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Biomedical subjects

F G Toback

Publications and source records attributed to F G Toback.

At least 37 records · Page 2Linked to original sources
PubMed1994

Glyceraldehyde-3-phosphate dehydrogenase modifier protein is associated with microtubules in kidney epithelial cells.

After exposure of monkey kidney epithelial cells to a reduced concentration of K, a known mitogenic signal, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD) is activated by a cytosolic protein whose function appears to be novel. A monospecific antibody was used as an immunoprobe to study the contribution of this G3PD modifier protein (MP) to signal transduction. Raising the extracellular Na concentration as well as lowering the K concentration of the medium increased the amount of MP in cytosol and also activated G3PD. Metabolic labeling of cells followed by preparation of detergent-soluble (cytosolic) and detergent-resistant (cytoskeletal) fractions, immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radiolabeled immune precipitates suggested that the protein was also associated with cytoskeleton. Depolymerization of the microtubules with colchicine or nocodazole increased cytosolic immunoreactive MP, whereas cytochalasin D had no effect. Taxol, which stabilizes microtubules, blocked the effects of colchicine or nocodazole. When tubulin, actin, and intermediate filament fractions of the cytoskeleton were prepared, blotted, and probed with specific antibodies, MP was found in the tubulin fraction. These observations suggest that MP is associated with the microtubules and can be displaced into the cytosol, wherein it could activate G3PD and thereby stimulate glycolytic production of ATP during mitogenic signal transduction.

Animals↗
PubMed1993

Regulation of renal epithelial cell endocytosis of calcium oxalate monohydrate crystals.

The earliest events in the formation of kidney stones are unknown. The most common crystal in kidney stones, calcium oxalate monohydrate (COM), when added to cultures of monkey kidney epithelial cells (BSC-1 line), was internalized by 19% of the cells after 30 min. COM crystal endocytosis was enhanced by serum, ADP, and epidermal growth factor, which are potent mitogens for these cells. Endocytosis of COM crystals was inhibited by diverse molecules including Tamm-Horsfall glycoprotein (THP), the tetrapeptide arginine-glycine-aspartic acid-serine, fibronectin, transforming growth factor-beta 2, and heparin. The capacity of THP, fibronectin, or heparin to inhibit endocytosis was mediated by an interaction of these molecules with cells, not by coating the crystals. Thus renal epithelial cell endocytosis of COM crystals can be regulated by diverse molecules including THP, the most common protein found in human urine. Crystal endocytosis and subsequent cellular responses could be important pathogenic steps in nephrolithiasis.

Animals↗
PubMed1993

Autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is an important cause of medical morbidity in the United States that affects one-half million persons and accounts for ESRD in about 10% of the chronic dialysis population. In addition to its effects on the kidney, the disease has important manifestations in the cardiovascular system (aneurysms, hypertension) and the gastrointestinal tract (hepatic cysts). Clinically important renal complications can develop as the disease progresses that require specialized attention, such as urinary tract infection, pain, and nephrolithiasis. The underlying cellular defect that causes ADPKD has eluded investigators thus far, but abnormalities in cellular proliferation, the tubular basement membrane, and cell fluid secretion appear important in pathogenesis. Factors that mediate progressive interstitial fibrosis and failure of renal function are undefined, although rigorous control of blood pressure appears to be an important therapeutic measure. Recent advances in molecular biology have localized the abnormal gene to chromosome 16 in 90% of families, making early genetic screening of asymptomatic family members possible in many cases. A positive diagnosis may have important effects on employment status, as well as health insurance, so that family members sometimes refuse to be assessed for the presence of the disease. Because of such complex social factors, counseling of an asymptomatic individual by his or her physician is required when considering the use of screening tests for ADPKD. Inadequate patient education may still represent an impediment to early detection, genetic counseling, and timely treatment of disease complications.

Female↗
PubMed1992

Cytokeratin reorganization induced by adenosine diphosphate in kidney epithelial cells.

