Growth kinetics of suspended microbial cells: from single-substrate-controlled growth to mixed-substrate kinetics

Growth kinetics, i.e., the relationship between specific growth rate and the concentration of a substrate, is one of the basic tools in microbiology. However, despite more than half a century of research, many fundamental questions about the validity and application of growth kinetics as observed in the laboratory to environmental growth conditions are still unanswered. For pure cultures growing with single substrates, enormous inconsistencies exist in the growth kinetic data reported. The low quality of experimental data has so far hampered the comparison and validation of the different growth models proposed, and only recently have data collected from nutrient-controlled chemostat cultures allowed us to compare different kinetic models on a statistical basis. The problems are mainly due to (i) the analytical difficulty in measuring substrates at growth-controlling concentrations and (ii) the fact that during a kinetic experiment, particularly in batch systems, microorganisms alter their kinetic properties because of adaptation to the changing environment. For example, for Escherichia coli growing with glucose, a physiological long-term adaptation results in a change in KS for glucose from some 5 mg liter-1 to ca. 30 microg liter-1. The data suggest that a dilemma exists, namely, that either "intrinsic" KS (under substrate-controlled conditions in chemostat culture) or micromax (under substrate-excess conditions in batch culture) can be measured but both cannot be determined at the same time. The above-described conventional growth kinetics derived from single-substrate-controlled laboratory experiments have invariably been used for describing both growth and substrate utilization in ecosystems. However, in nature, microbial cells are exposed to a wide spectrum of potential substrates, many of which they utilize simultaneously (in particular carbon sources). The kinetic data available to date for growth of pure cultures in carbon-controlled continuous culture with defined mixtures of two or more carbon sources (including pollutants) clearly demonstrate that simultaneous utilization results in lowered residual steady-state concentrations of all substrates. This should result in a competitive advantage of a cell capable of mixed-substrate growth because it can grow much faster at low substrate concentrations than one would expect from single-substrate kinetics. Additionally, the relevance of the kinetic principles obtained from defined culture systems with single, mixed, or multicomponent substrates to the kinetics of pollutant degradation as it occurs in the presence of alternative carbon sources in complex environmental systems is discussed. The presented overview indicates that many of the environmentally relevant apects in growth kinetics are still waiting to be discovered, established, and exploited.     

Inhibition of vinblastine efflux mediated by P-glycoprotein by grapefruit juice components in caco-2 cells

The authors describe a resource-allocation model developed in the Medical Care Clinical Center at the Baltimore Veterans Affairs Medical Center (a part of the VA Maryland Health Care System) and implemented in 1989. This model is a computer-based system that tracks the workload of each of the clinical center's specialty sections (e.g., cardiology) and calculates each section's workload as a percentage of the total clinical center workload. As the basis of this calculation, six activities of each section are tracked by the model (e.g., inpatient attending physicians' rotations; inpatient consultations; etc.) to determine what percentage of each activity of the entire clinical center was provided by each section. Each of these percentages is then recalculated according to a weighted average based on the relative value of the activity to the department; these averages are revised periodically as needed. The model provides an incentive for the specialty sections to increase productivity by generating competition among sections for physician salary support. Communication among all concerned at the clinical center and its associated medical school and teaching hospital has been the key to success in implementing the model, which is periodically reviewed and has been revised several times after meetings with section chiefs and division heads. The authors are confident that the use of the model has been at least partly responsible for increased productivity of clinical center physicians, especially in the areas of visits per physician and funded VA research dollars per physician. Perhaps equally important is the future potential of the model. Because of its simplicity and because it is generally seen to be fair and effective, it will continue to be used to reward activities most important to the clinical center, especially now that the center operates under a fully capitated system, and in this way wil help ensure the financial viability of the center.     

A novel method for direct and fluorescence independent determination of drug efflux out of leukemic blast cells

Multi drug resistance (MDR) is often due to an increased efflux of anti cancer drugs out of leukemic blast cells. Efflux assays are used to get an impression of functional resistance in those cells. Dyes like rhodamine 123 or 3'3'-diethyloxocarbocyanine iodide are commonly used for this purpose. A major known disadvantage is that dyes do not behave like cytotoxic drugs in efflux experiments. Assays using the self fluorescence of drugs like anthracyclines can not reveal a real impression of intracellular or effluxed drug due to quenching of the drug fluorescence in the nuclei of the cells. We have developed a reproducible and sensitive assay for direct and quantitative determination of drug efflux out of blast cells. This was done by a novel double radioactive labelling using a 3H-labelled drug and 14C-labelled sucrose as extracellular marker. So this assay can be applied to every drug of interest. Quenching of fluorescence is also by-passed with this technique as well as protracting washing or silicon oil procedures. As a model system we used the T-lymphoblastoid cell line CCRF CEM and its resistant sublines vincristine 100 and adriamycin 5000. The results were also transferable to clinical specimens of leukemic patients. In conclusion our assay may be used for precise and direct efflux measurement of a broad range of anti-cancer drugs in clinical MDR evaluation.     

