Biphasic Growth Research Articles

Effect of culture conditions on androgen sensitivity of the human prostatic cancer cell line LNCaP.

Several effects of androgens on LNCaP-FGC prostate tumor cells showed a biphasic pattern. Stimulation of growth and inhibition of secretion of prostatic acid phosphatase (PAP) was observed at low androgen concentrations (below 1 nM of the synthetic androgen R1881), and inhibition of growth and stimulation of PAP secretion was observed at higher concentrations. In contrast, prostate specific antigen (PSA) secretion did not show this biphasic response pattern. Comparable effects were found for two sublines of the LNCaP-FGC cells: an early (passage 20, androgen-dependent) and relatively late (passage 70, androgen-sensitive) passage of the cells. Culturing of both sublines in the presence of a high concentration of androgens (10 nM R1881) resulted initially in a decrease in growth rate, but the cells started to proliferate within 3 weeks. These cells became less sensitive to androgens, lost their biphasic response pattern, and showed reduced androgen receptor levels. Three weeks after removal of the excess of androgens, the passage 70 cells regained a biphasic growth response to androgens. Culture in medium without steroids but with EGF resulted in a decrease of both androgen sensitivity and androgen receptor level. In conclusion, rapid changes of the androgen sensitivity and receptor level of the LNCaP cells occurred under the influence of culture conditions. These changes were partly reversible and, therefore, were most likely due to adaptation of the cells.

Secondary Echinococcus multilocularis infection in severe combined immunodeficient (scid) mice: Biphasic growth of the larval cyst mass

E. multilocularis infection was suppressed in C.B-17 mice after intraperitoneal inoculation of protoscoleces, with larval cysts weighing no more than 1.0 g. In scid mice, which are genetically identical to C.B-17 except for a deficiency in functional lymphocytes, infection progressed and larval cysts reached a mass of 17.5 g at 15 weeks post-infection. The growth of the larval cyst mass in scid mice was similar to that in other susceptible mouse strains, with a biphasic pattern. Histological observations revealed giant cells and granulomatous inflammation in the C.B-17, but not in the scid mice. These results led to the conclusion that suppression of the growth of the larval cyst mass in the initial stage of infection in susceptible mice strains is caused by factors other than the host's lymphocytic immune response.

Selection and characterization of a copper-resistant subpopulation of Schizosaccharomyces pombe

Addition of 15 μM-CuSO4 to Schizosaccharomyces pombe 972 growing in a defined medium resulted in a biphasic growth curve and selection for a copper-resistant subpopulation. A clonal isolate of this subpopulation, designated strain SW40, had a decreased copper content per cell compared to the wild-type strain 972. The copper-resistance trait was stable in the absence of selection, and was the result of a single recessive chromosomal mutation. Copper resistance was not the result of metal ion efflux, extracellular chelation or an increased oxidative stress response. The results are consistent with a copper-resistance mechanism in which a modification of the cytoplasmic membrane results in decreased copper accumulation.

High glucose-induced proliferation in mesangial cells is reversed by autocrine TGF-beta.

