On-line prediction of the feeding phase in high-cell density cultivation of rE. coli using constructive neural networks.

Streptococcus pneumoniae (pneumococcus) is a bacterium responsible for a wide spectrum of illnesses. The surface of the bacterium consists of three distinctive membranes: plasmatic, cellular and the polysaccharide (PS) capsule. PS capsules may mediate several biological processes, particularly invasive infections of human beings. Prevention against pneumococcal related illnesses can be provided by vaccines. There is a sound investment worldwide in the investigation of a proteic antigen as a possible alternative to pneumococcal vaccines based exclusively on PS. A few proteins which are part of the membrane of the pneumococcus seem to have antigen potential to be part of a vaccine, particularly the PspA. A vital aspect in the production of the intended conjugate pneumococcal vaccine is the efficient production (in industrial scale) of both, the chosen PS serotypes as well as the PspA protein. Growing recombinant Escherichia coli (rE. coli) in high-cell density cultures (HCDC) under a fed-batch regime requires a refined continuous control over various process variables where the on-line prediction of the feeding phase is of particular relevance and one of the focuses of this paper. The viability of an on-line monitoring software system, based on constructive neural networks (CoNN), for automatically detecting the time to start the fed-phase of a HCDC of rE. coli that contains a plasmid used for PspA expression is investigated. The paper describes the data and methodology used for training five different types of CoNNs, four of them suitable for classification tasks and one suitable for regression tasks, aiming at comparatively investigate both approaches. Results of software simulations implementing five CoNN algorithms as well as conventional neural networks (FFNN), decision trees (DT) and support vector machines (SVM) are also presented and discussed. A modified CasCor algorithm, implementing a data softening process, has shown to be an efficient candidate to be part of an on-line HCDC monitoring system for detecting the feeding phase of the HCDC process.

Improved method to calculate the antibody avidity index.

The methods currently used to determine the immunoglobulin avidity index (AI) require the choice of a reference point in the ELISA titration curve. Since both curves, with and without denaturating reagents, seldom run in parallel, the AI value becomes highly dependent on this reference. The new method for AI calculation presented here takes into account the whole data of the ELISA titration curve in which the final numerical AI is the average of each point.

Introduction of air in the anaerobic culture of Streptococcus pneumoniae serotype 23F induces the release of capsular polysaccharide from bacterial surface into the cultivation medium.

AIM: An approach to increase Streptococcus pneumoniae capsular polysaccharide (CPS) in the culture medium during fed-batch cultivation in bioreactor. METHODS AND RESULTS: Streptococcus pneumoniae serotype 23F was cultivated in a 5-l bioreactor with nitrogen-sparging and followed by addition of air in the stationary phase. The amount of CPS released in the supernatant progressively increased under air sparging. The profile of cellular viability and optical density was similar in both cultures. Immunoelectron microscopy showed that the amount of tightly cell-bound CPS was higher in bacteria cultivated under nitrogen than under air. CONCLUSIONS: The stress caused by the addition of air at the stationary phase promoted a large increase of free CPS into the medium, as a consequence of the morphologic change in the capsule. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of air in the stationary phase of the culture would greatly simplify the subsequent downstream process, allowing CPS purification from the supernatant. The direct consequence of this process improvement is the reduction of vaccine production costs.

Polysaccharide production of Neisseria meningitidis (Serogroup C) in batch and fed-batch cultivations

Serogroup C polysaccharide from Neisseria. meningitidis constitutes the antigen for the vaccine against the disease caused by this bacterium. Aiming at enhancing the final polysaccharide concentration as well as the overall yield factor (polysaccharide/biomass), 20 cultivations were carried out in Frantz medium in a 13 L bioreactor at 35°C, 0.5 atm, 400 rpm and air flowrate of 2 L/min. A series of nine batch experiments was carried out under three different conditions (with control of dissolved oxygen at 10%, with control of pH at 6.5 and without dissolved oxygen and pH controls). Another set of runs consisted of 11 fed-batch cultivations without dissolved oxygen control, varying glucose concentration from less than 1.0-3.0 g/L, four of which performed controlling the pH at 6.5, and four under partial fed-batch conditions. The highest polysaccharide concentration (0.26 g/L) and the overall yield (0.16 g/g), were obtained in batch and partial fed-batch experiments when glucose concentration was maintained below 1.0 g/L. An empirical relation is proposed to relate the specific production rate of polysaccharide to glucose concentration during the stationary growth phase of the fed-batch runs. The obtained polysaccharide satisfies the molecular weight criterion, being a suitable antigen for vaccine production.

