Confluence Of Approaches Research Articles

Regulation of alpha 1 (I)-collagen gene expression in response to cell adhesion in Swiss 3T3 fibroblasts.

Nonadhesive conditions cause Swiss 3T3 fibroblasts to enter a quiescent state that is reversed upon reattachment to a surface. Previously, we demonstrated that adhesion in serum-free conditions is sufficient to activate suspension-arrested cells out of Go, with the induction of the growth-associated genes, c-fos, c-myc, and actin. In this study, we have employed this system to identify programs of gene expression that respond primarily to the adhesive state of the cell, rather than the growth state. We show that cells in different adhesive states can be distinguished by their patterns of protein synthesis. Analysis of one adhesion-responsive protein led to its identification as pro-alpha 1 (I)-collagen. Pro-alpha 1 (I)-collagen synthesis and mRNA levels are decreased up to 6-fold in suspension-arrested fibroblasts, but are enhanced up to 5-fold as cells approach confluence. This suggests that the reduced expression in suspension-arrested cells is not simply a result of quiescence. In addition, reattachment of suspended cells in serum-free conditions caused a 7-fold induction of collagen mRNA levels and a greater than 20-fold rise in the rate of procollagen synthesis. The expression of c-myc was induced during adhesion in serum-free medium as well as by serum addition to suspension-arrested cells. However, alpha 1 (I)-collagen gene expression was unaffected by serum in the absence of adhesion. These results indicate that collagen gene expression is directly responsive to cell adhesion, independent of the growth state.

Diagnostic models for engineering process management: A critical review of objectives, constraints and applicable tools

Abstract Automated and computer-based systems for the dynamic management of complex engineering processes have been the subject of active research in recent years. Many of these systems have adopted methodology and technology from the field of artificial intelligence and expert system research. In real-time process diagnosis and process management applications, where model based reasoning may be highly beneficial in terms of speed and functionality, a larger confluence of methods and approaches has started to form. The objective of these applications often includes, besides performing automated fault analysis and diagnosis, also establishing levels of confidence for the results that are obtained. Thus, both deterministic and uncertainty-based models and reasoning frameworks are often needed. Of interest among the deterministic modeling techniques that can be used in a PMS (Process Management System) are binary trees, rule networks, and graph networks, which in turn include influence diagrams, logic flowgraph methodology and signed directed graph. PMS uncertainty management can be based on formal probabilistic methods, such as Bayesian estimation and updating, or less traditional methods such as certainty factors, Dempster—Schafer theory and fuzzy-set theory. The main features, advantages and disadvantages of the approaches and methods that are suitable for use in a PMS are critically examined and discussed.

SEM characterization of a cellular layer separating blood vessels from endoderm in the quail embryo.

The associations between the developing blood vessels and both endoderm and splanchnic mesoderm in quail embryos at stages 9-11 were examined by using scanning electron microscopy. Embryos were pinned ventral-side up on agar plates and the endoderm was surgically removed prior to fixation and dehydration. This procedure exposes a netlike layer of cells closely apposed to the ventral surface of paraxial mesoderm and all visible blood vessels; we are calling this the subvascular layer. Development of this layer proceeds rostral-to-caudal, and lateral-to-medial, with the earliest stages of formation being visible over the unsegmented paraxial mesoderm of the segmental plate. The subvascular layer increases markedly in density slightly medial to the innermost boundary of the intraembryonic vascular plexus. Cells of this layer eventually establish a continuous sheet beneath the lateral plate and paraxial mesoderm and the notochord. With maturation, the cells of the subvascular layer approach confluence. The spatial and temporal patterns of development of the embryonic vascular tissues and the subvascular layer are closely correlated, suggesting a possible role for the subvascular layer in normal embryonic vascular development.

