Rifomycin: XIV. Production of Rifomycin B

The production of a fraction of the rifomycin complex (rifomycin B) by Streptomyces mediterranei has been described. Both qualitative and quantitative aspects of the fermentation are determined by suitable agitation-aeration parameters. In addition to conventional nutrient ingredients, the presence of some of the barbituric acid derivatives was found to be essential for rifomycin B production. An attempt was made to explain the mode of action of these compounds.     

Modelling mass transfer and agitator performance in multiturbine fermentors

A methodology for mathematically analyzing agitator performance and mass transfer in large multiturbine production fermentors is presented. The application of this approach provides a method for determining axial dissolved oxygen profiles under conditions of known mass transfer rates as a function of agitation-aeration characteristics. A stagewise approach is used which divides the fermentor into a series of mixing cells. This allows for each turbine and mixing cell to be individually optimized. The model also permits the determination of the mass transfer coefficient for each turbine based upon limited dissolved oxygen data. The primary limitation of this approach rests in the limited data and correlations available for multiturbine systems. The structure of the modelling approach can serve as a basis for testing single turbine correlations and adapting them to multiturbine systems. The step-by-step details of the mathematical analysis are presented and interpreted. A series of computer simulations demonstrate the effect of typical fermentor operating variables on the axial dissolved oxygen profile. Further simulations demonstrate the effect of modifying agitator blade numbers on the dissolved oxygen profile and agitator power requirement.     

Acetic acid production by immobilized acetobacter cells

Summary Using a pulsed gas and liquid flow and with cells directly adsorbed on to a suitably-formed support, aerobic transformations can be carried out in a fixed-cell reactor with significant gain in efficiency. Immobilized cells of Acetobacter on cordierite can produce acetic acid at a high rate which, at different dilution rates, may be limited either by product inhibition or by oxygen transfer requirements.     

The relative efficiency of acquisition of MHC:peptide complexes and cross-presentation depends on dendritic cell type

Intercellular exchange of MHC molecules has been reported between many cells, including professional and nonprofessional APCs. This phenomenon may contribute to T cell immunity to pathogens. In this study, we addressed whether the transfer of MHC class I:peptide complexes between cells plays a role in T cell responses and compare this to conventional cross-presentation. We observed that dsRNA-matured bone marrow-derived dendritic cells (BMDCs) acquired peptide:MHC complexes from other BMDCs either pulsed with OVA(257-264) peptide, soluble OVA, or infected with a recombinant adenovirus expressing OVA. In addition, BMDCs were capable of acquiring MHC:peptide complexes from epithelial cells. Spleen-derived CD8alpha(+) and CD8alpha(-) dendritic cells (DCs) also acquired MHC:peptide complexes from BMDCs pulsed with OVA(257-264) peptide. However, the efficiency of acquisition by these ex vivo derived DCs is much lower than acquisition by BMDC. In all cases, the acquired MHC:peptide complexes were functional in that they induced Ag-specific CD8(+) T cell proliferation. The efficiency of MHC transfer was compared with cross-presentation for splenic CD8alpha(+) and CD8alpha(-) as well as BMDCs. CD8alpha(+) DCs were more efficient at inducing T cell proliferation when they acquired Ag via cross-presentation, the opposite was observed for BMDCs and splenic CD8alpha(-) DCs. We conclude from these observations that the relative efficiency of MHC transfer vs cross-presentation differs markedly between different DC subsets.     

Measuring kLa with randomly pulsed dynamic method

This reports on the determination of the overall oxygen transfer coefficient in a mechanically agitated vessel using a randomly pulsed dynamic method. This method consists in exciting the system by randomly switching the inlet gas stream with air or nitrogen with an identical volumetric flow rate. A pseudo-random binary sequence was used. This procedure is routinely used in process control for the identification of system's transfer function. The pulsed dynamic method gives good reliability (as compared with the traditional gassing-out method) and reproducibility in water. However, further improvement is needed before it can be used to monitor on-line the k(L)a during a fermentation.     

A study of acetic acid production by immobilized Acetobacter Cells: Oxygen transfer

The immobilization of living Acetobacter cells by adsorption onto a large-surface-area ceramic support was studied in a pulsed flow reactor. The high oxygen transfer capability of the reactor enabled acetic acid production rates up to 10.4 g L(-1) h(-1) to be achieved. Using a simple mathematical model incorporating both internal and external mass transfer coefficients, it was shown that oxygen transfer in the microbial film controls the reactor productivity.     