Immune response to native NadA from Neisseria meningitidis and its expression in clinical isolates in Brazil

A mAb against the NadA protein from Neisseria meningitidis strain 3006 (serosubtype B : 2b : P1.2 : P5.2,8) demonstrated strong bactericidal activity against Brazilian epidemic serogroup B strain N44/89 (B : 4,7 : P1.19,15 : P5.5,7) and a serogroup C strain, IMC 2135 (C : 2a : P1.5,2), but not against another serogroup C strain, N1002/90 (C : 2b : P1.3 : P5.8). The immunogenicity of native NadA in an outer-membrane vesicle (OMV) preparation was also tested. Serum from mice immunized with OMV from serogroup B strain N44/89, which contains the NadA protein, showed bactericidal activity against serogroup B and C strains possessing NadA. In dot-blot analysis of 100 serogroup B and 100 serogroup C isolates from Brazilian patients, the mAb to NadA recognized about 60% of the samples from both serogroups. The molecular mass of the NadA protein from strain N44/89 determined by mass spectrometry was 37 971 Da and the peptide sequences were identical to those of NadA from N. meningitidis strain MC58.

Optimization of medium and cultivation conditions for capsular polysaccharide production by Streptococcus pneumoniae serotype 23F.

The influence of medium composition and culture conditions on Streptococcus pneumoniae serotype 23F cultivation was investigated in order to develop an industrial method for polysaccharide (PS) production. Acid-hydrolyzed casein (AHC) and dialyzed enzymatically hydrolyzed soybean meal (EHS) were investigated as nitrogen sources, and the vitamin solution of Hoeprich's medium and dialyzed yeast extract as vitamin sources. The influence of initial glucose concentration was also evaluated. In flask experiments, the best nitrogen source for PS production was AHC; EHS yielded small amounts of PS without interfering with bacterial growth. Dialyzed yeast extract provided an approximately 2-fold increase in PS production when compared to Hoeprich's vitamin solution. In a 5-l bioreactor, it was observed that the pneumococcus did not grow under aerobic conditions, CO(2) did not increase PS yield, glucose was inhibitory above 30 g l(-1), and the main glucose catabolism product was lactate, which had an inhibitory effect on cell growth. When anaerobic cultivation was performed under N(2) flow using the optimized medium, 240 mg l(-1) of soluble PS was obtained, which represents a 3-fold increase in yield as compared to that described in the published patent [Yavordios and Cousin (1983) European Patent 0 071515 A1]. Application of these results would considerably simplify upstream and downstream processes for PS production.

Optimization of medium and cultivation conditions for capsular polysaccharide production by Streptococcus pneumoniae serotype 23F

The influence of medium composition and culture conditions on Streptococcus pneumoniae serotype 23F cultivation was investigated in order to develop an industrial method for polysaccharide (PS) production. Acid-hydrolyzed casein (AHC) and dialyzed enzymatically hydrolyzed soybean meal (EHS) were investigated as nitrogen sources, and the vitamin solution of Hoeprich's medium and dialyzed yeast extract as vitamin sources. The influence of initial glucose concentration was also evaluated. In flask experiments, the best nitrogen source for PS production was AHC; EHS yielded small amounts of PS without interfering with bacterial growth. Dialyzed yeast extract provided an approximately 2-fold increase in PS production when compared to Hoeprich's vitamin solution. In a 5-l bioreactor, it was observed that the pneumococcus did not grow under aerobic conditions, CO2 did not increase PS yield, glucose was inhibitory above 30 g l-1, and the main glucose catabolism product was lactate, which had an inhibitory effect on cell growth. When anaerobic cultivation was performed under N2 flow using the optimized medium, 240 mg l-1 of soluble PS was obtained, which represents a 3-fold increase in yield as compared to that described in the published patent [Yavordios and Cousin (1983) European Patent 0 071515 A1]. Application of these results would considerably simplify upstream and downstream processes for PS production.

Identification of CYP3A4 as the enzyme involved in the mono-N-dealkylation of disopyramide enantiomers in humans.

