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Biomedical subjects

J Campisi

Publications and source records attributed to J Campisi.

At least 91 records · Page 5Linked to original sources
PubMed1984

Defective Ca2+ metabolism in Duchenne muscular dystrophy: effects on cellular and viral growth.

Normal fibroblasts in medium containing 0.02 mM CaCl2 arrested growth within 24 hr, whereas Duchenne muscular dystrophy fibroblasts continued to grow for 5 days, albeit at 40% of their rate in standard medium (1.8 mM CaCl2). Moreover, Duchenne cells in calcium-deficient medium showed an enhanced rate of protein synthesis (60% over the rate in standard medium), whereas normal cells were unaffected. Previously we described a general assay for detection of mutant cells by using herpes simplex virus I replication as a probe of cellular function. By altering the growth medium, one can elicit changes in viral DNA replication that depend upon cellular differences. Duchenne fibroblasts in calcium-deficient low-serum (0.5%) medium supported viral replication at a rate 7- to 10-fold greater than did normal cells infected under the same conditions. Using this viral assay, we have successfully identified all 10 samples of a blind coded set of Duchenne muscular dystrophy, normal, and heterozygote cells. In addition, differences of a lower magnitude were found between these cell strains as measured by cellular growth or protein synthesis. Therefore, a cell's ability to grow and support viral replication in calcium-deficient medium can be used to readily distinguish Duchenne muscular dystrophy fibroblasts from normal ones. These results suggest that the viral assay could be used as a prenatal diagnostic test. A defect related to calcium metabolism may be fundamental to this disease.

Calcium↗
PubMed1984

Post-transcriptional control of the onset of DNA synthesis by an insulin-like growth factor.

The control of eucaryotic cell proliferation is governed largely by a series of regulatory events which occur in the G1 phase of the cell cycle. When stimulated to proliferate, quiescent (G0) 3T3 fibroblasts require transcription, rapid translation, and three growth factors for the growth state transition. We examined exponentially growing 3T3 cells to relate the requirements for G1 transit to those necessary for the transition from the G0 to the S phase. Cycling cells in the G1 phase required transcription, rapid translation, and a single growth factor (insulin-like growth factor [IGF] I) to initiate DNA synthesis. IGF I acted post-transcriptionally at a late G1 step. All cells in the G1 phase entered the S phase on schedule if either insulin (hyperphysiological concentration) or IGF I (subnanomolar concentration) was provided as the sole growth factor. In medium lacking all growth factors, only cells within 2 to 3 h of the S phase were able to initiate DNA synthesis. Similarly, cells within 2 to 3 h of the S phase were less dependent on transcription and translation for entry into the S phase. Cells responded very differently to inhibited translation than to growth factor deprivation. Cells in the early and mid-G1 phases did not progress toward the S phase during transcriptional or translational inhibition, and during translational inhibition they actually regressed from the S phase. In the absence of growth factors, however, these cells continued progressing toward the S phase, but still required IGF at a terminal step before initiating DNA synthesis. We conclude that a suboptimal condition causes cells to either progress or regress in the cell cycle rather than freezing them at their initial position. By using synchronized cultures, we also show that in contrast to earlier events, this final, IGF-dependent step did not require new transcription. This result is in contrast to findings that other growth factors induce new transcription. We examined the requirements for G1 transit by using a chemically transformed 3T3 cell line (BPA31 cells) which has lost some but not all ability to regulate its growth. Early- and mid-G1-phase BPA31 cells required transcription and translation to initiate DNA synthesis, although they did not regress from the S phase during translational inhibition. However, these cells did not need IGF for entry into the S phase.

Animals↗
PubMed1984

Cellular oncogenes, growth factors, and cellular growth control.

In this article we relate the functioning of oncogenes, particularly myc and ras, to current ideas regarding regulation of mammalian cell growth by growth factors. Assuming the genetic basis of transformation to be alterations of several proto-oncogenes, the mechanisms by which transformation could diminish growth control are numerous. The oncogene could interact with a growth factor in several ways. Mutations could alter the quantity of an oncogene's product or its quality through primary structure or covalent modifications such as phosphorylations. Oncogenes could code for a receptor for a growth factor. Various alterations parallel to the above set could then affect growth factor function via receptor changes (including abolished requirement for the factor). Some oncogenes might operate during the chain of intracellular events that must follow growth stimulation. Introduction of such an oncogene (e.g., the coding region for a DNA virus T antigen) would bypass requirements for both growth factors and receptors. Various observations regarding the oncogenes, cell cycle-timed events, and growth factors have been presented in a way we hope will suggest experiments designed to provide a basis for understanding growth regulation at the genetic, biochemical, and cellular levels in normal cells and tumorigenic cells, which have deranged growth regulation.

Cell Cycle↗
PubMed1983

The study of DNA-repair defects using [125I]iododeoxycytidine incorporation as an assay for the growth of herpes simplex virus.

[125I]Iododeoxycytidine incorporation was used to measure herpes virus (HSV-1) DNA synthesis following specific DNA damage. Xeroderma pigmentosum fibroblasts were less able to replicate UV-irradiated viral DNA than were normal fibroblasts, indicating the necessity for excision repair for the survival of UV-irradiated virus. Because of its rapidity and ease of quantitation, this assay had advantages over standard viral mediated assays of DNA excision repair. It was possible to monitor viral replication as a function of the cellular cell cycle. Other genetic defects which have been proposed to reflect deficiencies in DNA-repair capacity were not detected by this assay. DNA-repair inhibitors, caffeine and 3-aminobenzamide, also did not show synergistic lethal effects on the replication of damaged viral DNA.