Quantitative studies of the kinetics of 5-aminolaevulinic acid-induced fluorescence in bladder transitional cell carcinoma

Photodynamic therapy is a potential treatment for superficial bladder cancer that utilizes photosensitizer drugs, which are activated by light to cause tissue destruction. However, first-generation photosensitizers cause prolonged phototoxicity, have poor tumour specificity and can accumulate within detrusor muscle, resulting in permanent loss of bladder capacity following treatment. A newer drug, called 5-aminolaevulinic acid (ALA), generates a sensitizer called protoporphyrin IX (PpIX) in situ and has been shown, qualitatively, to be more tumour specific. The fluorescence kinetics of ALA-induced PpIX was investigated in patient biopsies of bladder tumour, normal urothelium and detrusor muscle, both in vitro after incubation of specimens in ALA-rich culture medium for various times and in vivo after instillation of intravesical ALA before endoscopic resection. The fluorescence in tumour tissue was twice that of normal urothelium in vitro and up to tenfold in vivo. There was little ALA-induced fluorescence in detrusor muscle, both in vitro and in vivo. Most importantly, no patients experienced phototoxicity or other adverse events following intravesical instillation of ALA.     

Voltammetric studies on mechanisms of dopamine efflux in the presence of substrates and cocaine from cells expressing human norepinephrine transporter

The effects of substrates m-tyramine and beta-phenethylamine, as well as cocaine, on the DA efflux from a cell line stably expressing the human norepinephrine transporter (hNET) were investigated by using rotating disk electrode voltammetry. Both the substrates and cocaine induced apparent DA efflux in a concentration-dependent manner. Their EC50 values for inducing DA efflux were similar to their IC50 values for inhibiting DA uptake. The substrate-induced DA efflux was inhibited by various NET blockers, enhanced by raising the internal [Na+] with Na+,K+-ATPase inhibition, but was insensitive to membrane potential-altering agents valinomycin, veratridine, and high [K+]. The initial rate of m-tyramine-induced DA efflux was related to preloaded [DA] in a manner defined by a Michaelis-Menten expression. In contrast, DA efflux in the presence of cocaine displayed a much slower efflux rate, lower efficacy, was not stimulated by elevated internal [Na+], and was nonsaturable with preloaded [DA]. Single exponential kinetic analysis of the entire time course of the DA efflux showed that the apparent first-order rate constant for m-tyramine-induced DA efflux declined with increased preloaded [DA], whereas that for the DA efflux in the presence of cocaine was unchanged with varying preloaded [DA]. These results suggest that the substrates stimulate the NET-dependent DA efflux by increasing the accessibility of the NET to internal DA, whereas cocaine "uncovers" NET-independent DA efflux by reducing the accessibility of diffused/leaked external DA to the NET.     

Abnormal expression of a 170-kilodalton P-glycoprotein encoded by MDR1 gene, a metabolically active efflux pump, in CD4+ and CD8+ T cells from patients with human immunodeficiency virus type 1 infection

Peripheral blood CD4+ and CD8+ T cells from 16 patients with HIV-1 infection, 8 each with CD4+ T cell counts of > 200/mm3 (group I) and with CD4+ T cell counts of < 200/mm3 (group II), and 8 age- and sex-matched controls, were examined for the expression of P-glycoprotein (P-gp), a 170-kDa phosphoglycoprotein encoded by the MDR1 gene, using dual-color flow cytometric analysis. The function of P-glycoprotein was assessed by the accumulation of rhodamine-123 (Rh123) dye in the presence or absence of cyclosporin A (which inhibits Rh123 efflux). A significantly increased proportion of CD4+ T cells from patients with HIV-1 infection expressed P-glycoprotein as compared to controls, resulting in a significantly increased ratio of the proportions of CD4+P-gp+/CD8+P-gp+ cells. The ratio of CD4+P-gp+/CD8+P-gp+ in group II patients was significantly higher (p = 0.02) than in group I patients, suggesting a progressive increase in P-gp expression with the advancement of HIV-1 infection. The proportions of CD4+P-gp+ and CD8+P-gp+ T cells did not differ significantly between those who received AZT and those who were not treated with AZT. Contrary to expectation, both CD4+ and CD8+ T cells from patients accumulated significantly more Rh123 as compared to controls. Furthermore, cyclosporin A failed to increase intracellular accumulation of Rh123 in CD4+ and CD8+ T cells from patients. These data suggest a functionally defective P-gp expression in HIV-1 infection that appears to increase with the progression of HIV-1 infection. A study of a large number of patients with HIV-1 infection is needed to determine the effects of opportunistic infection and antiretroviral therapy on the expression of P-gp and to determine whether the expression of P-gp could serve as another surrogate marker for the progression of HIV-1 infection.     