To identify which cytochrome P-450 (CYP) isoform(s) are involved in the major pathway of disopyramide (DP) enantiomers metabolism in humans, the in vitro formation of mono-N-desalkyldisopyramide from each DP enantiomer was studied with human liver microsomes and nine recombinant human CYPs. Substrate inhibition showed that SKF 525A and troleandomycin potently suppressed the metabolism of both DP enantiomers with IC50 values for R(-)- and S(+)-DP of <7.3 and <18.9 microM, respectively. In contrast, only weak inhibitory effects (i.e., IC50 > 100 microM) were observed for five other representative CYP isoform substrates [i.e., phenacetin (CYP1A1/2), sparteine (CYP2D6), tolbutamide (CYP2C9), S-mephenytoin (CYP2C19), and p-nitrophenol (CYP2E1)]. Significant correlations (P <.01, r = 0.91) were found between the activities of 11 different human liver microsomes for mono-N-dealkylation of both DP enantiomers and that of 6beta-hydroxylation of testosterone. Conversely, no significant correlations were observed between the catalytic activities for DP enantiomers and those for the O-deethylation of phenacetin, 2-hydroxylation of desipramine, hydroxylation of tolbutamide, and 4'-hydroxylation of S-mephenytoin. Further evidence for involvement of CYP3A P450s was revealed by an anti-human CYP3A serum that inhibited the mono-N-dealkylation of both DP enantiomers and 6beta-hydroxylation of testosterone almost completely (i.e., >90%), whereas it only weakly inhibited (i.e., <15%) CYP1A1/2- or 2C19-mediated reactions. Finally, the recombinant human CYP3A3 and 3A4 showed much greater catalytic activities than seven other isoforms examined (i.e., CYP1A2, 2A6, 2B6, 2C9, 2D6, 2E1, and 3A5) for both DP enantiomers. In conclusion, the metabolism of both DP enantiomers in humans would primarily be catalyzed by CYP3A4, implying that DP may have an interaction potential with other CYP3A substrates and/or inhibitors.

High-level expression of tetanus toxin fragment C-thioredoxin fusion protein in Escherichia coli.

An insert of Clostridium tetani DNA corresponding to fragment C of tetanus toxin was amplified by PCR. This 1.4 kb fragment was cloned into the high-expression vector pET32a, under control of the T7 promoter. Expression of this plasmid in Escherichia coli BL21(DE3) resulted in the production of a fusion protein ( approximately 62 kDa) consisting of 112 amino acids of thioredoxin and approximately 450 amino acids of fragment C. This fusion protein was recognized by anti-tetanus toxoid antiserum in an ELISA and on immunoblots. The recombinant fragment-C-thioredoxin protein was purified significantly in one step by Ni(2+)-chelate Sepharose, the final yield being approximately 35 mg/l. Immunization of animals with the recombinant protein produced antibodies that were able to recognize the tetanus toxin. By using this gene-fusion expression system we produced soluble fragment C of tetanus toxin in a high yield, preventing many problems inherent in the use of other expression systems that produce either insoluble fragment C in inclusion bodies, or a soluble form, but in low yield, using E. coli as the expression host.

[Use of an expandable metallic stent in 4 patients with superior vena syndrome].

Recently developed expandable metallic stents have been adopted to superior vena cava syndrome with good results. We inserted stents into the superior vena cava in 4 patients with superior vena cava syndrome. The results were comfortable and no complications. We should consider the stenting as the first choice for superior vena cava syndrome.

Neisseria meningitidis serogroup C polysaccharide and serogroup B outer membrane vesicle conjugate as a bivalent meningococcus vaccine candidate.

Neisseria meningitidis serogroup C polysaccharide (PS C) was conjugated to serogroup B outer membrane vesicles (OMV) in order to test the possibility of obtaining a bivalent group B and C meningococcus vaccine. The conjugate and controls were injected intraperitoneally into groups of ten mice with boosters on days 14 and 28 after the primary immunization. The following groups were used as control: (i) PS C; (ii) PS C plus OMV; (iii) OMV; and (iv) saline. The serum collected on days 0, 14, 28 and 42 were tested by enzyme-linked immunosorbent assay (ELISA) for PS C and OMV, and by complement mediated bactericidal assay against serogroups B and C. ELISA for PS C as well as bactericidal titres against serogroup C meningococci of the conjugated vaccine increased eight-fold (ELISA) and 32 fold (bactericidal) after 42 days in comparison with the PS C control group. ELISA for OMV and bactericidal titre against serogroup B meningococci of the conjugate showed no significant difference in comparison with the OMV containing controls. Furthermore, Western Blot assay of the conjugate immune serum did not bind OMV class four protein which is related to the complement dependent antibody suppressor. The results indicate that the PS C-OMV conjugate could be a candidate for a bivalent vaccine toward serogroups B and C meningococci.

Aortic pseudoaneurysm in the L3-L4 disc space after lumbar disc surgery. A case report.

