RESUMO
Mycelial fragmentation in submerged cultures of the cephalosporin C (CPC) producing fungus Acremonium chrysogenum was characterized by image analysis. In both fed-batch and chemostat cultures, the proportion of mycelial clumps seemed to be the most sensitive morphological indicator of fragmentation. In a fed-batch fermentation culture, this declined from roughly 60% at inoculation to less than 10% after 43 h. Subsequent additions of glucose resulted in a sharp increase back to near the initial value, an increase that reversed itself a few hours after glucose exhaustion. Meanwhile CPC production continued to decline steadily. On the other hand, the addition of soybean oil enhanced CPC production, but had no significant effect on the morphology. Although it may sometimes appear that morphology and productivity are related in batch or fed-batch cultures, this study suggests that this is because both respond simultaneously to more fundamental physiological changes, dependent on the availability of carbon. In circumstances, such as supplementary carbon source addition, the relationship is lost. Chemostat cultures supported this belief, as CPC-production rates were hardly affected by the specific growth rate, but the morphology showed significant differences, i.e., lower dilution rates resulted in a lower proportion of clumps and in smaller clumps.
Assuntos
Acremonium/metabolismo , Cefalosporinas/biossíntese , Acremonium/crescimento & desenvolvimento , Acremonium/ultraestrutura , Carbono , Fermentação , Glucose , Processamento de Imagem Assistida por Computador , Óleo de Soja , Fatores de TempoRESUMO
Cosalane and its synthetic derivatives inhibit the binding of gp120 to CD4 as well as the fusion of the viral envelope with the cell membrane. The binding of the cosalanes to CD4 is proposed to involve ionic interactions of the negatively charged carboxylates of the ligands with positively charged arginine and lysine amino acid side chains of the protein. To investigate the effect of anion spacing on anti-HIV activity in the cosalane system, a series of cosalane tetracarboxylates was synthesized in which the two proximal and two distal carboxylates are separated by 6--12 atoms. Maximum activity was observed when the proximal and distal carboxylates are separated by 8 atoms. In a series of cosalane amino acid derivatives containing glutamic acid, glycine, aspartic acid, beta-alanine, leucine, and phenylalanine residues, maximum activity was displayed by the di(glutamic acid) analogue. A hypothetical model has been devised for the binding of the cosalane di(glutamic acid) conjugate to CD4. In general, the compounds in this series are more potent against HIV-1(RF) in CEM-SS cells than they are vs HIV-1(IIIB) in MT-4 cells, and they are least potent vs HIV-2(ROD) in MT-4 cells.
Assuntos
Fármacos Anti-HIV/síntese química , Ácido Aurintricarboxílico/síntese química , HIV-1/efeitos dos fármacos , Ânions/química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Ácido Aurintricarboxílico/análogos & derivados , Ácido Aurintricarboxílico/química , Ácido Aurintricarboxílico/farmacologia , Antígenos CD4/química , Linhagem Celular , Humanos , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
The binding of the anti-HIV agent cosalane to CD4 is thought to involve ionic interactions of negatively charged carboxylates of the ligand with positively charged residues on the surface of the protein. An investigation of the optimal anion distances for anti-HIV activity in a series of cosalane tetracarboxylate analogues has been completed, and maximal activity results when the two proximal and the two distal carboxylates are separated by eight atoms.
