Biomedical subjects

D J Franks

Publications and source records attributed to D J Franks.

At least 19 recordsLinked to original sources

PubMed2002

Platelet activating factor-induced apoptosis is inhibited by ectopic expression of the platelet activating factor G-protein coupled receptor.

The pro-inflammatory lipid mediator platelet activating factor (PAF: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) accumulates in ischemia, epilepsy, and human immunodeficiency virus-1-associated dementia and is implicated in neuronal loss. The present study was undertaken to establish a role for its G-protein coupled receptor in regulating neurotoxicity. PC12 cells do not express PAF receptor mRNA as demonstrated by northern analysis and RT-PCR. In the absence of the G-protein coupled receptor, PAF (0.1-1 micro m) triggered chromatin condensation, DNA strand breaks, oligonucleosomal fragmentation, and nuclear disintegration characteristic of apoptosis. Lyso-PAF (0.001-1 micro m), the immediate metabolite of PAF, did not elicit apoptotic death. Concentrations of PAF or lyso-PAF that exceeded critical micelle concentration had physicochemical effects on plasma membrane resulting in necrosis. Apoptosis but not necrosis was inhibited by the PAF antagonist BN52021 (1-100 micro m) but not CV3988 (0.2-20 micro m). Ectopic PAF receptor expression protected PC12 transfectants from ligand-induced apoptosis. PAF receptor-mediated protection was inhibited by CV3988 (1 micro m). These data provide empirical evidence that: (i) PAF can initiate apoptosis independently of its G-protein coupled receptor; (ii) PAF signaling initiated by its G-protein coupled receptor is cytoprotective to PC12 cells; (iii) the pro- and anti-apoptotic effects of PAF on PC12 cells can be pharmacologically distinguished using two different PAF antagonists.

Animals↗

PubMed2000

Characterization of prostaglandin endoperoxide H synthase-1 enzyme expression during differentiation of the megakaryocytic cell line MEG-01.

OBJECTIVE: Because the prostaglandin endoperoxide H synthase-1 (PGHS-1)-dependent formation of thromboxane A(2) is an important modulator of platelet function, this pathway represents a pharmacologic target for the inhibition of platelet function by aspirin. The objective of our research was to study how PGHS-1 expression is regulated in platelets. MATERIALS AND METHODS: Because platelets are anucleated, their protein content is a consequence of gene expression in precursor cells known as megakaryocytes. We used the immortalized human megakaryoblastic cell line MEG-01 as a model to study the expression of PGHS-1, because MEG-01 cells can be induced to differentiate into platelet-like structures by adding nanomolar concentrations of 12-0-tetradecanoylphorbol-13-acetate (TPA). We determined the expression profiles of PGHS-1 protein and mRNA in the cells comprising the three different populations of MEG-01 cultures: nucleated floating, nucleated attached, and platelet-like structures. RESULTS: We determined that PGHS-1 protein levels were higher in the nucleated adherent population than in the nucleated floating population. PGHS-1 protein levels were greatest in the anucleated platelet-like population. In contrast, we found that PGHS-1 mRNA levels were highest in the cells that comprised the nucleated adherent population. Addition of TPA induced the expression of PGHS-1 protein and mRNA in all three populations but did not change the relationship of the amount of PGHS-1 protein or mRNA expressed in a given population relative to the other two fractions. We measured the expression of PGHS-1 protein on a cell-by-cell basis in the nucleated MEG-01 populations. We found that the percentage of MEG-01 cells expressing PGHS-1 protein in the adherent population was greater than in the floating population. We measured a time-dependent increase in the percentage of cells that expressed PGHS-1 over a period of 8 days after singular addition of TPA (1.6x10(-8)M). Importantly, we observed that TPA treatment stimulated floating MEG-01 to adhere to the surface of the tissue culture vessel and that, after such treatment, only floating MEG-01 cells suffered a compromised viability. We found that a high percentage of control cells expressed glycoprotein IIb/IIIa and that TPA treatment did not significantly alter this percentage. We did not detect glycoprotein Ib in control cells but did measure a slight increase in the percentage of MEG-01 cells that expressed this antigen in the TPA-treated population. CONCLUSION: We established a correlation between the level of PGHS-1 expression and the overall level of differentiation of MEG-01 cells. PGHS-1 protein expression, which increases consistently over the full course of differentiation, now may be used as an additional and perhaps better index by which to survey megakaryocytes.