We investigated the effects of glucose concentration in serum-free media on the proliferative growth response of a cultured murine mesangial cell line. Raising the ambient D-glucose concentration from 100 mg/dl to 450 mg/dl stimulated cell proliferation after 24 to 48 hours but had a growth inhibitory effect after 72 to 96 hours of incubation. This biphasic proliferative response to high glucose concentration was not mediated by the elevated osmolarity of the medium and did not occur when L-glucose was used. The early phase of glucose-induced proliferation was associated with increased expression of the immediate early genes c-myc and egr-1 as well as with induction of the S-phase related proliferating nuclear cell antigen (PCNA). Several lines of evidence indicated that the late phase of glucose-induced growth inhibition was mediated by the bioactivation of endogenous transforming growth factor beta (TGF-beta). Neutralizing antibody against TGF-beta prevented the late inhibitory effects of glucose on proliferation. On the other hand, exogenous TGF-beta (1 ng/ml) significantly inhibited basal proliferation in mesangial cells. Furthermore, Northern blot analysis revealed that TGF-beta 1 mRNA was induced by 450 mg/dl glucose in the medium after 48 to 72 hours, but not after 24 hours. Cell cycle analysis demonstrated that mesangial cells incubated in high glucose for 24 hours have a higher percentage of cells in the S-G2 phase of the cell cycle compared with cells grown in normal glucose concentration. After 48 hours of culture in elevated glucose concentration, the percentage of cells in S-G2 phase was decreased, and became comparable to that of cells in normal glucose concentration. However, the addition of neutralizing anti-TGF-beta antibody stimulated the progression of cells towards S-G2 in high glucose medium after 48 hours. The findings of this study demonstrate a biphasic growth response of mesangial cells when they were cultured in high glucose concentration; initially there was a transient stimulation of replication for 24 to 48 hours followed by a sustained inhibition after longer incubation periods. This inhibition may be mediated by the glucose-induced synthesis and/or bioactivation of TGF-beta which can inhibit proliferation of mesangial cells in an autocrine fashion.

Growth of thermophilic bacteria in rapemeal‐derived media

K.H. YEOMAN AND C. EDWARDS. 1992. A clarified, soluble, protein‐rich medium was developed from rapemeal by boiling with 0·1 mmol/1 NaOH. The medium contained 78% of the original meal protein and supported the growth of a range of thermophilic micro‐organisms that included Bacillus, Streptomyces and fungi. Amino acid analysis of the medium showed a full range of amino acids, and that acidic ones such as glutamate predominated. Growth of a thermophilic Bacillus spp. (termed 1/15) in the medium was biphasic and significant amounts of extracellular protease were synthesized during the scond growth phase. Biphasic growth was probably due to preferential utilization of free amino acids during the first phase of growth followed by degradation of proteins during the second phase.

Degradation of mixtures of monochlorophenols and phenol as substrates for free and immobilized cells of Alcaligenes sp. A7-2

The biodegradation of the three isomeric monochlorophenols 2-(2CP), 3- (3CP) and 4-chlorophenol (4CP) and phenol by the constructed strain Alcaligenes sp. A7-2 was investigated. Mineralization took place in the order: phenol >4CP >2CP >3CP, whereas 3CP was mineralized only co-metabolically. In substrate mixtures with phenol, degradation of 4CP was decelerated but degradation of 2CP was accelerated. Free cells in batch culture showed biphasic growth with an equimolar mixture of 2CP and 4CP as substrates, perhaps due to diauxie. Degradation patterns obtained with free cells in batch culture were confirmed with immobilized cells in continuous culture. Immobilized cells of Alcaligenes sp. A7-2 built up a biofilm on the lava that was used as filling material in the packed-bed reactors. The continuous cultures remained stable despite increasing input rates of chlorophenol and phenol mixtures up to 1.16 mMo1.1−1.h−1 for several weeks.

Guanine nucleotide biosynthesis is regulated by the cellular p53 concentration.

As an approach to defining the role of p53 in cellular proliferation, murine cell lines were derived which contain a stably transfected temperature-inducible p53 expression system. Cell lines derived with the system exhibited a 3-6-fold physiologic elevation in the cellular p53 concentration when grown at 32.5 degrees C. A p53 induction phenotype was defined by examination of the growth properties of these lines at 32.5 degrees C. The induction phenotype had three main features: 1) a 2-4-fold increase in doubling time and biphasic growth kinetics; 2) delayed early S phase transit; and 3) complete reversibility either by growth at 37 degrees C or by growth in the presence of added hypoxanthine or xanthosine. The reversal of the induction phenotype by these purine salvage precursors implicated the purine nucleotide biosynthetic pathway as the cellular target for the antiproliferative action of p53. Subsequent genetic and biochemical analyses identified a p53 induction-related purine pathway defect which was localized to the step of inosine 5'-monophosphate conversion to xanthosine 5'-monophosphate. This enzymatic step catalyzed by inosine 5'-monophosphate dehydrogenase (EC 1.2.1.14) is the rate-limiting step for GTP synthesis. Extracts from p53-inducible cells growing at the induction temperature show a specific reduction in inosine 5'-monophosphate dehydrogenase enzymatic activity. These findings define p53 as a cellular regulator of the synthesis of GTP, a key regulatory nucleotide for many important cellular processes. Moreover, observations of the growth behavior of p53-inducible cells suggest that by regulating the production of GTP, p53 can control cellular quiescence.