Ataxia Telangiectasia↗
PubMed1982

Differences in growth regulation of normal and tumor cells.

Normal cells cannot initiate DNA synthesis under inadequate external conditions, yet after growth has started they complete their division cycle under these conditions. The sensitive biochemical event for a growing cell is proposed to be accumulation of a labile protein which in adequate amounts permits entry into S phase, after about 2 hr, and completion of the cycle. Instability of this protein (half-life about 2.5 hr) creates a dynamic state so that its accumulation depends on rates of both synthesis and degradation. Neoplastic cells may show poorly regulated growth either by synthesizing this protein more rapidly or degrading it less rapidly, under conditions that limit normal cells' growth. Known mechanisms of overproduction include: more copies of the protein's structural gene per cell, an adjacent high-activity promoter, or autoproduction of growth factors. Less rapid degradation could result from less protease activity or from stabilizing modifications of the protein. Thus, derangement in the control of a labile growth-regulatory protein acting by any one of these diverse mechanisms could lead to neoplasia.

Animals↗
PubMed1982

Restriction point control of cell growth by a labile protein: evidence for increased stability in transformed cells.

It has been proposed that animal cells must accumulate a labile protein(s) before they can pass the restriction (R) point in the G1 phase of the cell cycle [Rossow, P. W., Riddle, V. G. H. & Pardee, A. B. (1979) Proc. Natl. Acad. Sci. USA 76, 4446--4450]. Here, we present evidence that this R protein acquires increased stability in transformed 3T3 cells, thereby allowing these cells to continue growth under conditions that arrest untransformed cells. Low doses of cycloheximide or histidinol drastically reduced the rate at which normal 3T3 (A31) fibroblasts in early G1 could enter DNA synthesis. These drugs had less effect on entry of two tumorigenic A31 derivatives, BPA31 and SVA31, in S, although measurement of [3H]leucine incorporation showed that the inhibitors were equally effective in the three cell lines. The hypothesis is that the transformed lines are less sensitive because moderate inhibition of their R protein synthesis is compensated by lower rates of protein degradation. To test this idea, we completely inhibited cytoplasmic protein synthesis for several hours shortly before A31 and BPA31 cells had reached the R point. After removal of inhibitor, A31 cells showed delays in the onset of S that were in excess of the inhibitor pulse, consistent with decay of labile protein during the pulse. BPA31 cells showed no excess delays, suggesting a much more stable R protein. The half-life of the R protein was estimated as 2.5 hr in A31 cells, indicating that, in these cells, R protein synthesis starts at the beginning of G1. In the BPA31 cells the R protein showed no signs of decay for at least 8 hr.

Animals↗
PubMed1980

Calcium-induced decrease in membrane fluidity of sea urchin egg cortex after fertilization.

Fertilization of the sea urchin egg is a dramatic example of cell activation resulting from the interaction of an external stimulus, the spermatozoon, with the cell surface. Growing and quiescent cells may have different membrane states. Here we report membrne fluidity measurements on a surface membrane fraction, the cortex, isolated from unfertilized and fertilized eggs. The fluidity of the fertilized egg cortex, measured by electron spin resonance (ESR) spectroscopy using 5-doxylstearate as a probe, is less than that of the unfertilized cortex. In the intact egg the intracellular CA2+ to the cortex fraction isolated from unfertilized eggs triggers a fluidity decrease in vitro. The fluidity decrease seems to represent a Ca2+-induced change in membrane structure rather than a direct interaction of Ca2+ with phospholipid headgroups.

Animals↗
PubMed1978

Fertilization-induced changes in membrane fluidity of sea urchin eggs.

By use of a spin label fatty acid, 5-doxylstearate, an increase in bulk membrane fluidity was observed after fertilization of two species of sea urchin eggs. Eggs partially activated by ammonia showed a similar effect. The data suggest that a structural change involving membrane lipids accompanies activation.

Ammonia↗
PubMed1976

Novel mutation that causes a structural change in a lipoprotein in the outer membrane of Escherichia coli.

A novel mutation which caused a structural change in a lipoprotein in the outer-membrane has been found in Escherichia coli K-12. The lipoprotein of the wild-type strain is known to have a peculiar amino terminal structure: glycerylcysteine with two fatty acids attached by ester linkages and one fatty acid by an amide linkage. In contrast to the wild-type lipoprotein, the mutant lipoproteins is isolated from the E. coli envelope as a dimer of molecular weight of about 15,000. The dimer can be reduced by mercaptoethanol to the lipoprotein monomer of molecular weight of about 7,500. The monomer has a free thiol group which is susceptible to monoiodacetie mutant lipoprotein is extremely low in comparison with that into the wild-type lipoprotein. These results suggest that the mutant is defective in transferring a glycerol group to the thiol group of the amino terminal cysteine residue of the lipoprotein. The gene responsible for this modification reaction has been located at 36.5 min on the E. coli chromosome.

Bacterial Proteins↗