Spectral evidence for non-calcium interactions of intracellular Indo-1

Indo-1 is widely used to measure intracellular free calcium, [Ca2+]i, by comparing the fluorescence emission at 2 or more wavelengths with the emissions, which are assumed to be known, of Indo-1 when it is fully calcium-bound and when it is fully calcium-free. Accurate quantitation requires that these "reference" values be obtained on intracellular dye, and the full spectra of this study show that the reason is a significant spectral shift of the calcium-free peak, but not the calcium-bound. A mathematical analysis shows that the new peak must be a new state of the Indo-1 molecule, since it cannot be simply due to residual calcium in the cell. When intracellular "reference" spectra were used in the data analysis, [Ca2+]i could be calculated from whole spectra or from the ratio of observations at two wavelengths with good agreement. When extracellular "reference" spectra were used, the value calculated by the ratio method depended on the choice of wavelengths.     

Release of periplasmic enzymes and other physiological effects of beta-lactamase overproduction in Escherichia coli

When Escherichia coli containing the plasmid ptac11 is induced with 10(-4) M isopropyl-beta-thiogalactopyranoside (IPTG), 90% of the beta-lactamase activity of an overnight culture is present in the medium. The high extracellular activity of beta-lactamase does not result from cell lysis but from an increase in the permeability of the outer membrane. The excreting cells release several other periplasmic enzymes into the extracellular fluid and are more sensitive to lysis by detergents. It was also shown that in these cells the level of two membrane proteins, OmpA and OmpC, is decreased. None of these phenomena were observed with the plasmid pDW17, which has a mutation in the tac promoter that reduces its activity to one fourth of the tac promoter.     

Design of a system for the control of low dissolved oxygen concentrations: critical oxygen concentrations for Azotobacter vinelandii and Escherichia coli

The physiological activity of microorganisms in environments with low dissolved oxygen concentrations often differs from the metabolic activity of the same cells growing under fully aerobic or anaerobic conditions. This article describes a laboratory-scale system for the control of dissolved oxygen at low levels while maintaining other parameters, such as agitator speed, gas flowrate, position of sparger outlet, and temperature at fixed values. Thus, it is possible to attribute in dilute nonviscous fermentations all physiologic changes solely to changes in dissolved oxygen. Experiments were conducted with Azotobacter vinelandii and Escherichia coli. Critical oxygen concentrations for growth (that value of oxygen allowing growth at 97% of mu max) were measured as 0.35 +/- 0.03 mg/L for A. vinelandii and 0.12 +/- 0.03 mg/L for E. coli. These values are significantly different from the commonly quoted values for critical oxygen concentrations based on respiration rates. Because of the superior dissolved oxygen control system and an improved experimental protocol preventing CO2 limitation, we believe that the values reported in this work more closely represent reality.     

Environmental parameters influencing phenolics production by batch cultures of Nicotiana tabacum

The influence of temperature, illumination, hormonal levels (2,4-D and kinetin), carbon to nitrogen ratios, antibiotics, and precursor feeding on phenolics production by Nicotiana tabacum (tobacco) was studied. This plant cell system was chosen as a model system to learn more about secondary product formation in plant cell tissue cultures. This is the first study to manipulate all of these environmental parameters with a single plant cell system. The most striking results were with 2,4-D manipulation. The removal of 2,4-D resulted in significant phenolics production during the stationary phase, while normal levels strongly suppressed phenolics production during the stationary phase. The addition of phenylalanine stimulated phenolics production per gram of cells but strongly inhibited growth.     

Multistage continuous culture to examine secondary metabolite formation in plant cells: Phenolics from Nicotiana tabacum

The feasibility of operating a multistage continuous culture of plant cells was demonstrated for Nicotiana tabacum. Cells in the second stage of a two-stage chemostat were morphologically distinct from cells in the first stage or cells in a single-stage unit with a holding time equal to the combined holding times in the two-stage system. Cells in the second stage produced much higher levels of phenolics per unit weight of cells than cells in either the first-stage or single-stage unit. The steady-state was reproduced. When a glucose side stream was fed to the second stage, an increase in apparent cell division was observed with a simultaneous decrease in phenolics productivity. When the toxic precursor phenylalanine was pulsed into the reactor, the quantity of biomass decreased temporarily while phenolic productivity increased. These experiments demonstrate that multistage continuous culture may be useful in increasing secondary metabolite formation in cells and in exploring mechanisms controlling secondary metabolite formation.     

Impetigo—Bacteriologic Features and Renal Involvement

One hundred children with impetigo were studied with particular emphasis upon the organism causing the infection and associated renal complications. In 50 per cent of cases, Group A beta-hemolytic streptococcus grew on cultures of material from the lesions, and evidence of recent infection with this organism as shown by an elevation of antistreptolysin O titer was present in an additional 17 per cent of cases.Acute glomerulonephritis developed in three of the 66 children with bacteriologic or serologic evidence of streptococcal infection. Four other children in this group and nine children with staphylococcal impetigo had unexplained microscopic hematuria.All children with nephritis already had evidence of the disease when first seen. In most of those with unexplained hematuria, this condition was detected at the first visit. Hematuria developed in others while they were receiving systemic antibiotics. The significance of isolated microscopic hematuria is uncertain, but is seen often in association with cutaneous infection with both staphylococcus and streptococcus. Microscopic hematuria as defined is apparently not prevented by antibiotic therapy.If acute glomerulonephritis that follows streptococcal cutaneous infection is to be prevented, streptococcal impetigo will have to be treated promptly after onset.     

Effect of Trichoplusia ni BTI-Tn 5B1-4 cell density on human secreted alkaline phosphatase production

Summary The effect of Tn 5B1-4 cell density at infection on the production of human secreted alkaline phosphatase (SEAP) was investigated using a split-flow air-lift bioreactor. Volumetric productivities of SEAP were highest when Tn 5B1-4 cells were infected at about 3 × 10⁵ cells/cm². Cell-to-cell contact inhibition is an important factor responsible for the reduced protein production at high cell densities. Other factors such as oxygen or nutrient limitation, or build up of metabolic inhibitors do not appear to be as important.     

Continuous, high level production and excretion of a plasmid-encoded protein by Escherichia coli in a two-stage chemostat

The stable continuous overproduction of a plasmidencoded protein, beta-lactamase, for at least 50 days by Escherichia coli K-12, RB791(pKN), with release into the culture medium has been demonstrated in two-stage chemostats. The second-stage culture was continuously induced with 0.1 mM IPTG. Continuous expression of beta-lactamase could not be sustained with this strain in a single-stage chemostat because of cell death and selection for lac(-1) cells. beta-Lactamase production in the second stage was sensitive to the second-stage dilution rate and the distribution of the limiting substrate (i.e., glucose) between the first and second stages. The fraction of viable, excreting cells and the average copy number in the induced culture was measurably higher under those conditions of dilution rate and substrate distribution which yielded high beta-lactamase levels. The best operating conditions found at 20 degrees C were a first-stage dilution rate of 0.12 h(-1), a second-stage dilution rate of 0.03 h(-1), and equal glucose feed supplied to each stage. Enzymatically active beta-lactamase was produced at a level of 25% of total cellular protein with 90% excretion yielding 300 mg beta-lactamase/L that was 50% pure at an OD(600) < 6. (c) 1993 Wiley & Sons, Inc.