Flow cytometric measurement of fluorescence resonance energy transfer on cell surfaces. Quantitative evaluation of the transfer efficiency on a cell-by-cell basis

A method has been developed for the determination of the efficiency (E) of the fluorescence resonance energy transfer between moieties on cell surfaces by use of a computer-controlled flow cytometer capable of dual wavelength excitation. The absolute value of E may be calculated on a single-cell basis. The analysis requires the measurement of samples stained with donor and acceptor conjugated ligands alone as well as together. In model experiments HK 22 murine lymphoma cells labeled with fluorescein-conjugated concanavalin A (Con A) and/or rhodamine conjugated Con A were used to determine energy transfer histograms. Using the analytic solution to energy transfer in two dimensions, a high surface density of Con A binding sites was found that suggests that the Con A receptor sites on the cell surface are to a degree preclustered . We call this technique flow cytometric energy transfer ( FCET ).     

The localized coupling of bacterial photophosphorylation: Effect of antimycin a and N,N-dicyclohexylcarbodiimide) in chromatophores from Rhodopseudomonas sphaeroides,Ga, studied by single turnover event analysis

1. The inhibition by antimycin A of the cyclic electron transfer has been studied in chromatophores from Rhodopseudomonas sphaeroides Ga following an approach based on the analysis of the relaxation kinetics of the reaction center optical changes in pulsed light. The recovery kinetics of the bacteriochlorophyll redox state have been found to be clearly biphasic. The half-times of the fast phase (13 ms) and slow phase (about 400 ms) were not modified by antimycin in a range of concentrations from 0.1 to 9 μM. On the other hand the percentage extent of the fast phase, which reflects the rate of the cyclic electron transfer, was monotonically decreased by increasing concentrations of the inhibitor. This indicates that antimycin decreases progressively the fraction of the photosynthetic units, active in cyclic electron transfer. 2. The ATP yield per flash observed under conditions of controlled inhibition of electron flow was strongly dependent upon the amount of active redox cycles. On the other hand, the amplitude of the carotenoid band shift, which has been demonstrated unequivocally to be correlated to the ATP yield per flash in uninhibited chromatophores, was not affected by antimycin up to a 40% inhibition of electron flow. 3. The effect of a progressive limitation by DCCD in the number of active ATP synthetase complexes on flash-induced phosphorylation has been examined. The decrease in ATP yield observed over a wide range of flash frequencies is related simply to the ATPase activity and to phosphorylation in continuous light, irrespective of the value of the membrane potential, which appears to be stabilized by this inhibitor. 4. As a whole, the results obtained at low concentrations of antimycin and under conditions of partial inhibition by DCCD evidence a localized coupling between the redox reactions and phosphorylation.     

Laser impact assessment in a biofilm-forming bacterium Pseudoalteromonas carrageenovora using a flow cytometric system

Impact by pulsed laser irradiations from an Nd:YAG laser on the marine biofilm-forming bacterium Pseudoalteromonas carrageenovora has been studied using a flow cytometric system. The biofilm-forming bacteria in the planktonic state have been irradiated while flowing, and the mortality and bacterial attachment have been determined by exposing TiN coupons in the system. Coupons suspended in the non-irradiated bacterial flow were treated as the control. The fluence used in the study was 0.1 J/cm(2). Three flow rates (14, 28, and 42 cm/min) and two exposure durations (15 and 30 min) were tested. The results showed the increase in bacterial mortality with the decrease in flow rate. The maximum mortality of 27.5% was observed when the flow rate was 14 cm/min. The bacterial attachment increased with the increase in flow rate and exposure duration. The area of bacterial attachment on the experimental coupons exposed to the irradiated sample was significantly lesser than that for the nonirradiated sample. The results thus show in a flowing system, low power pulsed laser irradiations could reduce the bacterial attachment even though it did not cause significant mortality.     

Highly efficient transfer and stable integration of foreign DNA into partially digested rice cells using a pulsed electrophoretic drive

A new method has been developed to introduce foreign DNA into rice cells. Gene delivery occurred when an electrophoretic drive with cycles of intervallic electric field was applied to a mixture containing partially digested small cell groups (SCGs) and plasmid DNAs. Gene transfer efficiency was evaluated by the detection of -glucuronidase (GUS) activity resulting from expression of a chimaeric plasmid DNA. The optimal combination of treatment conditions (3 V/cm, 30 s pulse and 30 min electrophoretic run) produced a frequency of up to 8.2% of blue cells in transformed microcalluses 40 days after culture of treated SCGs without selection for kanamycin resistance. Southern hybridization showed that the foreign gene had integrated into the chromosomal DNA. These results demonstrate that pulsed electrophoretic drive is applicable to the transfer of foreign genes into plant cells.     