Assuntos
Ácido Aurintricarboxílico/síntese química , Ácido Aurintricarboxílico/farmacologia , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacologia , Ácido Aurintricarboxílico/análogos & derivados , Ácido Aurintricarboxílico/metabolismo , Antígenos CD4/metabolismo , Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/metabolismo , Ácidos Carboxílicos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Humanos , Modelos Moleculares , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
The effect of biomass concentration and mycelial morphology on fungal fermentation broth rheological properties has been investigated. In previous work it had been shown that commonly used rheological parameters, such as the power law consistency and flow behavior indices, could be correlated successfully with the broth biomass concentration and clump morphological parameters of roughness and compactness. More recent work on a broader range of data showed a correlation between roughness and compactness; consequently, it was not correct to use both of these morphological variables simultaneously in rheological parameter correlations. Furthermore, earlier correlations were only made using clump morphological parameters, as clumps were found to be around 90% of the biomass in batch fermentations. In the present work it was found that the percentage of clumps fell to around 30% to 40% of a sample during the later stages of fed-batch fermentations. No clear relationship between the flow behavior index and biomass concentration was found, at least for those phases of the fermentation in which the viscosities were high enough for the rheology to be characterized by a disk turbine rheometer. The mean value of the flow behavior index was found to be 0.35 +/- 0.1 (standard deviation) throughout both batch and fed-batch fermentations, although some significant deviations from this value were observed early and very late in the fermentations. Correlations for the consistency index, measured using a disk turbine rheometer, were based on the biomass concentration and the mycelial size (represented by the mean projected area or the mean maximum dimension of all the mycelia). These correlations were reasonably successful for both fed-batch and batch fermentations. The correlation using the mean maximum dimension was preferred to that using the mean projected area, as the former is independent of magnification. The proposed correlation is: where K is the consistency index (Pa. s(n>)), C(m) is the biomass concentration as dry cell weight (g L(-1)), and D is the mean maximum dimension (microm). It should be noted that small changes in the exponent on the biomass concentration (alpha) may dramatically affect any predictions. Consequently, caution in the use of this correlation (and that based on mean projected area) is advocated, although its accuracy may be suitable for operational or design purposes.
Assuntos
Biomassa , Fermentação , Penicillium chrysogenum/crescimento & desenvolvimento , Reologia , Reatores Biológicos , Meios de CulturaRESUMO
Apoptosis is a form of cell death in which the dying cell plays an active part in its demise. At the morphological level, it is characterised by cell shrinkage rather than the swelling seen in necrotic cell death. In cell culture, apoptosis limits the yield of economically and medically important products, and can result in synthesis of imperfect molecules. Therefore, this process must be identified, monitored and fully understood, so that a means to regulate it can be developed. We have developed a new automatic image analysis assay for detecting apoptosis in animal cell culture on the basis of the annexin-V affinity assay. The results of this assay were compared with data generated by flow cytometry and manual scoring. All three methods were found to correspond well but image analysis like flow cytometry offers operator-independent results, and can be used as a tool for rapid monitoring of viable cell number, apoptosis and necrosis in animal cell culture. Furthermore, reduction in cell size was measured and was found to precede the appearance of phosphatidylserine on the cell surface.
Assuntos
Anexina A5/análise , Apoptose , Animais , Linhagem Celular , Citometria de Fluxo , Processamento de Imagem Assistida por Computador , Camundongos , NecroseAssuntos
Fungos , Processamento de Imagem Assistida por Computador , Previsões , Fungos/citologia , Fungos/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Processamento de Imagem Assistida por Computador/tendências , Saccharopolyspora/citologia , Saccharopolyspora/fisiologia , EsporosRESUMO
Image analysis is now well established in quantifying and characterising microorganisms from fermentation samples. In filamentous fermentations it has become an invaluable tool for characterising complex mycelial morphologies, although it is not yet used extensively in industry. Recent method developments include characterisation of spore germination from the inoculum stage and of the subsequent dispersed and pellet forms. Further methods include characterising vacuolation and simple structural differentiation of mycelia, also from submerged cultures. Image analysis can provide better understanding of the development of mycelial morphology, of the physiological states of the microorganisms in the fermenter, and of their interactions with the fermentation conditions. This understanding should lead to improved design and operation of mycelial fermentations.