Exogenous adenosine diphosphate (ADP), the most potent mitogen for nontransformed African green monkey kidney epithelial cells of the BSC-1 line, rapidly alters the appearance of the cell monolayer. Examination of the cells with indirect immunofluorescence using monoclonal antibodies reveals a considerable reorganization of cytokeratin filaments without a major change in the pattern of microtubules or microfilaments. In untreated confluent cells, cytokeratin filaments are predominantly confined to a star-like spot in the perinuclear area, but these can be seen to begin to spread within 2 min after addition of ADP. The effect is particularly notable using anti-cytokeratin 8 antibodies. At 6 h this process is complete and produces a well-developed filamentous network throughout the cell. By 12 h, the network appears to collapse, so that the filaments again form a spot in the perinuclear area, a process that is complete by 24 h. Immunoblotting of total cellular proteins reveals no apparent alterations in the amounts of several species of cytokeratins, including cytokeratin 8 and 18, at 3 or 24 h after exposure to ADP. Other purine and pyrimidine nucleotides which do not stimulate DNA synthesis in these cells fail to alter cytokeratin organization, and there is no apparent alteration in the distribution of vimentin, another intermediate filament protein. The rapid ADP-induced cytokeratin reorganization appears to coincide with the induction of early growth-response gene transcription in these cells and may be correlated with the capacity of ADP to subsequently initiate DNA synthesis. This dramatic and reversible cytokeratin reorganization immediately after exposure to ADP may be an important step in the mitogenic signal transduction pathway.

Adenosine Diphosphate↗
PubMed1992

Endocytosis of calcium oxalate crystals and proliferation of renal tubular epithelial cells in a patient with type 1 primary hyperoxaluria.

A patient with primary hyperoxaluria who received a liver-kidney transplant is presented. A postoperative renal biopsy showed apparent tubular cell endocytosis of calcium oxalate crystals and cell proliferation, indicating that renal epithelial cells do not perceive urinary crystals as inert. Such cellular responses to crystals may have a role in nephrolithiasis.

Adult↗
PubMed1992

Calcium oxalate monohydrate crystals are endocytosed by renal epithelial cells and induce proliferation.

Monkey kidney epithelial cells of the nontransformed BSC-1 line were used as a model system with which to search for biological responses to urinary crystals commonly found in renal stones. Calcium oxalate monohydrate (COM), the most common urinary crystal, was avidly internalized, initiated DNA synthesis, and stimulated cell multiplication. The increase in DNA synthesis observed after exposure to COM crystals was equivalent in magnitude to that of 10% calf serum, but occurred 8 h later. Maximal stimulation of DNA synthesis by COM was associated with crystal endocytosis by 50% of the cell monolayer. COM crystals also stimulated DNA synthesis and multiplication of canine kidney epithelial cells (MDCK line). As COM stimulated growth of both monkey and canine renal cells but not fibroblasts, the mitogenic effect of this crystal appeared cell-type specific. Hydroxyapatite also enhanced multiplication of BSC-1 cells, whereas brushite, another calcium-containing urinary crystal, did not. In the presence of nephrocalcin (NC), a glycoprotein in normal human urine that inhibits nucleation, aggregation, and growth of COM crystals, the capacity of these crystals to initiate DNA synthesis was blocked. This is the first demonstration that specific calcium-containing urinary crystals can induce proliferation of renal epithelial cells and that NC can inhibit this effect.

Animals↗
PubMed1992

Phenol red inhibits growth of renal epithelial cells.

Phenol red coeluted with a novel kidney cell growth factor during its isolation from conditioned medium by high-performance liquid chromatography. The possibility that phenol red rather than the putative factor mediated the growth-promoting activity was tested, because this pH indicator is known to possess estrogenic properties including a mitogenic effect. Unexpectedly, phenol red at the concentration found in Dulbecco's modified Eagle's medium (DMEM) inhibited growth of monkey (BSC-1) and canine kidney (MDCK) epithelial cells by 20-30%, but stimulated multiplication of 3T3 fibroblasts. The growth-inhibitory effect of phenol red for BSC-1 cells was reversible, concentration dependent, and cell-density independent when multiplication was examined in the presence and absence of the dye. Phenol red partially masked the mitogenic effect of calf serum and epidermal growth factor and was additive to the growth-inhibitory effect of transforming growth factor-beta 2. These observations indicate that phenol red at the concentration in DMEM both underestimates the potency of mitogens and overestimates the strength of an inhibitor of kidney epithelial cell growth and suggest that the dye be omitted from the culture medium when a new growth-regulatory compound is under study.