Stimulation of taurine efflux from human placental tissue by a hypoosmotic challenge

A statistical model for predicting a woman's lifetime risk of hip fracture using her bone mineral density at menopause has been proposed by Black et al. (1992b). We made an additional assumption concerning the correlation of bone mineral density between any two ages among postmenopausal women and applied the modified model to baseline ages between 50 and 85 years and any bone mineral density level likely to be observed in the population. The results are displayed in a form more convenient for application of this model in the clinical setting.     

(Sp)-8-chloroadenosine 3'',5''-cyclophosphate induced differentiation on human leukemia HL-60 cells

The structure and effects of sulfonylurea derivatives were described. Today the second generation of sulfonylureas rather is prescribed because of its lower dosage and better peripheral activity. The non-insulin-dependent diabetes after weight reduction and failure of other oral therapy is the main indication to introduction of sulfonylurea derivates.     

(-)-Deprenyl protects human dopaminergic neuroblastoma SH-SY5Y cells from apoptosis induced by peroxynitrite and nitric oxide

In Parkinson's disease the cell death of dopamine neurons has been proposed to be mediated by an apoptotic death process, in which nitric oxide may be involved. This article reports the induction of apoptosis by nitric oxide and peroxynitrite in human dopaminergic neuroblastoma SH-SY5Y cells and the antiapoptotic activity of (-)-deprenyl. After the cells were treated with a nitric oxide donor, NOR-4, or a peroxynitrite donor, SIN-1, DNA damage was quantitatively studied using a single-cell gel electrophoresis (comet) assay. NOR-4 and SIN-1 induced DNA damage dose-dependently. Cycloheximide and alkaline treatment of the cells prevented the DNA damage, indicating that the damage is apoptotic and that it depends on the intracellular signal transduction. Superoxide dismutase and the antioxidants reduced glutathione and alpha-tocopherol protected the cells from the DNA damage. (-)-Deprenyl protected the cells from the DNA damage induced by nitric oxide or peroxynitrite almost completely. The protection by (-)-deprenyl was significant even after it was washed from the cells, indicating that (-)-deprenyl may activate the intracellular system against apoptosis. These results suggest that (-)-deprenyl or related compounds may be neuroprotective to dopamine neurons through its antiapoptotic activity.     

Direct assessment of triploid cells in mosaic human fetuses by fluorescence in-situ hybridization

Villous tissues from 30 spontaneous abortions and the same number of artificial abortions were obtained and analysed for the frequency of polyploid cells. Single cell suspensions were made from these tissues without culture and the ploidy of > 100 cells was analysed. Trisomies of chromosomes 17 and 4 have rarely been reported in villous cells of spontaneous abortions, suggesting that the presence of more than three copies of chromosomes 17 and 4 per cell indicates polyploidy. The number of chromosomes 17 and 4 was detected by fluorescence in-situ hybridization analysis using centromeric probes D17Z1 and D4Z1. Most villous cells from cases of spontaneous and artificial abortions had two D17Z1 or D4Z1 signals per cell, with very small percentages of cells (0.5 +/- 0.4%) showing three signals per cell. However, in four cases of spontaneous abortions, 2-12% of cells had three D17Z1 or D4Z1 signals per cell. This indicates the presence of triploid cells in these cases of spontaneous abortion, at a significantly higher frequency compared to artificial or the remaining 26 cases of spontaneous abortion. In addition, three cases contained 0.2-0.4% of cells showing six signals, indicating that these cells were dividing triploid cells. The low frequency of mosaicism reported here would not be detectable by conventional chromosomal analysis.     