Effect of carbon source and oxidative metabolism on secondary metabolism inStreptomyces thermoviolaceus

The effect of carbon source on a number of cultural parameters was investigated inStreptomyces thermoviolaceus. Glucose-grown cultures produced the antibiotic granaticin during pH-controlled growth in a fermenter. Biphasic growth occurred for all the carbon sources tested at 45°C, the inflexion of which occurred at a biomass density of between 0.5 and 0.6 gL−1 and which coincided with the onset of appearance of secondary metabolites. Maximum antibiotic production occurred in proline-grown cultures, which also had the slowest growth rates during the secondary phase of growth. Respiratory chain activity was probed by measuring NADH oxidation in membrane preparations exposed to a range of cyanide concentrations. Modulation of the terminal oxidase activity was apparent so that membranes prepared from cultures in the antibiotic-producing phase were less sensitive to KCN than those prepared from the early exponential phase of growth. The probable reason for this difference was the synthesis of cytochrome oxidased during later stages of growth. These changes in respiratory activity are discussed in relation to patterns of growth and timing of the appearance of secondary metabolites synthesised byS. thermoviolaceus.

Development of mammalian endothermic metabolism: quantitative changes in tissue mitochondria

The development of energy metabolism of mammalian tissues was assessed in the tammar wallaby Macropus eugenii by the measurement of mitochondrial parameters in the liver, heart, kidney, and brain. Tissues taken from wallabies (n = 27) ranging from 10-day-old pouch young (weighing approximately 4 g) to adults (averaging 6.2 kg) were weighed and fixed, and mitochondrial volume and mitochondrial membrane surface area (MMSA) were determined by quantitative electron microscopy techniques. Developmental changes in these parameters were analyzed chronologically and allometrically. Relative growth rates of all four tissues decreased during development. Liver and heart showed constant allometric growth throughout development, whereas kidney and brain showed biphasic allometric growth. Tissue metabolic intensity assessed by MMSA (m2/cm3 tissue) was constant in liver, showed a threefold increase in brain during pouch life, showed a fourfold increase in the heart between 100 and 200 days of age, and showed a twofold increase in the kidney at the end of pouch life. In all tissues, adult levels of tissue metabolic capacity were present at pouch exit. In all four tissues, total MMSAs were at "reptilian" levels at birth and gradually increased to "mammalian" levels. Each tissue exhibited a different developmental timetable. When the total MMSAs for all four tissues were summed there was a similar pattern of allometric development between summed MMSA and whole animal metabolic rate.

Extracellular protease activity in antibiotic-producingStreptomyces thermoviolaceus

Production of secondary metabolites was investigated in the thermophilic streptomyceteStreptomyces thermoviolaceus grown at 45°C in a fermenter. Extracellular protein was secreted into the culture medium at the same time as an antibiotic granaticin; both were synthesized during the second slower phase of biphasic growth, which is most apparent at 45°C for this organism. Protease, assayed as azocaseinase, was identified as one component of, and marker for, the excreted protein. The effects of different growth temperatures revealed that the synthesis of extracellular protein, like that of the antibiotic, was maximal in cultures grown between 37° and 45°C, whereas protease activity was greatest in 50°C grown cultures. A method was devised, based on acetone precipitation, for concentrating the protease activity from culture supernatants. Characterization of the concentrated activity using inhibitors suggested the presence of a serine and a metallo-type protease. A peptide substrate specific for the metallo-protease showed that it had a pH optimum for activity of 6.5–7.0. Approximately 50% of the activity was lost after 80 min of incubation at 70°C. Although calcium (5 mM) promoted increased thermotolerance such that around 65% of the activity remained after 100 min at 70°C, it seems that manganese and/or zinc may be more important for enzyme activity.