Cell Adhesion↗

PubMed1998

The presence of an unusual PKC isozyme profile in rat liver cells.

We have previously shown that protein kinase C (PKC) is involved in the mitogenic response of T51B cells to epidermal growth factor. In fact, epidermal growth factor was an excellent mitogen, even after prolonged pretreatment of cells with TPA, suggesting that the PKC isoform implicated in proliferation is not down-regulated by 12-O-tetradecanoyl phorbol-13-acetate (TPA). We have now determined that the PKC isozymes -alpha, -beta, -delta, -epsilon, and -zeta are present in T51B cells. All five isoforms are associated with the plasma membrane and the cytoplasm and are either in or around the nucleus. PKC-beta I has a slightly different subcellular profile from that of the other isoforms in that it is clearly and strongly associated with the nuclear membrane. Also, a unique and novel pattern is obtained from immunoblots with anti-PKC-beta I. PKC-beta I is detected as a single band of 70 kDa in the cytosolic fraction and as a doublet of 65 and 77 kDa in the membrane fraction. PKC-alpha, -delta, and -epsilon were down-regulated by pretreatment of cells with TPA, while PKC-zeta was unaffected. Of particular interest was the fact that TPA did not down-regulate PKC-beta I. In fact, the amount of this isoform associated with the plasma membrane increased. These findings indicate that it is probably PKC-beta I that is involved in the mitogenic response of T51B cells to epidermal growth factor. Since PKC-zeta is also not down-regulated by TPA, the possible involvement of this isoform needs to be resolved.

Animals↗

PubMed1997

Estimation of total collagen and types I and III collagen in canine rotator cuff tendons.

The collagen composition of the supraspinatus, infraspinatus, and subscapularis tendons, which form part of the rotator cuff of the shoulder, was determined. Tendons were obtained from adult, male beagle dogs and total collagen was estimated by measurement of hydroxyproline. There was little variation in collagen content among the three major cuff tendons and the quantity approximated that cited in the literature for other tendons. However, the collagen content in the insertion zone of the supraspinatus tendon was significantly higher than in the tendon proper. NaCl fractionation of supraspinatus collagen indicated that type I was the predominant collagen but significant amounts of type III and possibly some type II and type V were also present. Interestingly, there appeared to be more type III collagen in the insertion zone than in the tendon proper, cyanogen bromide digestion and peptide mapping confirmed this finding. The differential collagen composition of the supraspinatus tendon may contribute to the high incidence of tear that is associated with this rotator cuff tendon.

Animals↗

PubMed1995

Modifications in cytokeratin and actin in cultured liver cells derived from griseofulvin-fed mice.

BACKGROUND: Hepatocytes from mice fed griseofulvin (GF) for 8 months form Mallory bodies (MBs), which represent a pathologic state of intermediate filaments (IFs). The cellular mechanisms that lead to MB formation are not known. EXPERIMENTAL DESIGN: This study was aimed to investigate if MB formation could be related to modification in cytokeratin (CK) metabolism. Primary cultures of hepatocytes from control and GF livers were studied. Immunofluorescence microscopy was used to study the organization of the cytoskeleton in these cells. The hepatocytes were labeled with [35S]methionine or [32P]orthophosphate to study, respectively, the level of amino acid incorporation into IF proteins (CK 8 and CK 18) and their phosphorylation levels. The response to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate stimulation of the phosphorylation of CK 8 and CK 18 was also elicited in contrast to control hepatocytes. RESULTS: We found that there was a change in the organization of actin and the IF network in the hepatocytes from GF-treated animals. This was associated with an increase in labeled amino acid incorporation into CK 8 and CK 18 as well as in actin. Although there was no significant difference in the absolute level of CK phosphorylation, we found modifications in the phosphorylated isomers of CK 8, the more phosphorylated isomers becoming more prominent. The treatment of the hepatocytes with 12-O-tetradecanoyl-phorbol-13-acetate did not induce changes in the level of CK phosphorylation in GF-pretreated hepatocytes. CONCLUSIONS: These results suggest that the modification of the IF network and MB formation are the consequences of increased CK synthesis and the modification of phosphorylation. They could alter the normal interaction of the IFs with different cellular components, which results in conformational changes of CKs and the reorganization of the IF network to the form of MBs.