Animals↗
PubMed1992

Adenine nucleotides stimulate migration in wounded cultures of kidney epithelial cells.

Adenine nucleotides speed structural and functional recovery when administered after experimental renal injury in the rat and stimulate proliferation of kidney epithelial cells. As cell migration is a component of renal regeneration after acute tubular necrosis, we have used an in vitro model of wound healing to study this process. High density, quiescent monkey kidney epithelial cultures were wounded by mechanically scraping away defined regions of the monolayer to simulate the effect of cell loss after tubular necrosis and the number of cells that migrated into the denuded area was counted. Migration was independent of cell proliferation. Provision of adenosine, adenine nucleotides, or cyclic AMP increased the number of migrating cells and accelerated repair of the wound. Other purine and pyrimidine nucleotides were not effective. Arginine-glycine-aspartic acid-serine peptide, which blocks the binding of extracellular fibronectin to its cell surface receptor, completely inhibited migration in the presence or absence of ADP. Very low concentrations of epidermal growth factor (K0.5 approximately 0.3 ng/ml) stimulated migration, whereas transforming growth factor-beta 2 was inhibitory (Ki approximately 0.2 ng/ml). Thus, adenosine and/or adenine nucleotides released from injured or dying renal cells, or administered exogenously, may stimulate surviving cells in the wounded nephron to migrate along the basement membrane, thereby rapidly restoring tubular structure and function.

Adenine Nucleotides↗
PubMed1991

Antimitogenic and mitogenic actions of interleukin-1 in diverse cell types are associated with induction of gro gene expression.

Interleukin-1 (IL-1) is an antiproliferative factor for growing human melanoma A375-C6 cells. To define the molecular basis for the action of IL-1, we set out to identify early genes induced by the cytokine in the absence of de novo protein synthesis. cDNA libraries were constructed from A375-C6 cells that were exposed or unexposed to IL-1 plus cycloheximide. Subtractive hybridization was used to prepare a library that was enriched for IL-1-induced clones. Two of these clones were shown by Northern analysis to represent IL-1-inducible genes. Nucleotide sequencing identified these genes as gro/melanoma growth stimulatory activity, which encodes a cell secretory product, and c-jun, which encodes a transcription factor. IL-1 caused persistent steady state elevation of gro mRNA but only transient induction of c-jun. Northern analysis using gene probes for the transcription factors c-fos and Egr-1 revealed that IL-1 induced c-fos but not Egr-1 expression in these cells. This indicates that differential early gene expression characterizes the growth-inhibitory action of IL-1. In contrast, serum, which is mitogenic for these cells, induces c-jun, c-fos, and Egr-1, but not gro expression. These data imply that in A375-C6 cells, both growth-inhibitory and stimulatory signals can channel their action through c-fos and c-jun genes. As gro induction was specifically associated with the antimitogenic action of IL-1, we studied the effect of the cytokine on gro gene expression in other types of cells. IL-1 was mitogenic for human glioblastoma and monkey kidney epithelial cells and induced gro whereas other mitogens did not. Thus, IL-1 can induce gro gene expression in diverse cell types, whether it acts to stimulate or inhibit proliferation. Like other cytokines gro may play diverse cell-specific roles in growth control.

Animals↗
PubMed1991

Signals that release growth factors from renal epithelial cells.