We report a patient who developed an aortic pseudoaneurysm in the L3-L4 disc space after lumbar disc surgery. The diagnosis was made by MRI and aortography, and repair using a prosthetic graft and anterior fusion was successful. We discuss the predisposing factors, the clinical picture and management of vascular injuries during disc excision.

The early phase of colon tumorigenesis induced by dimethylhydrazine in ICR mice.

In order to study the influence of fiber supplements on dimethylhydrazine induction of colon tumorigenesis six-week-old CD1 (ICR): Crj mice were injected i.m. at a dimethylhydrazine (DMH) dose 10 mg/kg body weight once weekly for 10 weeks with or without dietary supplementation with 3% polydextrose, lactosucrose or cellulose, or 3% polydextrose and 3% cellulose in combination. There were no significant differences in colon tumor induction among the groups. However, microadenomas were observed 10 weeks after the first treatment of DMH so that this protocol may be useful for studies of the early phase of colon carcinogenesis in mice.

Two types of stretch-activated channels coexist in the rabbit corneal epithelial cell.

Ion channels contribute to the regulation of cellular function through control of the membrane potential and intracellular concentration of various ions. We examined stretch-activated channels in the corneal epithelial cell. Patch clamping was applied to enzymatically dissociated corneal epithelial cells to characterize their stretch-activated ion channels. The plasma membrane was stretched by applying suction to the patch pipette in cell-attached or inside-out patch configuration. The ion selectivity, voltage-dependence, and stretch-dependence were examined. Two kinds of stretch-activated channel events were observed; the previously-reported large conductance (L) channel and a novel small conductance (S) channel. The probability of recording L vs. S channels in the cell-attached configuration was about 2:1. The L channel was potassium selective with single channel conductance (gamma) of about 160 pS under the symmetrical (150 mm K+) solution. The S channel was permeable to Na+ and K+ with gamma of about 20 pS under the same conditions. Both L and S channels showed little activity in the absence of suction applied to the recording pipette. Channel activity was evoked by suction (negative pressure) stronger than -20 mmHg in both channels. The open probability (Po) and the mean current increased in proportion to further applied stretch and did not saturate for applied suction as strong as -80 mmHg, the pressure at which the gigaseal started to break. Thus, two types of stretch-activated channels coexist in corneal epithelial cells; a potassium-selective L channel and non-selective S channel. The contribution of these channels to the membrane potential is discussed.

Colon tissue implanted into the glandular stomach in rats lack susceptibility to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) carcinogenesis.

The present study was undertaken to determine the response of colon mucosa implanted into the fundus of stomach in 6-week old male F344 rats to oral administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Samples of colonic tissue about 8 mm in diameter were obtained from various colon sites and surgically implanted into the anterior wall of the fundus by isografting. MNNG was chronically administered at a concentration of 100 mg/l in the drinking water for 16 weeks starting 4 weeks after the operation and the grafted colon mucosa was examined at 12 months after the operation. Control rats received a sham-operation and the same amount of MNNG. In the MNNG administered groups, only one adenoma containing Paneth cells was noted in the implanted colon tissue whereas over 40% incidence of gastric tumors was observed in the pyloric mucosa. In the operated rats not given MNNG no gastric tumors were observed in either the grafted site or the pylorus.

[Comparison of long-term clinical results of the three models of the Björk-Shiley valve prosthesis and the Omnicarbon valve prosthesis].

A multi-institutional cooperative study, that was a comparison of long-term results of the replacement of the four models of the oblique disc valve prosthesis which had been implanted on aortic and mitral position alone or double from December 1976 to September 1992 in the eight national hospitals in Japan was performed. Seven hundred and thirty-four patients and 765 prostheses that consisted of 582 patients and 610 prostheses of the Björk-Shiley (BS) valve, including 326 patients of the Spherical disc (SP) valve (49 aortic, 259 mitral, and 18 double aortic-mitral), 103 patients of the Convexoconcave disc (CC) valve (45, 51, 7) and 153 patients of the Monostrut (MS) valve (101, 49, 3), and of 152 patients (71, 78, 3) and 155 prostheses of the Omnicarbon (OC) valve were compared with their mortality and morbidity in every valve position respectively according to the approval by STS-guideline. The MS valve and the OC valve showed 0% to 11.3% of operative mortality, 0.3%/py to 1.8%/py of valve related mortality, 85.5% +/- 5.6% to 98.4% +/- 1.6% of actuarial survival rate at 10 years, and 58.5% +/- 6.4% to 82.7% +/- 5.7% of actuarial free rate of all mortality and morbidity at 10 years in every valve position. Structural deteriorations occurred in two patients of the CC valve in the mitral position only, and its rate of all valve positions showed 0.04%/py. Significant differences were seen in actuarial survival rate at 10 years after aortic valve replacement, and in operative mortality rate, improved degree of NYHA class, structural deterioration rate and actuarial free rate of all mortality and morbidity after mitral valve replacement between the group of the MS and OC valve and the group of the SP and CC valve. Therefore the MS valve or the OC valve should be selected to implant rather than the SP valve or the CC valve, and the patient who had been implanted with the CC valve should be treated considering valve position, valve position, valve size, age and activity of the patient and the manufactured date of the prosthesis.