Actins↗

PubMed1994

1,25-Dihydroxyvitamin D3 translocates protein kinase C beta to nucleus and enhances plasma membrane association of protein kinase C alpha in renal epithelial cells.

1,25-Dihydroxycholecalciferol (1,25-(OH)2-D3) increases membrane-associated protein kinase C (PKC) activity and immunoreactivity in renal epithelial (Madin Darby bovine kidney, MDBK) cells (Simboli-Campbell, M., Franks, D. J., and Welsh, J. E. (1992) Cell Signalling 4, 99-109). We have now characterized the effects of 1,25-(OH)2-D3 on the subcellular localization of three individual isozymes by immunofluorescence and immunoblotting. Although the total amount of PKC alpha, PKC beta, and PKC zeta are unaffected by 1,25-(OH)2-D3, this steroid hormone induces subcellular redistribution of both PKC alpha and PKC beta. Treatment with 1,25-(OH)2-D3 (100 nM, 24 h) enhances plasma membrane association of PKC alpha and induces translocation of PKC beta to the nuclear membrane. The effects of 1,25-(OH)2-D3 appear to be limited to the calcium-dependent PKC isozymes, since 1,25-(OH)2-D3 has no effect on the calcium independent isozyme, PKC zeta. In contrast to rapid transient PKC translocation seen in response to agents which interact with membrane receptors to induce phospholipid hydrolysis, modulation of PKC alpha and PKC beta is observed after 24 h treatment with 1,25-(OH)2-D3. In MDBK cells, the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) (100 nM, 24 h) down-regulates PKC alpha and, to a lesser extent, PKC zeta, without altering their subcellular distribution. TPA also induces translocation of PKC beta to the nuclear membrane. MDBK cells treated with 1,25-(OH)2-D3, but not TPA, exhibit enhanced phosphorylation of endogenous nuclear proteins. In addition to the distinct effects of 1,25-(OH)2-D3 and TPA on PKC isozyme patterns, 1,25-(OH)2-D3 up-regulates both the vitamin D receptor and calbindin D-28K, whereas TPA down-regulates the expression of both proteins. These data support the involvement of PKC in the mechanism of action of 1,25-(OH)2-D3 and specifically implicate PKC beta in 1,25-(OH)2-D3-mediated nuclear events.

Animals↗

PubMed1994

The epidermal growth factor mitogenic signal is modulated by protein kinase C in T51B rat liver cells.

The regulation of cell proliferation involves a complex interplay between several signal transduction pathways. The effect of EGF on DNA synthesis in serum starved quiescent, synchronized T51B cells was investigated by [3H]thymidine incorporation and flow cytometry. 1 nM EGF or readdition of serum initiated G1 progression and entry into S phase by 18 h and DNA synthesis reached a maximum by 28 h. Low concentrations of EGF markedly stimulated DNA synthesis, but EGF was not as potent as readdition of serum. The effect of EGF on DNA synthesis was only partially blocked by the tyrosine inhibitors genistein and tyrphostin, suggesting that other signalling pathways play a role in EGF-stimulated mitogenesis. 1 nM EGF caused a rapid, transient increase in the activity of membrane-associated protein kinase C (PKC) followed by a longer sustained increase that continued into S phase. TPA (12-O-tetradecanoyl-phorbol-13-acetate) did not mimic EGF, rather it caused a slight stimulation of membrane-associated PKC activity within 1 h followed by a dramatic downregulation of PKC within 4 h. TPA was without effect on DNA synthesis alone, but when added along with EGF or serum TPA caused a significant enhancement of DNA synthesis. Pretreatment of quiescent, serum-deprived T51B cells with TPA reduced the basal level of DNA synthesis; however, under these conditions EGF became as potent a mitogen as serum. We hypothesize that EGF via activation of PKC regulates the activity of its receptor by switching from high affinity to low affinity states. Downregulation of PKC by long term treatment with TPA removes this regulation thus rendering T15B cells more sensitive to exogenous EGF.