CAMP levels in proliferating rat mammary epithelial cells in vitro and in vivo

Agents that elevate intracellular cAMP levels are required for growth of many cell types in culture including normal rat mammary epithelial (RME) cells. To determine if the intracellular levels of cAMP that result from stimulation by agents such as cholera toxin (CT) or prostaglandin E-1 (PGE-1) are within the physiological range, cAMP levels were determined in RME cells growing in primary culture and compared to levels measured in freshly isolated mammary epithelium. The results indicate that the cAMP levels of mammary epithelial organoids obtained from 45-day-old virgin rats are 4 to 6 pmol/10 6 cells. Growth of RME cells in primary culture in the presence of CT results in cAMP levels of approximately 15 to 20 pmol/10 6 cells early in culture when cells are proliferating rapidly. As cells approach confluence, cAMP concentrations decrease to levels observed in fresh organoids. CT-stimulated cAMP levels appear to be within the range of those found in pregnant mammary epithelium in vivo. Growth of RME cells in medium supplemented with PGE-1 instead of CT results in cAMP levels equivalent to those found in fresh mammary epithelial organoids and under these conditions the growth rate is approximately half that found in CT-stimulated cells. These results indicate cAMP to be a positive regulator of cell growth in vivo at levels that are within the physiological range.

Rehabilitation: small goals, sustained interventions.

Rehabilitation to restore and maintain functioning in older people is relatively new. Traditionally, ageist prejudices and the medical preoccupation with cure have led to neglect of chronic disabilities in older people. With a focus on an aging population, however, a new approach to rehabilitation is taking shape at the confluence of geriatric and psychosocial approaches. Significant components of this emerging approach are: setting small goals as incentives for motivating the patient, the timing of the disability within the individual's life course, coping behavior and the way the disability is defined, the importance of social support to the recovery potential, and the incorporation of sustained interventions into daily routines and life-styles. This article illustrates this approach with examples from common disabling conditions, proposing that the quality of aging can be markedly enhanced for older people suffering from functional disability.

Serum deprivation of vascular smooth muscle cells enhances collagen gene expression.

Vascular smooth muscle cells produce the bulk of connective tissue protein in major arteries. Previously, we demonstrated that collagen synthesis by cultured bovine aortic smooth muscle cells in exponential growth is low and increases as cells approach confluence and growth slows. To evaluate more directly the effects of the proliferative state, subconfluent cultures of smooth muscle cells were incubated in medium supplemented with 0.5% fetal calf serum; under these conditions of growth factor deprivation, cells become quiescent within 72 h. The mRNA levels for type I and III collagens increased markedly within 12 h; at 48 h, they were between 2- and 15-fold higher than in cells growing exponentially. Upon addition of serum to quiescent cultures, cells re-entered the cycle, and collagen mRNA levels decreased. Expression of alpha 1(I), alpha 2(I), and alpha 1(III) mRNAs differed significantly, indicating differential gene regulation. In nuclear run-off assays, changes detected in collagen transcription were insufficient to account for changes in mRNA levels, suggesting additional post-transcriptional sites of control. The inverse response of collagen expression to the proliferative state suggests that exposure of smooth muscle cells in vivo to factors which influence their growth state could have profound effects on the structure of the arterial wall.

Possible role of c- fos, c-N- ras and c- mos proto-oncogenes in muscular development

Time course analyses of various proto-oncogene transcripts compared with cytoskeleton-specific and muscle-specific messenger RNAs (mRNAs) were carried out during growth and differentiation of a clonal line of rat myoblasts that retain the capacity to form non-contractile fibres in vitro. Throughout their growth phase, these cells express consistent levels of c- fos, c- myc, c-Ki- ras and c-N- ras RNA and no c- mos RNA. When the cultures approach confluency the level of c- fos RNA rises sharply 3–4-fold, peaks, and rapidly declines when muscle-specific transcripts start accumulating, to become negligible in myotube-forming cells. These changes occur whatever the concentration in seric factors. By contrast, the level of c-N- ras RNA rises up to 3-fold and both c- myc and c-Ki- ras RNAs are slowly eliminated during the myogenic process, whereas no c- mos RNA is detectable. However, skeletal muscles from prenatal fetuses and adult animals were reproducibly found to contain both low and high levels of c- mos RNA respectively. These data and the demonstration that inactivation of the c- fos gene correlates with the loss of myogenic capability in six lines of neoplastic myoblasts, including four lines transformed by the v- fos oncogene, suggest a physiological function for this proto-oncogene during early stages of myogenesis and for the c-N- ras and c- mos genes in later stages of muscular development.