Effect of a proteinase inhibitor from the plasma of Bothrops jararaca on coagulant and myotoxic activities of Bothrops venoms.

A proteinase inhibitor purified from the plasma of Bothrops jararaca was tested for the neutralization of total blood clotting activity, prothrombin activating factor and thrombin-like activity of B. jararaca venom and also myotoxic activity of B. jararacussu venom. This inhibitor was also tested for neutralization of the lethality induced by the venom of B. jararaca and B. jararacussu. The inhibitor markedly reduced the total blood clotting activity (95%) and the prothrombin activating factor (98%), and partially decreased the thrombin-like activity of B. jararaca venom. The proteinase inhibitor failed to neutralize the myotoxic activity of B. jararacussu. Lethality induced by the venom of B. jararaca in mice was inhibited by the proteinase inhibitor, whereas the protection on B. jararacussu venom was only partial. These results show that the proteinase inhibitor purified from the plasma of B. jararaca inhibits different proteinases but not myotoxins of Bothrops venoms, and consequently neutralizes the toxic effect due to these proteins.

Cushing's syndrome due to primary pigmented nodular adrenocortical disease with cardiac myxomas and mucocutaneous lentigines.

A young Japanese female demonstrated unusual features of Cushing's syndrome, cardiac myxomas and mucocutaneous lentigines. At the age of 12 years she presented with growth failure and obesity. The dexamethasone suppression test, the metyrapone test and low corticotropin concentrations indicated a primary adrenal disorder. At surgery, the adrenal glands were not enlarged (the right, 4.0 g; the left; 4.5 g) but had numerous small dark brown nodules. The pathological findings showed multiple small black cortical nodules containing large cells with eosinophilic cytoplasm and lipofuscin, and internodular cortical atrophy. These abnormalities were consistent with primary pigmented nodular adrenocortical disease. At age 22 years she complained of fatigue and palpitations associated with mid-chest pain. Four cardiac myxomas, suspected from the echocardiogram, were surgically removed. Because Cushing's syndrome and cardiac myxomas are life-threatening conditions, an awareness of the complex is important.

Neutralization of myotoxic activity of Bothrops venoms by antisera to purified myotoxins and to crude venoms.

A phospholipase myotoxin (MOO-1) and a non-phospholipase myotoxin (JSU-5) were studied for their antigenic cross-reactivity and neutralization by different antisera. Antisera against JSU-5 and MOO-1 reacted equally with both myotoxins in ELISA assays. The specificity of these antisera was also similar, recognizing the same 14,000-18,000 mol. wt components in the venoms of Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi and Bothrops pradoi. Using creatine kinase assays, JSU-5 myotoxicity was completely neutralized by B. jararacussu antivenom or anti-JSU-5 antibodies and partially neutralized by B. moojeni antivenom or anti-MOO-1 antibodies. MOO-1 myotoxicity was completely neutralized by antisera against JSU-5 and MOO-1 and B. jararacussu antivenom, and only partially neutralized by B. moojeni antivenom. B. jararacussu venom induced high titres of antibodies against purified myotoxins. This antiserum completely inhibited the myotoxicity of the homologous venom and significantly reduced the myotoxicity of the remaining myotoxin-containing venoms. It is suggested that B. jararacussu venom is a good immunogen to induce antibodies against myotoxins present in the venoms of the different species of Bothrops.

Purification of a proteinase inhibitor from the plasma of Bothrops jararaca (jararaca).

A proteinase inhibitor was isolated from the plasma of Bothrops jararaca by three chromatographic steps: DEAE Sephacel, Phenyl Sepharose and Bio Gel P200. It inhibited caseinolytic and hemorrhagic activity of the whole venom of B. jararaca. Proteolytic activity of bothropasin and J protease, both metalloproteinases of the venom, were neutralized by the inhibitor. The J protease-inhibitor complex was isolated by gel filtration chromatography in HPLC and the electrophoresis pattern of this complex showed that the interaction between enzyme and inhibitor is not covalent.