Induction of NG108-15 cells differentiation by human bone marrow stromal cells

The survival and differentiation of neuronal cells is dependent on factors such as neurotrophins, cytokines and components of extracellular matrix. Bone marrow stromal cells have been shown to support the growth and differentiation of neuroblastoma cells. In an attempt to study the effects of bone marrow stromal cells on neuronal differentiation, we have co-cultured neuroblastoma x glioma hybrid NG108-15 cells with human bone marrow stromal cells. After co-culturing, clones exhibiting morphological differentiated phenotype and high level of neurofilament expression were isolated. Interestingly, these clones maintain their ability to proliferate in contrast to differentiated NG108-15 cells induced by dibutyryladenosine 3',5'-cyclic monophosphate. These results suggested that bone marrow stromal cells can induce partial differentiation of NG108-15 cells.     

(R)-5-fluoro-5,6-dihydrouracil: kinetics of oxidation by dihydropyrimidine dehydrogenase and hydrolysis by dihydropyrimidine aminohydrolase

The biologically active isomer of 5-fluoro-5,6-dihydrouracil [(R)-5-fluoro-5,6-dihydrouracil, R-FUH2] was synthesized to study the kinetics of its enzymatic oxidation and hydrolysis by homogeneous dihydropyrimidine dehydrogenase (DPDase) and dihydropyrimidine aminohydrolase (DPHase), respectively. DPDase catalyzed the slow oxidation of R-FUH2 at pH 8 and 37 degrees with a Km of 210 microM and a kcat of 0.026 sec-1 at a saturating concentration of NADP+. The catalytic efficiency (kcat/Km) of DPDase for R-FUH2 was 1/14th of that for 5,6-dihydrouracil (UH2). In the opposite direction, DPDase catalyzed the reduction of 5-fluorouracil (FU) with a Km of 0.70 microM and a kcat of 3 sec-1 at a saturating concentration of NADPH. Thus, DPDase catalyzed the reduction of FU 30,000-fold more efficiently than the oxidation of R-FUH2. In contrast to the slow oxidation of R-FUH2 by DPDase, R-FUH2 was hydrolyzed very efficiently by DPHase with a Km of 130 microM and a kcat of 126 sec-1. The catalytic efficiency of DPHase for the hydrolysis of R-FUH2 was approximately twice that for the hydrolysis of UH2. Because R-FUH2 is hydrolysis of R-FUH2 was approximately twice that for the hydrolysis of UH2. Because R-FUH2 is hydrolyzed considerably more efficiently than it is oxidized and because the activity of DPHase was 250- to 500-fold greater than that of DPDase in bovine and rat liver, the hydrolytic pathway should predominate in vivo.     

Characteristics of a human cell line transformed by DNA from human adenovirus type 5

One hundred and fifty lungs from the cases below 15 years of age with various congenital heart diseases and 80 controls were used for histometrical and histological studies. Cases with congenital heart disease were divided into two groups of the increased and the decreased pulmonary blood flow. In the former group, the thickness of the pulmonary arterial media was the same as that of controls in the neonatal period, and through the wall thickness gradually decreased in a pattern seen in controls, the thickness was constantly larger than that of controls. In some cases, the media increased gradually within 6 months after birth. Pneumonia and massive pulmonary hemorrhage were seen in a higher incidence in autopsy cases. Pneumonia in younger infants was histologically characteristic and possibly more correlated to their death. In the latter group, most of the cases were with the thinner medias of the pulmonary arteries. Massive pulmonary hemorrhage was not common in the latter group.     

Analysis of human primary bone cells by fluorescence activated cell scanning: methodological problems and preliminary results

We describe the development of flowcytometrical methods to analyse human primary osteoblast-like cultures obtained from trabecular bone explants in comparison to the human osteosarcoma cell line HOS 58. Two antigens typical of osteoblasts were studied: bone alkaline phosphatase and collagen/procollagen I; the non-specific attachment protein fibronectin served as control. The morphology of all different antigens is shown by immunocytochemistry before flowcytometrical analysis. The establishment of flowcytometry is described in detail. While all antigens tested were nearly 100% positive in the HOS 58 cells in immunocytochemistry and flowcytometry, in primary osteoblast-like cells results varied widely between both methods. Cell permeabilisation before flowcytometry improved the homogeneity of results, probably by increasing the accessibility of the specific antibody to intracellular compartments. Though up to 80% of cells were lost during preparation the ratio of positive versus negative cells in specific experiment was not dependent on the cell recovery. Therefore, the cells finally analysed seemed to be representative of the total population.     