Structure and expression of murine malic enzyme mRNA. Differentiation-dependent accumulation of two forms of malic enzyme mRNA in 3T3-L1 cells.

Many murine cells express two mRNAs with markedly different sizes (2.0 and 3.1 kilobases (kb)) that hybridize with cDNA probes for cytosolic malic enzyme ((S)-malate NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40). A series of overlapping cDNA clones corresponding to 3129 nucleotides of malic enzyme mRNA was isolated and sequenced to determine the relationship between the two mRNAs and establish the primary structure of mouse malic enzyme. The larger mRNA has an open reading frame of 1716 nucleotides followed by a 3' untranslated region of 1348 nucleotides. The sequence of an exceptionally G/C-rich (88%) portion (65 nucleotides) of the 5' noncoding region was also established. An uncommon poly A addition signal (AUUAAA) is used during the processing of the 3.1-kb mRNA. The 2.0-kb mRNA results from the utilization of another poly A addition signal that truncates the 3' noncoding sequence by approximately 1 kb. The mRNA coding sequence indicates that the malic enzyme subunit contains 572 amino acid residues and has a Mr of 64,000. Two putative components of an NADP-binding domain are located between residues 100 and 165. During the differentiation of 3T3-L1 preadipocytes into adipocytes both the rate of synthesis and relative mRNA concentration for malic enzyme and another lipogenic enzyme, ATP-citrate lyase, are coordinately increased 5-7-fold. However, as preadipocytes approach confluence, the mRNA levels for both lipogenic enzymes transiently increase 3-4-fold, whereas the rates of synthesis of the two proteins are only slightly elevated. Thus, lipogenic enzyme expression is controlled at a pretranslational level during adipogenesis, but the accumulation of the same enzymes may also be subject to translational control in the fibroblast-like preadipocytes. In contrast, mRNA coding for a third enzyme required for lipogenesis, glycerol-3-phosphate dehydrogenase, is not detected in 3T3-L1 preadipocytes, but rapidly accumulates during adipocyte development.

The regulation of proteolysis in normal fibroblasts as they approach confluence. Evidence for the participation of the lysosomal system.

The effect of the lysosomotropic agent NH4Cl and the proteinase inhibitors leupeptin, Z-Phe-Ala-CHN2 (benzyloxycarbonylphenylalanylalanyldiazomethane) and pepstatin on the degradation of intracellular proteins in Swiss 3T3 mouse and normal human fibroblasts in both the exponential and stationary (confluent) growth phases in nutritionally complete conditions was investigated. Inhibitory effects of all four agents on degradation in both growth states were detected. The increase in proteolysis normally occurring as cells approach confluence could be completely blocked by NH4Cl, by Z-Phe-Ala-CHN2, or by pepstatin in the presence of leupeptin. These results suggest that the lysosomal system is responsible for the regulation of proteolysis at confluence and further confirm its role in 'basal' proteolysis in growing cells.