Assuntos
Actinomycetales/ultraestrutura , Fermentação , Fungos/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Esporos/ultraestrutura , Actinomycetales/fisiologia , Fungos/fisiologia , Processamento de Imagem Assistida por Computador/instrumentação , SoftwareRESUMO
The molecular diversity of inhibitor-resistant TEM (IRT) enzymes was explored using a strategy which involved DNA amplification by polymerase chain reaction (PCR), analysis of restriction fragment length polymorphism (RFLP), and direct nucleotide sequencing. The study of plasmid-borne genes from 27 strains, resistant to amoxicillin and beta-lactamase-inhibitor combinations, identified mutations resulting in amino acid change at positions 69, 244, 275, and 276 known to be associated with the IRT phenotype and a mutation at nucleotide position 162 in the promoter region. These mutations were found to lie on two different gene sequences, described here as "TEM-1B like" and "TEM-2 like" restriction linkage groups. Further analysis, of nucleotide sequences of promoter and coding regions of the beta-lactamases, confirmed that a given mutation causing IRT phenotype could be associated with two different gene sequence frameworks and two different causal mutations could lie on identical gene sequence framework. These data argue in favor of convergent phenotypic evolution of IRT enzymes under the selective pressure imposed by the intensive clinical use of beta-lactam-beta-lactamase inhibitor combinations.
Assuntos
Antibacterianos/farmacologia , Ácidos Clavulânicos/farmacologia , Escherichia coli/genética , Resistência beta-Lactâmica/genética , beta-Lactamases/genética , Amoxicilina/farmacologia , Ácido Clavulânico , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Evolução Molecular , Genes Bacterianos/genética , Variação Genética/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNARESUMO
The influence of the agitation conditions on the morphology of Penicillium chrysogenum (freely dispersed and aggregated forms) was examined using radial (Rushton turbines and paddles), axial (pitched blades, propeller, and Prochem Maxflow T), and counterflow impellers (Intermig). Culture broth was taken from a continuous fermentation at steady state and was agitated for 30 min in an ungassed vessel of 1.4-L working volume. The power inputs per unit volume of liquid in the tank, P/V(L), ranged from 0.6 to 6 kW/m(3). Image analysis was used to measure mycelial morphology. To characterize the intensity of the damage caused by different impellers, the mean total hyphal length (freely dispersed form) and the mean projected area (all dispersed types, i.e., also including aggregates) were used. [In this study, breakage of aggregates was taken into account quantitatively for the first time.]At 1.4-L scale and a given P/V(L), changes in the morphology depended significantly on the impeller geometry. However, the morphological data (obtained with different geometries and various P/V(L)) could be correlated on the basis of equal tip speed and two other, less simple, mixing parameters. One is based on the specific energy dissipation rate in the impeller region, which is simply related to P/V(L) and particular impeller geometrical parameters. The other which is developed in this study is based on a combination of the specific energy dissipation rate in the impeller swept volume and the frequency of mycelial circulation through that volume. For convenience, the function arising from this concept is called the "energy dissipation/circulation" function.To test the broader validity of these correlations, scale-up experiments were carried out in mixing tanks of 1.4, 20, and 180 L using a Rushton turbine and broth from a fed-batch fermentation. The energy dissipation/circulation function was a reasonable correlating parameter for hyphal damage over this range of scales, whereas tip speed, P/V(L), and specific energy dissipation rate in the impeller region were poor. Two forms of the energy dissipation/circulation function were considered, one of which additionally allowed for the numbers of vortices behind the blades of each impeller type. Although both forms were successful at correlating the data for the standard impeller designs considered here, there was preliminary evidence that allowing for the vortices would be valuable. (c) 1996 John Wiley & Sons, Inc.