Differential effects of calmodulin antagonists on indol-3-ylacetic Add-and gibberellic acid-induced biphasic growth responses

The effects of chlorpromazine and calmidazolium on rapid IAA- and GA3-induced changes in growth rate of etiolated pea stems were measured. During the initial period of up to 160 min after hormone application, the responses to both IAA and GA3 were seen to be biphasic, showing two acceleration peaks. Chlorpromazine or calmidazolium applied simultaneously with the hormones blocked the first IAA-induced acceleration peak, but did not affect the second one. In contrast, the first GA3-induced peak was not prevented by chlorpromazine or calmidazolium, while the second one was completely abolished. The results support the concept that the active calmodulin-calcium complex may be an essential component of hormone-controlled stem elongation. They also point to differential mechanisms of IAA and GA3 actions.

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In general, wild-type gram-negative enteric bacteria are not able to utilize D-amino acids as the precursors of respective L-amino acids. We found, however, that an L-histidine auxotroph mutant, TA100, derived from Salmonella typhimurium strain LT 2 and used in the Ames test, showed a biphasic growth curve in the presence of both L- and D-histidine at concentrations of 5 micrograms/ml and 100 micrograms/ml, respectively. L-histidine may be utilized preferentially and then, after a short lag, D-histidine may be utilized. The short lag time is therefore considered to be the time required for induction of such an enzyme that converts D-histidine to L-histidine and for uptake of D-histidine by the bacterial cells.

The effects of gonadotrophin releasing hormone analogues in prostate cancer are mediated through specific tumour receptors

We have investigated the possibility of a direct regulatory effect of gonadotrophin releasing hormone (GnRH) analogues on prostatic cancer cell growth. Here we report high affinity binding (Kd = 50 nM) of a GnRH analogue resulting in biphasic growth modulation of the human androgen-sensitive prostatic cancer cell line LNCaP. In contrast, the human androgen-insensitive prostatic cancer cell line DU145 showed low-affinity (Kd = 10 microM) binding without any biological response to the GnRH analogue. A GnRH-specific radioimmunoassay demonstrated GnRH-like immunoreactivity in the concentrated culture medium from both cell lines. Seventy-six human benign and malignant tumours were assayed following surgical resection. Nineteen of 22 (86%) malignant tumours and 49 of 54 (91%) benign tumours, exhibited high affinity GnRH-analogue binding. Fourteen of 19 (74%) malignant tumours and 17 of 49 (35%) benign tumours exhibiting high affinity binding contained GnRH-like immunoreactivity, suggesting that this system may be involved in prostatic epithelial cell growth in vivo.

Acral Melanoma

Clinical and histopathologic analyses of 28 cases of acral melanoma in the last 24 years were carried out in this study. Of our 81 patients with cutaneous malignant melanoma, 28 (35%) had acral melanoma. The mean patient age was 59 years old. The two most commonly involved sites were the soles and toes. The preferential sites for metastasis were the lymph nodes, followed by the lungs, bone, and liver in descending order. Histologically, all skin biopsy specimens showed biphasic growth patterns and consisted of either spindle cells or epithelioid cells or both. The treatment was in all cases mainly surgical and, in patients at advanced stage, chemotherapy was added. The mean survival time for the series was 34.03 months, while the 3-year survival rate was only 29%.

Indirect interaction in milk between proteolytic and isogenic non-proteolytic strains of Lactococcus lactis. I. Effect of pre-culturing by a non-proteolytic variant