Animals↗

PubMed1994

Modulation of a renal estrogen receptor by protein kinase C.

In this paper we demonstrate the existence of specific, high affinity 17-beta estradiol binding in MDBK cells (a normal non-transformed renal cell line). Scatchard analysis revealed binding characteristics typical of the estrogen receptor (ER). Only unlabelled 17-beta estradiol, diethyl stilbestrol and estrone effectively competed with [3H] 17-beta estradiol for binding, other steroids did not compete. Short term TPA treatment of MDBK cells with TPA increased PKC activity and immunoreactivity and caused a transient increase in 17-beta estradiol binding, while longer term treatment with TPA decreased PKC activity and immunoreactivity. The inactive phorbol ester 4 alpha PDD was without effect on PKC activity and the ER. TPA did not affect the affinity of the ER for the nucleus nor did it increase degradation of the receptor. We hypothesize that the renal ER may be a substrate for PKC and its properties can be modified by PKC similarly to the vitamin D receptor.

Animals↗

PubMed1992

TPA decreases 1,25(OH)2D3 binding and calbindin D-28K in renal (MDBK) cells.

The effect of the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) on vitamin D receptors (VDRs) was studied in MDBK cells, a normal bovine renal epithelial cell line. 24 h treatment of MDBK cells with TPA resulted in down-regulation of VDR number, with no change in the binding affinity for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or approximate molecular weight determined by fast protein liquid chromatography (FPLC). TPA treatment also reduced the level of calbindin D-28K, a vitamin D-dependent renal protein. 4 alpha-Phorbol 12,13-didecanoate (4 alpha-PDD), an inactive phorbol ester, did not affect either 1,25(OH)2D3 binding or calbindin D-28K levels. TPA elicited a significant decrease in membrane-associated protein kinase C (PKC) activity which coincided with the reduction in VDR number and calbindin D-28K. These data support a link between TPA, PKC activity and vitamin D actions in kidney.

Animals↗

PubMed1992

1,25(OH)2D3 increases membrane associated protein kinase C in MDBK cells.

To determine whether 1,25-dihydroxycholecalciferol [1,25(OH)2D3] affects protein kinase C (PKC) activity in kidney, as has been demonstrated in HL-60 cells we measured 1,25(OH)2D3 binding, PKC activity and PKC immunoreactivity in Madin Darby bovine kidney (MDBK) cells, a normal renal epithelial cell line derived from bovine kidney. Our data demonstrate that MDBK cells exhibit specific high affinity binding for 1,25(OH)2D3, indicating the presence of the vitamin D receptor (VDR). Treatment of MDBK cells with 1,25(OH)2D3 for 24 h increased membrane PKC activity and immunoreactivity. The effect of 1,25(OH)2D3 was dose-dependent, with a peak effect observed at 10(-7)M 1,25(OH)2D3. The 1,25(OH)2D3 induced increase in membrane PKC was paralleled by a comparable decrease in cytosolic PKC activity and amount. Although time course studies were consistent with a VDR mediated effect of 1,25(OH)2D3 on PKC protein synthesis, total PKC activity was not increased by 1,25(OH)2D3, suggesting an effect on PKC translocation or localization. These results suggest that 1,25(OH)2D3 modulates PKC mediated events in kidney, a classic target for this steroid hormone.

Animals↗

PubMed1992

Differential phosphorylation of CK8 and CK18 by 12-O-tetradecanoyl-phorbol-13-acetate in primary cultures of mouse hepatocytes.

