized by the relationships and the correlation between the two enzymes and suggest that T-ALL and T-LL appear to be the group of lymphoid malignancies with a high degree of incoordination between ADA and PNP activities.