Direct observation of laser generated free radicals from a myocardium target site.

The first direct observation of unstable free radicals generated by laser irradiation of myocardium samples has been performed by EPR at 100 K. Characteristic iron signals are measured for pulsed laser ablation of the sample. The EPR results are discussed in relation to the relative efficiency of the various types of lasers to produce free radicals. A comparison is made of the types of free radicals produced by the action of pulsed vs continuous wave (cw) laser energy.     

Modeling of processes of heat transfer in whole-body hyperthermia

The method of whole-body hyperthermia in which the body temperature for a short time reaches values up to 43-44 degrees C holds currently much promise. However, at body temperatures above 42 degrees C, the risks associated with the hemodynamic instability and the appearance of arrhythmia in the patient increase. A model of heat transfer has been created to increase the efficiency and safety of the immersion-convectional method of whole-body hyperthermia. This model takes into account changes in the skin blood flow and the dynamics of pulse rate depending on body temperature. The model of heat transfer adequately reflects processes of heating of the organism and can form a basis for the calculation of distribution of heat inside the organism.     

Application of scale-down experiments in the study of kinetics of oxytetracycline biosynthesis

Scale-down experiments in antibiotic biosynthesis were performed by transferring the corresponding amounts of fermentation broth from industrial to laboratory and pilot-plant fermentors where the cultivation process was continued at different cultivation conditions. A previously proposed mathematical model was used to explain the experimental results. The effects of temperature, agitation-aeration intensity, and medium addition during the process were investigated. Computer simulation data were fitted to the experimental data, and good agreement was found. As a consequence of increasing temperature up to 37 degrees C, increases in the specific growth and autolysis rates as well as the specific rates of antibiotic synthesis and carbohydrate utilization were in evidence. Temperature increases of up to 40 degrees C caused a lower oxytetracycline yield. The effect of increased oxygen transfer rate on oxytetracycline biosynthesis was more pronounced at higher temperatures than at lower cultivation temperatures. Culture differentiation (strain segregation) was also studied; it was found that the increased cultivation temperature could be favorable for the growth of biomass active in oxytetracycline biosynthesis. Results of experiments at the pilot-plant scale showed that fed batch and repeated fed batch cultures could be successfully applied and the period of intensive antibiotic synthesis could be prolonged significantly.     

Venous ultrasound catheter-tip technique for evaluation of arterial hemodynamics.

A variety of devices has been used for measuring flow properties of deep-lying arteries, but many have limitations. This paper describes a relatively nontraumatic intravenous approach which uses a catheter in connection with a pulsed ultrasonic Doppler velocity meter (PUDVM) and an ultrasound echo track. The venous ultrasound catheter (VUC) has permitted measurements of local instantaneols blood velocity, flow, and wall motion in the abdominal aorta and iliac arteries of beagle dogs; evaluation studies have been conducted to compare the VUC recordings with an independent method for measuring blood flow and wall motion. Coupling of this catheter-tip device with the PUDVM and echo track provides chronic measurements of hemodynamic parameters in these deep vessels which were virtually impossible to obtain previously. This technique may prove useful in monitoring vessel pathology longitudinally as well as in basic experimental situations requiring flow and arterial wall mechanical properties.     

Non-invasive mass transfer measurements in complex biofilm-coated structures

We demonstrate a novel application of (13)C pulsed field gradient (PFG) NMR to monitor mass transfer, due to both flow and diffusion, in a 3D complex porous support structure modified by biofilm growth. This enables timescales an order of magnitude larger than previously possible to be accessed with respect to displacement probability distribution (propagator) measurements. The evolution in the propagator shape with observation time to the Gaussian asymptote (constant dispersion coefficient) is consequently well resolved. We also simulated the measured displacement propagators with good agreement between experiment and prediction. The methodology has significant potential for the selective characterization of the transport of nutrients, metabolic products, pollutants and biocides in such complex biofilm-containing structures.     