RESUMO
A structured kinetic model describing growth, differentiation, and penicillin production in submerged Penicillium chrysogenum fermentations is reported. The filamentous hyphae are divided into four distinct regions on the basis of the activities and structure of hyphal compartments, viz., actively growing (mainly apical) regions, nongrowing or penicillin producing regions, vacuoles, and degenerated or metabolically inactive regions. A mechanistic approach is taken to give quantitative descriptions of differentiation and degeneration as a consequence of vacuolation. The growth and degeneration of vacuoles are expressed in the form of a population balance. The model assumes that newly generated vacuoles appear by differentiation of healthy regions, grow in size with limitation of available substrate, and eventually give rise to empty hyphal compartments. In the model the penicillin production is related to the amounts of the nongrowing regions of the hyphae. The model is used for successful predictions of the amounts of the four hyphal regions and the penicillin G production rate throughout the fed-batch fermentations of an industrial P. chrysogenum strain under different glucose feeding regimes. Quantitative information on proportions of the hyphal regions was obtained from image analysis measurements and the parameters of the kinetic model were identified. When the glucose feed rate to the production culture is switched between a high and a low value, the model can successfully predict the dynamic changes of differentiation and the resulting penicillin production caused by the variations in the nutrient conditions. The use of image analysis to characterize differentiation as a basis for structured modeling of the penicillin fermentation appears to be very powerful, and the method has great potential for use in process simulation and control of antibiotic fermentations.
RESUMO
A link between vacuolation and fragmentation of Penicillium chrysogenum mycelia in stirred tank submerged fermentations is reported. Quantitative information on vocuolation and morphology was obtained by image analysis. In fed-batch fermentations the coincidence of the events of rapid vacuolation and the fall of the mean total and main hyphal lengths suggests that hyphal fragmentation is not necessarily due to "shear" alone. The physiological state of the hyphae, characterized by the proportions of vaccuoles, was found to have a significant influence on the breakage of mycelial hyphae, It was found that the fragmentation was greater when the hyphae became heavily vacuolated following nutrient limitation in the culture, i.e., during the switch from the rapid growth to the production phase.
RESUMO
Fungal spores are used in the laboratory for culture maintenance and at laboratory and other scales as inocula for fermentations. The spore swelling and germination processes constitute a major part of the lag phase, and the subsequent culture morphology and productivity can be greatly influenced by the initial concentration and condition of the spores. An image analysis method has been developed for assessing the viability and the germination characteristics of fungal spores in submerged cultures. Structural variations during germination, i.e., swelling, germ tube formation, and germ tube elongation, are measured in terms of distributions of spore volumes and of germ tube lengths and volumes. These measurements are fully automatic and give a very rapid assessment of spore viability. This image analysis method might be used as a tool in culture maintenance and for determining the quality of inocula for fungal fermentations.
RESUMO
A clinical isolate of Escherichia coli, strain CB-134, recovered in 1986 from an abdominal abscess, exhibited resistance to penams, oxyimino-beta-lactams including broad-spectrum cephalosporins (cefotaxime, ceftriaxone, ceftazidime), and aztreonam but remained susceptible to cephamycins (cefoxitin, cefotetan) and to moxalactam and imipenem. Clavulanate (2 micrograms/ml) restored the susceptibility of the strain to broad-spectrum cephalosporins and aztreonam. A beta-lactamase with an isoelectric point (pI) of 5.9 was detected in strain CB-134, and the corresponding gene was transferred by conjugation to E. coli together with the associated aminoglycoside resistance determinant [AAC(3)-II] and tetracycline, trimethoprim, and sulfonamide resistance. The beta-lactamase efficiently hydrolyzed cefotaxime and ceftriaxone but only moderately hydrolyzed ceftazidime and was inhibited by clavulanate and sulbactam (1 microM) and by anti-TEM-1 and anti-TEM-2 sera. This extended-spectrum beta-lactamase, conferring resistance to cefotaxime, ceftriaxone, ceftazidime, and aztreonam, was comparable to CTX-1 (TEM-3) but differed from it by pI. Agarose gel electrophoresis of the plasmid DNA indicated that this new enzyme was coded by pUD16, a plasmid of 220 kilobases which belongs to the Inc6 incompatibility group. Hybridization with an intragenic probe for TEM-1 revealed that this beta-lactamase derives from TEM-type beta-lactamases and hence it was named TEM-4.