Prostaglandin production by 3T3-L1 cells in culture

Rapidly growing cultures of 3T3-L1 preadipocytes produce large quantities of prostaglandins when they are either stimulated with the calcium ionophore A23187 or incubated with arachidonic acid. The main prostaglandin produced under all conditions was prostaglandin E 2. Prostaglandin production in response to ionophore stimulation or incubation with arachidonic acid decreased markedly, however, as the cultures approached confluence, were maintained in the confluent state, or were stimulated to differentiate. Enrichment of confluent, differentiated cultures with arachidonic acid did not enhance prostaglandin production. Recovery of prostaglandin production occurred when logarithmic growth was reinstituted by reseeding confluent cultures at low cell densities, but sparse cultures maintained in a low-growth phase did not recover the ability to produce large amounts of prostaglandin E 2. Therefore, the decline in prostaglandin synthetic capacity appears to be associated with the decrease in growth rate as the cells approach confluence. Media conditioned by confluent cells reduced prostaglandin E 2 production when added to rapidly growing cells, suggesting that an inhibitor of prostaglandin synthesis may be formed by the confluent cultures. Nondifferentiating 3T3 fibroblasts, which similarly release mainly prostaglandin E 2, also exhibited a decrease in prostaglandin production as the cultures became confluent. The amounts of prostaglandins produced by 3T3 cells in the confluent state were much greater, however, than those produced by confluent or differentiated 3T3-L1 cultures. These findings suggest that the low capacity to produce prostaglandins may be involved in either the induction or maintenance of differentiation in 3T3-L1 cells.

Towards a Confluence of Quantitative and Qualitative Approaches to Curriculum Evaluation

(1980). Towards a Confluence of Quantitative and Qualitative Approaches to Curriculum Evaluation. Journal of Curriculum Studies: Vol. 12, No. 4, pp. 367-370.

Depletion of cellular glycogen during the early logarithmic growth phase of human fibroblasts

Human diploid fibroblasts deplete 50% of their cellular glycogen by day 4 after subcultivation in 100 mg% glucose medium. The glycogen content increases again as the cells approach confluency. Growth of cells in low glucose medium results in rapid glycogen depletion and indicates that stored glycogen has a limited potential as an energy source.

The role of adenosine 3′, 5′‐cyclic monophosphate in the density‐dependent regulation of growth and tyrosinase activity of b‐16 melanoma cells

Incubation of cultured B-16 melanoma cells with 1-methyl-3-isobutyl xanthine (MIX) produced a sustained rise in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) which preceded an increase in the specific activity of tyrosinase (EC 1.10.3.1). Cultures of two clones of melanoma cells, one having a mean population doubling time twice that of the other, showed density-dependent inhibition of growth. The tyrosinase activity of each line increased progressively during logarithmic growth, reaching maximal values shortly after the cultures achieved confluence. Intracellular cAMP levels fell during logarithmic growth, being minimal in confluent cultures. The stimulatory effects of MIX and confluence on tyrosinase activity were additive. Cells plated at high density had a lower tyrosinase activity than cells allowed to achieve a similar density by successive division from sparsely planted cultures although the intracellular cAMP levels of such cultures were not different. We support the observations of other investigators that agents which increase intracellular cAMP concentrations can both inhibit cell division and stimulate tyrosinase activity. There are, however, mechanisms for increasing tyrosinase activity and inhibiting cell division which are expressed as B-16 melanoma cells approach confluence and which are not mediated by an increase in intracellular cAMP concentrations.

Isolation and characteristics of HeLa surface proteins

HeLa 71 and 65 cells grown in attached culture possess a coat of extracellular proteins that can be released from the cell by mild EDTA-detachment, with no significant effect on cellular integrity. This suggests that these surface proteins are weakly associated with the cell, possibly through divalent cations. The high affinity of surface proteins for critical divalent cations, shown by their high precipitability by Zn 2+, Ca 2+ and Mg 2+, supports this assumption. Since surface proteins appear to be phosphoproteins, as suggested by significant incorporation of 32Pi in vitro, it is possible that binding occurs through M 2+− PO = 4· The amount of surface protein on HeLa 65 cells grown in suspension culture is greatly reduced compared with cells grown in monolayer culture. This may be related to impaired availability of Ca 2+ in suspension culture medium. In monolayer grown HeLa cells surface proteins are predominantly distributed underneath the cells. The highest amount of these proteins is found on cells prior to growth initiation and steadily decreases as cells approach confluency. As shown by radioactive leucine protein labeling, surface proteins are primarily comprised of proteins synthesized within HeLa cells and released to the outer cell surface. The presence of serum proteins in surface protein matrix is physiologically significant.