The phosphorylation of cytokeratin was investigated in primary cultures of hepatocytes. The two hepatocyte cytokeratins CK8 and CK18 (55,000 and 49,000 M(r) respectively) were phosphorylated, CK8 being more phosphorylated than CK18. Treatment of the hepatocytes with 150 nM 12-O-tetradecanoyl-phorbol-13-acetate (TPA) an activator of protein kinase C induced a transient increase in the level of phosphorylation of CK8 but not CK18. This effect was maximal after 15 min of TPA treatment and was maintained for up to 3 h. After 22 h of treatment with TPA, which down-regulates protein kinase C, CK8 phosphorylation was returned to the basal level. Further addition of TPA to the 22-h treated cells did not cause an increase in CK8 phosphorylation. Indirect immunofluorescence microscopy with a monoclonal antibody to CK8 indicated that while the addition of TPA induced the formation of granular cytokeratin aggregates in some hepatocytes, in most hepatocytes no major changes in the intermediate filament network were observed. Staining for actin showed that actin microfilaments were rapidly reorganized after the treatment and a loss of stress fibres were observed. We propose that CK8 is an in vivo substrate for protein kinase C and that the specific phosphorylation of CK8 plays a role in protein kinase C signal transduction.

Animals↗

PubMed1992

Activation of protein kinase C modulates dihydroxycholecalciferol synthesis in rat renal tubules.

1,25(OH)2D3, the biologically active metabolite of vitamin D, is produced from 25(OH)D3 by the renal mitochondrial 25(OH)D3 1 alpha-hydroxylase. Several studies have implicated reversible phosphorylation and a possible role for protein kinase C (PKC) in acute regulation of 1,25(OH)2D3 production. In the experiments described here, we studied 1,25(OH)2D3 production in freshly isolated rat renal tubules treated with activators and inhibitors of PKC. In this mammalian system, TPA, but not its inactive analogue 4 alpha PDD, inhibited 1,25(OH)2D3 production in a dose-dependent fashion within 20 min. The acute inhibition of 1,25(OH)2D3 production by TPA exposure was preceded by an increase in membrane associated PKC activity, which was paralleled by a decrease in cytosolic PKC activity. Pre-incubation of tubules with staurosporine, a PKC inhibitor, abolished the inhibitory effect of TPA on 1,25(OH)2D3 production. Chronic (18 h) exposure of tubules to high dose TPA resulted in down regulation of both membrane and cytosolic PKC activity and re-exposure to TPA did not affect PKC translocation or 1,25(OH)2D3 production in down regulated tubules. Our data strongly suggest that modulation of renal PKC activity may be an important mechanism for acute regulation of 1,25(OH)2D3 production.

Alkaloids↗

PubMed1992

Cementless revision of cemented stem failures associated with massive femoral bone loss. A technical note.

A revision technique of the failed cemented femoral stem was developed using a standard cementless porous-coated prosthesis. The advantages of this technique are that the prosthesis has apposition to autogenic bone rather than allograft; problems of long stem can be avoided; supplementary allograft provides enough strength to allow early weight bearing; it facilitates the extraction of the distal cement; and further revision is relatively easy in the healed, viable bone bed.

Aged↗

PubMed1990

The role of signal-transducing events in the proliferative response of cells to a mitogenic viral K-ras protein.

Activated oncogenic ras proteins are powerful mitogenic agents which by themselves can initiate and maintain the proliferation of quiescent cells in the absence of any exogenous growth factors. In an attempt to understand how ras proteins induce proliferation we examined the early events in the G0 to G1 transition caused by the activation of a thermolabile K-ras protein in quiescent, serum-starved tsKSV-transformed NRK cells. We show that ras reactivation, in the absence of exogenous growth factors, triggered a rapid surge in free cytosolic Ca2+ and diacylglycerol production, which led to a transient increase in membrane-associated protein kinase C (PKC) activity which was necessary for G1 transit. Unlike TPA-stimulated PKC activity, the ras-induced increase in PKC was readily extracted from membranes by EGTA. These signal transducing events occurred despite the fact that ras activation did not induce the tyrosine phosphorylation of any known surface receptor. The results indicate that the K-ras protein triggers the G0 to G1 transition by an intracellular mechanism and not indirectly via autocrine stimulation.