The indirect interaction between proteolytic and lsogenic non-proteolytic strains of Lactococcus lactis was studied by performing sequential cultures in skim milk. After pre-culturing with a non-proteolytic strain for specifie periods of time, the milk was pasteurized, re-inoculated with a second strain and then re-incubated. This second strain was either the same non-proteolytic strain, or the parental proteolytic strain. ln the control milk, bath proteolytic parental and non-proteolytic strains grew exponentially at the same rate. Whereas the non-proteolytic variant stopped growing, its parental strain continued to grow exponentially, but at a lower growth rate than in the first exponential growth phase. When grown in milk pre-cultured by the non-proteolytic strain, the same strain exhibited a slower growth rate and a lower maximum population than when grown in control milk. While abundant growth of the pre-culture strain led to lower maximum populations of the second culture, the combined cell populations in the first and second cultures were constant. Furthermore, this sum was equal to the maximum population attained by the non-proteolytic strain in control milk. When grown in pre-cultured milk, the proteolytic strain exhibited biphasic exponential growth and attained the same maximum population as when cultured in control milk. While the growth rates during the 2 exponential phases were not affected, the shift from the first to the second growth rate occurred earlier in the pre-cultured than in the control milk, with the shift in growth rates occurring earlier in milk containing higher populations of pre-culture organisms. This apparent inhibitory effect of pre-culturing milk with a non-proteolytic variant was suppressed by adding sorne non-proteic nitrogen sources to the milk, thus indicating that the pre-cultured milk was depleted in non-proteic nitrogen sources. The result of this depletion was an indirect competition in milk between isogenic proteolytic and non-proteolytic strains for non-proteic nitrogen. This indirect competition was also observed using 11 other isogenic pairs of proteolytic and non-proteolytic strains belonging to the species L lactis.

Production of Juvenile Atlantic Salmon (Salmo salar) in Two Acidic Rivers of Nova Scotia

Densities, growth, and production of juvenile Atlantic salmon (Salmo salar) were estimated at sites in two acidic rivers of different pH, the Westfield and North rivers, in Nova Scotia, Canada, from 1982 to 1984. Annual production was significantly lower in the more acidic Westfield River (< 0.4 g∙m−2∙yr−1 at pH 4.7–5.4) than in the North River (1.4 g∙m−2∙yr−1 at pH 5.6–6.3). Low production rates in the more acidic river were attributable to the lower densities and poorer survival of juveniles at the lower pH levels. Low densities of parr in the more acidic river were a function of the high initial mortality of postemergent fry, and the mortality of parr during periods of pH minima such as over winter. Marking and recapture information and trapping at a weir for counting migrating fishes indicated that there was little emigration of parr other than in the spring when some movements occurred preceding and during smoltification. Average potential yield of 2-yr smolts was 0.8 smolt/100 m2 in the Westfield River and in the range of 2.7–6.6 smolts/100 m2 at higher pH levels in the North River. Growth was not limiting to production at the lower pH levels. After the first winter, 1-yr-old parr were much larger at the lower pH than they were at less acidic pH levels, the faster growth possibly resulting from the lower density of parr at the lowest pH. A temperature-related biphasic growth of 1-yr-old parr with periods of maximum growth in spring and autumn was recorded. Reduced production, a result of deleterious effects of low pH on survival and density in the Westfield River was probably sufficient to prevent the continued maintenance of a naturally produced salmon population in that river, whereas production at higher pH levels in the North River was comparable to that in near-neutral waters of other areas.

Inhibition of normal rat kidney cell growth by transforming growth factor-β is mediated by collagen

Abstract We have investigated the mechanism of inhibition of the serum-free monolayer growth of normal rat kidney (NRK) cells by transforming growth factor-beta (TGF-beta). NRK cells grown on fibronectin-coated dishes exhibited a biphasic response to TGF-beta. Monolayer growth was slightly stimulated by subpicomolar concentrations, while picomolar concentrations of TGF-beta inhibited NRK cell growth in the presence or absence of epidermal growth factor. NRK cells exhibited a similar biphasic growth response to exogenous type I collagen. TGF-beta induced a 3-5-fold increase in the deposition of type I collagen-like proteins into the extracellular matrix of NRK cells during serum-free growth. Type I collagen-like proteins were identified by their sensitivity to degradation by purified bacterial collagenase and by Western blot analysis. The TGF-beta dose-response curves for induction of extracellular matrix-localized collagen and inhibition of NRK cell growth were similar. Finally, the inclusion of a purified bacterial collagenase, which did not degrade TGF-beta or TGF-beta receptors, or alter control NRK growth, prevented exogenous collagen or TGF-beta from inhibiting the serum-free growth of NRK cells. Our results demonstrate that an increase in collagen secretion plays an important role in the inhibition of the growth of NRK cells by TGF-beta.