Insulin receptors in 3T3 fibroblasts: Relationship to growth phase, transformation and differentiation into new cell types

The amount of [ 125I]insulin binding per 2 × 10 6 cells is measured in three lines of mouse embryonic 3T3 fibroblast at different growth stages. Insulin binding is found to be lowest in growing cells of all three types, increasing as cells reach stationary phase. Binding in 3T3-M cells approaches a plateau as cells become stationary. Insulin binding in 3T3-L cells, many of which differentiate into adipocytes following cessation of growth, show further increase in insulin binding post-confluence, in parallel with their differentiation into adipocytes. Binding of insulin in spontaneously transformed cells is higher at all phases of growth than the other two lines, rising to a much higher eventual plateau at approx. 17 days post-confluence. Scatchard plots of insulin binding tend to reflect the same degree of relative insulin binding in these three cell lines. Previously starved cells of all three types exhibit a drop in insulin binding following their first feeding, which corresponds with a second growth spurt in response to nutrients in fresh serum. These results suggest that insulin, as reflected by binding per cell, may play only a minor role in actively growing adequately fed cells of all three types, its major role developing as these cells approach confluence. It is also suggested that higher insulin binding in transformed vs non-transformed cells may indicate a special role for insulin in the loss of contact inhibition, by preserving transport of limiting nutrients in dense, nutrient-depleted transformed cultures.

Regulation of hypoxanthine transport by cyclic nucleotides in cultured Chinese hamster lung fibroblasts

The regulation of hypoxanthine transport activity by Chinese hamster lung fibroblasts grown in culture was examined in wild-type clones and 8-azaguanine-resistant mutant clones which lack hypoxanthine-guanine phosphoribosyltransferase. Hypoxanthine transport activity increases with increased rates of cellular growth expressed as viable cell number, total cell protein, and DNA synthesis. The transport activity for hypoxanthine declines when the fibroblasts approach confluence or after exposure to cycloheximide or actinomycin D. In vivo incubation of either fibroblast subline with 100 μ m dibutyryl cyclic AMP decreases transport activity over 50%, whereas exposure to 10 μ m dibutyryl cyclic GMP increases hypoxanthine uptake by 40%. A synergistic effect is observed when fibroblasts are incubated with a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine or theophylline) plus glucagon, an adenylate cyclase stimulator. Such additions result in a 70% decrease in the cellular transport capacity. Stimulation of hypoxanthine transport by 40% is observed following incubation with insulin. Addition of all agents produces maximum changes in the rate of hypoxanthine transport only after a 6-h in vivo incubation with the fibroblasts. These findings suggest that hypoxanthine transport is regulated by the intracellular concentration of cyclic nucleotides. This control may occur at the level of gene expression for a hypoxanthine transport protein.

Regulation of amino acid transport activity and growth rate of animal cells in culture.

Balb/3T3 mouse cells grown in cell culture contain the A and L systems for neutral amino acid transport. L-Alanine was used as a test amino acid for the A system and L-leucine for the L system. Transport activities for both transport systems are altered as the growth conditions for the cells are altered. When cell growth was arrested by allowing cells to approach confluency, or by the removal of serum or by the addition of protein synthesis inhibitors, System A transport activity decreased and System L activity increased significantly. The content of the cellular amino acids increased 2- to 3-fold whenever cell growth was arrested by the conditions stated above. Increased cellular amino acids, therefore, are associated with decreased transport activity of System A and increased activity of System L. Altering cellular amino acids by loading or depleting the cells produced the predicted responses for the rapidly exchanging L-system but System A was less responsive. It is possible that regulatory mechanisms operate for the A system other than the direct action of alterations in cellular amino acid levels. The implications of the amino acid transport activity changes observed here on various studies on the role of transport in cell growth regulation and malignant transformation are discussed.