Animals↗

PubMed1989

A novel method for measuring protein kinase C activity in a native membrane-associated state.

Physiological activation of protein kinase C (PKC) is believed to occur by redistributing soluble enzyme to the phospholipid environment of membranes. Currently available in vitro methods of measuring PKC activation all involve prior extraction of membrane-associated enzyme and its reconstitution in an artificial phospholipid environment or modification (such as partial trypsinization) of the enzyme itself. Here we report a novel method which, for the first time, allows measurement of active PKC still in its native, membrane-associated state using a specific, physiological substrate. Thus, with this new method PKC activity can be measured while still in an environment that approximates the in vivo situation.

Animals↗

PubMed1989

Protein kinase C and a viral K-RAS protein cooperatively enhance the response of adenylate cyclase to stimulators.

The protein kinase C stimulator TPA (12-O-tetradecanoyl phorbol-13-acetate) enhanced the responsiveness of adenylate cyclase to IPR (isoproterenol) and PGE1 (prostaglandin E1) in quiescent tsKSV-NRK cells at the nonpermissive 41 degrees C. Reactivating the thermolabile mitogenic/oncogenic K-ras protein in tsKSV-NRK cells by dropping the temperature to 36 degrees C also enhanced the responsiveness of adenylate cyclase to IPR and PGE1. The enhancement was transient and peaked at 6 hours after the temperature shift. This enhanced responsiveness was specifically due to the reactivated viral K-ras protein rather than the temperature shift because the same temperature shift did not affect adenylate cyclase responsiveness in uninfected NRK cells, nor was it a result of the mitogenic stimulus since reacting the mitogenic pp60v-src protein in tsASV-NRK cells did not affect adenylate cyclase responsiveness. The increased responsiveness of adenylate cyclase at 6 hours after the temperature shift was not a result of elevated membrane-associated PKC activity. However, the reactivated viral K-ras protein strongly increased the stimulability of membrane-associated PKC by TPA and it further increased TPA's ability to enhance the responsiveness of adenylate cyclase to IPR and PGE1. Thus, a viral K-ras protein and membrane-associated protein kinase C can cooperate to increase the responsiveness of adenylate cyclase to agonists.

Adenylyl Cyclases↗

PubMed1989

Activation of protein kinase C sensitizes the cyclic AMP signalling system of T51B rat liver cells.

Activation of protein kinase C (PKC) by phorbol esters (TPA) results in a modification of the cyclic AMP system leading to either attenuation or amplification of the cyclic AMP signal. In the non-neoplastic T51B rat liver cell line, TPA, when added to intact cells, had no effect on the basal level of cyclic AMP synthesis but caused a 1.5 fold amplification of the stimulation induced by beta-adrenergic agents, cholera toxin and forskolin. The effect appeared to be mediated by PKC since diacylglycerols caused the same amplification as did TPA while inactive phorbol esters were without effect. Phosphorylation of Gs or the catalytic subunit of adenylate cyclase by PKC is likely to be responsible for the enhancement of cyclic AMP synthesis. TPA also caused translocation of PKC; however, the time course of the translocation was longer than the time course of the enhancement of adenylate cyclase activity. Thus, the ability of TPA to amplify cyclic AMP synthesis is probably mediated by activation of PKC that is already present in the membrane.

Animals↗

PubMed1987

A viral K-RAS protein increases the stimulability of adenylate cyclase by cholera toxin in NRK cells.

Incubation at 41 degrees C stops the proliferation of tsK-NRK rat kidney cells in serum-deficient medium by inactivating the mitogenic/oncogenic thermolabile viral K-RAS protein that is produced in these cells. Dropping the temperature to 36 degrees C reactivates the viral K-RAS protein which stimulates the serum-starved quiescent cells to resume proliferating without added serum factors. Here it is shown that while the reactivated viral protein does not by itself significantly stimulate adenylate cyclase, it greatly increases the stimulability of adenylate cyclase by cholera toxin. The data suggest that the viral K-RAS protein directly or indirectly affects adenylate cyclase by inactivating the Gi inhibitory component of the membrane associated enzyme.

1-Methyl-3-isobutylxanthine↗