Detection of receptor clustering by flow cytometric fluorescence anisotropy measurements

BACKGROUND: Perrin equation suggests an alternative way for the accurate energy transfer determination on a cell-by-cell basis by measuring polarized donor intensities in a conventional flow cytometer. METHODS: The relationship between energy transfer and fluorescence anisotropy of the donor was investigated by flow cytometric generation of Perrin-lifetime plots of fluorescent antibody-labeled MHC class I and class II molecules on the surface of living cells. The energy transfer reduced the fluorescence lifetime of the donor. RESULTS: Perrin plots have proven to be sensitive to the segmental mobility of the labeling dye and that of antibodies of different isotypes, and homo-transfer due to the multiple labeling of antibodies. A method demonstrating the feasibility of energy transfer determination by measuring anisotropy enhancement of the donor is presented. Flow cytometric histograms of the donor anisotropy and of the deduced energy transfer efficiency are shown, indicating clustering of MHC class I and class II molecules on the surface of human T lymphoblasts. In the Appendix, a method for the simultaneous determination of both energy transfer efficiency and donor fluorescence anisotropy, without need for G-factor measurement, is also presented. CONCLUSIONS: We demonstrate that energy transfer efficiency, i.e., proximity, between suitably selected donor and acceptor, and the rotational relaxation of the donor, i.e., donor mobility, can be simultaneously measured in a flow cytometer.     

Design of a bubble-swawm bioreactor for animal cell culture

A stationary bubble-swarm has been used to aerate a mammalian cell culture bioreactor with an extremely low gas flow rate. Prolonging the residence time of the gas bubbles within the medium improved the efficiency of the gas transfer into the liquid phase and suppressed foam formation. An appropriate field of speed gradients prevented the bubbles from rising to the surface. This aeration method achieves an almost 90% transfer of oxygen supplied by the bubbles. Consequently, it is able to supply cells with oxygen even at high cell densities, while sparging with a gas flow of only 0.22·10^–3–1.45·10^–3 vvm (30–200 ml/h).The reactor design, the oxygen transfer rates and the high efficiency of the system are presented. Two repeated batch cultures of a rat-mouse hybridoma cell line are compared with a surface-aerated spinner culture. The used cell culture medium was serum-free, either with or without BSA and did not contain surfactants or other cell protecting agents. One batch is discussed in detail for oxygen supply, amino acid consumption and specific antibody production.     

Proximity of lectin receptors on the cell surface measured by fluorescence energy transfer in a flow system.

Molecules of the lectin concanavalin A have been labeled separately with the fluorescein and rhodamine chromophores and jointly bound to the surface of transformed Friend erythroleukemia cells. The two dyes constitute an ideal donor-acceptor pair for fluorescence resonance energy transfer thereby permitting the determination of the proximity relationships between bound ligand molecules and the corresponding surface receptors. The transfer efficiency at saturation (about 57%) was measured in a multiparameter flow system using laser excitation at 488 nm and detection of fluorescein and rhodamine emission intensities as well as the emission anisotropy of the rhodamine fluorescence for each cell. The degree of energy transfer was estimated from the quenching of donor emission, the sensitization of acceptor emission, and the depolarization of acceptor fluorescence. The system has been modeled according to a formalism developed by Gennis and Cantor (Biochemistry 11: 2509, 1972). We estimate the separation between the surfaces of bound lectin molecules at saturation to be 0-40 A, a range possibly characteristic for micropatches induced by ligand binding.     

Pulsed Doppler ultrasound system for the measurement of velocity distributions and flow disturbances in arterial prostheses

For the investigation of flow through prosthetic arteries a pulsed Doppler ultrasound system has been characterized. Preliminary in vitro experiments using this system are described; they verify its suitability for making velocity profile and flow disturbance measurements. The output from a frequency tracker is compared with spectral analysis of Doppler signals for both laminar and turbulent flow regimes and the root mean square fluctuations on the tracker output signal are used to identify transition from laminar to turbulent flow. In addition, the turbulent itensity of poststenotic flow is quantified at several axial locations and for different rates of flow. Finally, we present velocity profile measurements which were obtained using a deconvolution technique to account for the finite size of the sample volume.     

Hydredynamic bench for pulsatile flows. Application to hemodynamic studies (author's transl)

Oscillatory or pulsatile flows are produced on a hydrodynamic bench by a volumetric gear pump. The moto-pump group is an original device which is very different from other systems. It is driven by a wave synthesizing generator which enables any flow rate to be produced especially oscillatory, pulsatile or physiological flow. The synthesizing generator delivers a periodic wave with variable amplitude, shape and frequency. The moto-pump group is controlled by an amplifying comparator; it produces a flow whose rate follows the control signal. A study of the modifications of flow patterns induced by models of vascular stenoses has been performed with this bench. The study of the velocity distribution has been effected with a pulsed Doppler ultrasonic velocimeter.