Contact inhibition of growth is restored to malignant melanocytes of man and mouse by a hamster protein

Contact inhibition of growth, an in vitro property whose loss has been correlated with increasing tumorigenicity in vivo, is restored to hamster malignant melanocytes by a glycoprotein previously isolated from culture medium of a contact-inhibited hamster melanocytic cell line. Such growth inhibition is characterized by changes in cultures from a disoriented, multilayered, pleomorphic morphology to well oriented monolayers of fibroblast-like cells, with significant reduction in saturation densities. The present studies show that: 1. 1. The morphologic and growth inhibitory effects transcend species barriers, occurring also in malignant melanoma cell lines of human and murine origin. 2. 2. Arrest of growth occurs in the G1 (G0) phase of the cell cycle, as in the classical case of diploid fibroblasts. 3. 3. Cessation of growth is not due to depletion of essential constituents of the growth medium, but occurs only as cells approach confluence. The melanocyte contact inhibitory factor (MCIF) may be the prototype for a class of surface-associated proteins concerned with regulation of normal cell-cell interactions leading to feedback inhibition of growth.

Plasma membrane glycoprotein which mediates adhesion of fibroblasts to collagen.

DEFECTS in cell behaviour associated with malignancy may be causally related to alterations in the plasma membrane of the cell on transformation1. Much interest has focused on a protein found on the surface of normal but not transformed cells 2–7. This protein, designated SF (ref. 5), LETS (ref. 8), band 1 (ref. 9), L1 (ref. 3) or CSP (ref. 10), is a glycoprotein7,9,11 with a molecular weight of 210,000–250,000 (refs 7, 8 and 12). It is present in the serum12 and is sensitive to removal from the surface of normal fibroblasts by low levels of trypsin2,3,9. Several biological properties of this protein have been described. Normal fibroblasts arrested in mitosis lack this protein8,9. As normal cells approach confluency, there is a quantitative increase in the amount of this protein present on the cell surface8,9, whereas confluent normal cells, stimulated by the addition of serum, have lowered levels8. In conflict with these results, implying a growth regulatory function, is the finding that stimulation of growth-arrested normal cells by thrombin leads to thymidine incorporation without a concomitant decrease in the levels of this protein13. We now present evidence which points to a functional role for the protein as a mediator of the adhesion of fibroblasts to collagen. The protein is probably identical to a serum factor previously reported to be necessary for the attachment of SV40-transformed 3T3 fibroblasts to collagen-coated dishes14. Based on this functional role, we propose the name cell adhesion factor (CAF) for this protein.

Cooperative cell interactions in pure and mixed culture

The population growth and cell morphology of mouse A9s and human foreskin fibroblasts (HFFs) were used to investigate cooperative cell interactions in pure and mixed culture. It was found that neither cell line employed the bacterial growth optimization strategy whereby the lag phase is used to build up stockpiles of materials which serve as bottlenecks to limit growth. Instead, a strategy is employed which requires colony formation and cooperative cell interaction. The behavior of cell growth rates as a function of population density and time after seeding is more complicated than generally appreciated. At sparse density, shortly after seeding, there is a period of growth rate acceleration which is dependent upon population density. As moderate densities are achieved, this acceleration ceases and the growth rate levels off at a high value. As the cultures approach confluency, contact inhibition of growth occurs, and the growth rate decelerates to a lower but non-zero value. During non-confluency the growth rate of cultures is inversely proportional to the percent of singlets (individual cells not in contact with other cells) within a population. Moreover, there is a hysteresis-like effect in that the growth properties of cells depend not only upon momentary conditions, but upon the prior history of cell interactions as well. These effects are not the result of medium conditioning. Instead, they appear to involve either direct cell contact or at least the very close proximity of cells. During non-confluency the growth kinetics of A9s and HFFs in mixed culture are independent of one another. At near confluency, the A9s unilaterally inhibit further HFF proliferation, but themselves continue to reproduce past confluency by invading a second niche—the culture fluid overlying the mixed monolayer. Inhibition of HFF growth by A9s involves territorial exclusion, whereby HFFs are excluded from local areas heavily (but non-confluently) populated by A9s. Locally confluent domains of A9s expand by proliferation, and apparently cause nearby HFFs to retreat before them. As this encroachment progresses, HFFs seem to be herded together by the A9s into ribbon-shaped enclaves which cease proliferation but pile up into multi-layers.