Characterization of the three tyrosine residues of delta 5-3-ketosteroid isomerase by time-resolved fluorescence and circular dichroism

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni converts delta 5-3-ketosteroids to delta 4-3-ketosteroids via an enolic intermediate. Site-specific mutagenesis has identified Tyr-14 and Asp-38 as the catalytically essential general acid and base, respectively. Three tyrosine residues (Tyr-14, Tyr-55, and Tyr-88) are the only significant fluorophores in the wild-type isomerase. Recent studies of the steady-state fluorescence of the wild-type enzyme and all six mutant enzymes in which one or two tyrosine residues have been mutated to phenylalanine show that the fluorescence intensity of Tyr-14 is very high, that of Tyr-88 is very low, and that of Tyr-55 is intermediate and comparable to that of N-acetyltyrosine amide in solution (Li, Y.-K., Kuliopulos, A., Mildvan, A.S., & Talalay, P. (1993) Biochemistry 32, 1816-1824). Extension of these experiments by time-resolved fluorescence and fluorescence anisotropy measurements demonstrates that Tyr-14, which is in a hydrophobic environment, has an unusually long fluorescence lifetime (4.6 ns) as compared to Tyr-55 (2.0 ns) or Tyr-88 (0.8 ns) and to most protein tyrosine residues (0.2-2 ns). The F?rster distances obtained from the absorption and emission of these tyrosines predict that total quenching of Tyr-14 fluorescence by Tyr-55, and to a lesser degree by Tyr-88, would occur if their orientations were favorable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of c-myc translocation in human lung cancer cells by fluorescence in situ hybridization (FISH)

c-myc gene amplification has been found in lung cancer, however, it can not explain all cases of lung cancer with c-myc gene overexpression. Gene translocation is one of the ways by which oncogene is activated. But the old methods for detecting gene mutations are not so effective for the detection of gene translocation, especially in solid tumors. Fluorescence in situ hybridization (FISH) can be used to detect gene translocation more efficiently. Using FISH, we discovered c-myc gene translocation in a lung adenocarcinoma cell line GLC-82 and SV40T-transformed human bronchial epithelial cells. In GLC-82, c-myc gene translocated to the short arm of a C group marker chromosome. In the SV40T-transformed epithelial cells, c-myc gene translocated to 14q32, which was the same as that found in Burkitt's lymphomas. Translocation was related to oncogene activation. c-myc translocation may play an important role in the carcinogenesis of lung cancer.     

Widespread expression of MRP8 and MRP14 in human cerebral malaria by microglial cells

Recently, laser treatment of the prostate has been added to the urologist's armamentarium for the treatment of bladder outlet obstruction secondary to benign prostatic hyperplasia (BPH). Until now, limited data on long-term outcome are available notwithstanding the fact that such information is crucial in determining the ultimate role of laser prostatectomy in the treatment of BPH. We now have 3-year data of a comparative study using the Urolase and Ultraline fiber in Nd:YAG sidefiring laser prostatectomy. The study was performed to compare laser prostatectomy using a pure coagulation (Urolase fiber) and a combination of a coagulation and vaporization (Ultraline fiber). In a period of 15 months, 93 men were randomized for laser treatment with the Ultraline fiber (N = 44) or the Urolase fiber (N = 49). Symptom scores, maximal uroflow, postvoiding residual volume, and sexual history were noted over a 3-year period. Adverse events and retreatments were also recorded. The mean postoperative catheterization time was 18 days, without significant difference between the two groups. After 3 years, we demonstrated a durable improvement in maximal flow rate, from 7.8 to 13.9 mL/sec in the Urolase group and from 7.9 to 13.6 mL/sec in the Ultraline group. In both groups, however, a considerable decrease in the maximal flow rate was noted after 3 years compared with 3 months after treatment, from 18.7 to 13.9 mL/sec in the Urolase group and from 20.0 to 13.6 mL/sec in the Ultraline group. The symptom scores showed marked and lasting improvement. The postvoiding residual urine volume became very low in the early postoperative period but did significantly increase after 3 years; nevertheless, it was still only 50% of the preoperative value. Although after 3 years, the maximal uroflow rate was still significantly improved compared with baseline, a considerable decrease was noted when compared with the early postoperative value. The same considerable and lasting improvement in subjective outcome (symptom scores) was seen in both groups. Although the Ultraline fiber also causes vaporization of prostatic tissue, no differences could be noted in the clinical outcome obtained with the two fibers.