Glucose as a substrate in recombinant strain fermentation technology

Glucose supplements to complex growth media of Escherichia coli affect the production of a recombinant model protein under the control of a temperature-sensitive expression system. The bacterial “Crabtree effect”, which occurs in the presence of glucose under aerobic conditions, not only represses the formation of citric acid cycle enzymes, but also represses the formation of the plasmid-encoded product even though the synthesis of this protein is under the control of the temperature-inducible lambda P R-promoter/cl857-repressor expression system. When the recombinant E. coli is grown at a moderate temperature (35° C) with protein hydrolysate and glucose as substrates, a biphasic growth and production pattern is observed. In the first phase, the cells grow with a high specific growth rate, utilizing glucose and forming glutamate as a byproduct. The intracellular level of recombinant protein is very low in this phase. Later, glutamate is consumed, indicating an active citric acid cycle. The degradation of glutamate is accompanied by the intracellular accumulation of high amounts of recombinant protein.

A detailed analysis of Saccharomyces cerevisiae growth kinetics in batch, fed-batch, and hollow-fiber bioreactors

The use of the yeast Saccharomyces cerevisiae has increased greatly over the past few years for the production of pharmaceuticals, specialty chemicals, and other commodities. One reason for this is the many advantages it offers for synthesis and secretion of recombinant DNA products. However, the growth characteristics of this yeast are quite complex and only recently has detailed analysis been available to provide additional insight into such phenomena as biphasic growth and catabolic repression. This paper summarizes and organizes current literature and also details the experimental behavior of on-line and off-line variables of S. cerevisiae in glucose-limited-batch, fed- batch, and hollow-fiber bioreactors. The observed behavior is interpreted using knowledge of yeast catabolism biochemistry and physiology. Special focus is placed on the interpretation of off-gas behavior with respect to TCA enzyme activity and the saturation level of oxygen utilization capacity.

Biphasic intrauterine growth in insulin-dependent diabetic pregnancies.

Early fetal growth delay (7-14 weeks of gestation) has been reported in insulin-dependent diabetic (IDD) pregnancies and in several animal models. Macrosomia is a classic feature of the infant of the IDD mother. We hypothesized therefore that a biphasic pattern of fetal growth exists in IDD pregnancies. We compared fetal growth measurements [biparietal diameter (BPD) and abdominal circumference (AC)] obtained sonographically from 106 IDD pregnancies (Class B-RT) to similar data obtained from 117 normal, nondiabetic patients. The goals for diabetic glycemic control were: fasting blood sugar less than or equal to 100 mg/dl and postprandial blood sugar less than 140 mg/dl. From one to five ultrasonographic measurements were performed at varying gestational ages in all study patients. For data analysis, one examination from each pregnancy was randomly selected by computer. Gestational age (GA) was calculated from last menstrual period and corroborated by infant physical examination (Ballard score) at birth. BPD growth pattern was biphasic in the diabetic group, described by a cubic equation: BPD = 4.99 - 0.567GA + 0.037(GA)2 - 0.0005(GA)3, R2 = 0.935. Such a biphasic pattern did not exist in the control population [BPD = -3.0323 + 0.473(gestation) - (-0.0040)(gestation)2, R2 = 0.9173]. Early growth delay was greater in fetuses that subsequently developed macrosomia (p less than 0.01). Similar results were found for AC measurements. We conclude that fetal growth delay occurs in the first half of the IDD pregnancy, followed by a phase of increased growth. The mechanism of the early growth delay is unclear. We speculate that early growth delay may be due to a "toxic" effect of glucose or other metabolite; and subsequent increased growth relates to fetal hyperinsulinism which develops from weeks 15 to 20 of gestation.