Monkey kidney epithelial cells of the nontransformed BSC-1 line have been used as a model system to investigate growth control. Renal growth in K depletion nephropathy was studied in culture by reducing the K concentration of the medium, which accelerated cell proliferation. This response appeared to be mediated by release of a growth-promoting activity that has an apparent molecular weight of 12,000 to 30,000. Growth stimulation was also observed when the Na concentration of the medium was reduced and was associated with the appearance of two growth-promoting factors (apparent molecular weight, 6,200 and 9,000) that exhibited cell-type specificity. Thus, modest reductions in the extracellular concentration of K or Na result in rapid appearance of autocrine factors that could modulate cell function along the nephron. The most powerful mitogen for BSC-1 cells is adenosine diphosphate (ADP). This nucleotide stimulates expression of several cell cycle-specific genes and proto-oncogenes, and induces secretion of a platelet-derived growth factor-like protein that is not mitogenic for BSC-1 cells. Release of this growth factor by renal epithelial cells in vivo would represent a paracrine mechanism to initiate proliferation of neighboring stromal or vascular cells.

Animals↗
PubMed1990

Kidney epithelial cells release growth factors in response to extracellular signals.

The growth of nontransformed monkey kidney epithelial cells in culture appears to be regulated by the interplay of positive and negative autocrine growth factors. Reduction of the potassium or sodium concentration of the medium induces rapid release of novel growth-promoting activities, whereas addition of the mitogen adenosine diphosphate stimulates the appearance of a platelet-derived growth factor-like protein which could function in a paracrine manner. These observations suggest that autocrine and paracrine growth factors could play an important role in physiological and pathological states in the kidney.

Epithelial Cells↗
PubMed1990

Macula densa cells of mouse kidney do not synthesize epidermal growth factor precursor mRNA.

Epidermal growth factor (EGF) has previously been found in cells of the thick ascending limb of Henle's loop (TALH) and the first part of the distal convoluted tubule (DCT) of mouse kidney. To define the cellular localization of the mRNA that encodes the protein precursor of EGF, we used high-resolution in situ hybridization. EGF precursor cDNA was hybridized to both paraffin-embedded and frozen sections of mouse kidney. EGF precursor mRNA was detected in cells comprising the TALH and the first part of the DCT. In contrast, tubular cells in the macula densa did not express this specific mRNA. The results indicate that expression of the EGF precursor gene is restricted to specific types of cells in the distal nephron.

Animals↗
PubMed1990

A human early response gene homologous to murine nur77 and rat NGFI-B, and related to the nuclear receptor superfamily.

When a human fetal muscle cDNA library was screened with the human thyroid hormone receptor alpha 2 cDNA at low stringency, we found a weakly hybridizing cDNA. The sequence of the insert was 2498 basepairs, with an open reading frame of 1794 basepairs encoding a protein of 598 amino acids and a predicted molecular mass of 64 kDa. The DNA-binding domain and the ligand-binding domain are similar to those of steroid and thyroid hormone receptors. Moreover, this cDNA is highly homologous to mouse nur77 and rat NGFI-B, which are early response genes induced by nerve growth factor and other serum growth factors. We designated this gene NAK1. The modulation of expression of NAK1 during stimulation of cell growth was studied. The mRNA of NAK1 was induced rapidly and transiently by growth-stimulating agents, such as adenosine diphosphate, in monkey kidney cells (BSC-1), by phytohemagglutinin in human lymphocytes, and by serum stimulation of arrested fibroblasts. It is expressed in human fetal muscle and adult liver, brain, and thyroid. NAK1 could be a nuclear receptor. It will be of great interest to determine the ligand for NAK1 and the genes that are regulated by it.

Amino Acid Sequence↗
PubMed1988

An alternate method utilizing small quantities of ligand for affinity purification of monospecific antibodies.

An alternate method was designed to couple a limited quantity of protein to an affinity support when a conventional technique was unsuccessful. This was achieved through the introduction of a small number of sulfhydryl groups to the ligand by reaction with 2-iminothiolane which resulted in a limited number of reactive sites on the protein. Amino groups on an AH-Sepharose 4B matrix were linked to sulfhydryl groups on the ligand using the heterobifunctional agent m-maleimidobenzoyl sulfosuccinimide ester (sulfo-MBS). This method was employed to prepare an affinity support using a cytosolic protein that activates glyceraldehyde-3-phosphate dehydrogenase as a ligand. Monospecific antibody purified from the affinity column recognized only this protein on a Western blot of a cytosolic extract of kidney epithelial cells.

Animals↗