Assuntos
Cefalosporinas/metabolismo , Resistência Microbiana a Medicamentos/genética , Escherichia coli/efeitos dos fármacos , Plasmídeos , beta-Lactamases/metabolismo , Ácidos Clavulânicos/farmacologia , Conjugação Genética , DNA Bacteriano/genética , Eletroforese em Gel de Ágar , Escherichia coli/enzimologia , Escherichia coli/genética , Hidrólise , Focalização Isoelétrica , Cinética , Testes de Sensibilidade Microbiana , Peso Molecular , Hibridização de Ácido Nucleico , beta-Lactamases/genéticaRESUMO
Oligonucleotide probes specific for detection of the TEM-1 and OXA-1 beta-lactamase genes were compared with isoelectric focusing in 114 gram-negative beta-lactamase-producing strains representing at least 16 species. Correlations of 96 and 100% with isoelectric points were found for the TEM-1 and OXA-1 probes, respectively.
Assuntos
Bactérias/enzimologia , Oligonucleotídeos , beta-Lactamases/análise , Resistência Microbiana a Medicamentos , Focalização IsoelétricaRESUMO
A novel type of oxacillin-hydrolyzing beta-lactamase, termed OXA-4, has been detected in three Pseudomonas aeruginosa strains isolated in Paris between 1977 and 1981. The strains contained similar plasmids that determined resistance to carbenicillin, chloramphenicol, gentamicin, kanamycin, streptomycin, sulphonamide, tetracycline, tobramycin, sodium borate, and mercuric chloride, had a size of approximately 150 megadaltons, and belonged to the P-5 incompatibility group. Compared to reference OXA-1 beta-lactamase produced by plasmid RGN238, OXA-4 beta-lactamase produced by these plasmids had a similar substrate profile, similar response to inhibitors, and identical immunological reactions but differed in isoelectric point. In a more recent survey of 10 French hospitals plasmid-determined OXA-4 beta-lactamase production was found in P. aeruginosa isolates from four hospitals in the Paris area.
Assuntos
Plasmídeos , Pseudomonas aeruginosa/enzimologia , beta-Lactamases/isolamento & purificação , Resistência Microbiana a Medicamentos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Hidrólise , Focalização Isoelétrica , Pseudomonas aeruginosa/genética , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
CARB-4, a novel carbenicillin-hydrolyzing beta-lactamase with an isoelectric point of 4.3, was discovered in a strain of Pseudomonas aeruginosa from France. It was determined by a multiresistant transmissible plasmid belonging to the P-2 incompatibility group.
Assuntos
Carbenicilina/metabolismo , Plasmídeos , Pseudomonas aeruginosa/genética , beta-Lactamases/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologiaRESUMO
The properties of PSE-2 beta-lactamase have been examined by using two new PSE-2-producing plasmids, pMG33 and pMG74, as well as plasmid R151, found in Pseudomonas aeruginosa. PSE-2 beta-lactamase resembled other PSE enzymes in activity against carbenicillin, but it also resembled OXA enzymes, such as OXA-1, in rapid hydrolysis of oxacillin, cloxacillin, and methicillin and in inhibition by sodium chloride but not by cloxacillin. Antisera that inactivated TEM-1, TEM-2, OXA-1, or PSE-1 and PSE-4 beta-lactamase failed to cross-react with PSE-2, which thus appears to be immunologically distinct. The plasmids determining PSE-2 varied in geographical origin, size, transfer proficiency, and incompatibility specificity, but all determined resistance to carbenicillin, gentamicin, kanamycin, streptomycin, spectinomycin, sulfonamide, and tobramycin. From a pUZ8-R151 recombinant plasmid in Escherichia coli, the PSE-2 beta-lactamase gene could be transposed to a second plasmid in a 6.4-megadalton unit together with resistance to gentamicin, kanamycin, streptomycin, spectinomycin, sulfonamide, and tobramycin. Transposition was recA independent. We propose the designation Tn1404 for this unit, which, like transposons carrying OXA-1, PSE-1, PSE-4, and some transposons determining TEM-1, includes genes for beta-lactam, aminoglycoside, and sulfonamide resistance.