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J F Whitfield

Publications and source records attributed to J F Whitfield.

At least 19 recordsLinked to original sources
PubMed2004

Treatment with parathyroid hormone hPTH(1-34), hPTH(1-31), and monocyclic hPTH(1-31) enhances fracture strength and callus amount after withdrawal fracture strength and callus mechanical quality continue to increase.

The influence of intermittent hPTH(1-34)NH2, hPTH(1-31)NH2, and monocyclic [Leu27]cyclo (Glu22-Lys26)hPTH(1-31)NH2 treatment on callus formation, mechanical strength, and callus tissue mechanical quality of tibial fractures in rats was investigated after 8 and 16 weeks of healing. In the 8 weeks of healing animals, the PTH-peptides were injected subcutaneously during the entire observation period (15 nmol/kg/day [hPTH(1-34)NH2: 15 nmol = 60 microg]), and control animals with fractures were given vehicle. In the 16 weeks of healing animals, the PTH-peptides were injected only during the first 8 weeks of healing (15 nmol/kg/day), after which the animals were left untreated during the rest of the healing period. After the first 8 weeks of healing, increased fracture strength and callus volume were seen in the PTH-treated rats (ultimate load 66%, ultimate stiffness 58%, callus volume 28%), and the three peptides were equally effective. No difference in callus tissue mechanical quality was found between PTH and vehicle animals. After 16 weeks of healing, no differences in fracture strength, callus volume, or callus tissue mechanical quality were seen between PTH and vehicle. When comparing PTH-treated animals at 8 and 16 weeks, fracture strength and callus tissue mechanical quality continued to increase after the withdrawal of PTH (ultimate load 23%, ultimate stress 88%, elastic modulus 87%) and external callus volume declined during this period (27%).

Animals↗
PubMed2004

The neuronal primary cilium--an extrasynaptic signaling device.

Many, but likely most, neurons in the central nervous system have a nonmotile "primary" cilium extending like an antenna or finger from one of the pair of centrioles in the cell's centrosome into the extracellular space. Since their discovery over 100 years ago, these organelles have been either dismissed as functionless relicts of a bygone era or more often simply ignored. However, it has long been known that the photoreceptor-bearing outer segments of retinal rods and cones are modified primary cilia and it has recently been found that kidney cells' primary cilia are sensitive flowmeters the disabling of which causes polycystic kidney disease. It has also been recently shown that somatostatin sst3 receptors and serotonin 5-HT(6) receptors are selectively sited on neurons in various parts of the rat brain. It seems likely that these selectively receptored neuronal primary cilia will turn out to be the forerunners of a family of cell-signaling devices that help drive various brain functions by sending signals into their own cells and into adjacent cells through gap junctions and via conventional chemical synapses.

Animals↗
PubMed2001

Structural requirements for conserved arginine of parathyroid hormone.

Arg-20 is one of two residues conserved in all peptides known to activate the parathyroid hormone (PTH) receptor. Previous studies have failed to find any naturally encoded analogues of residue 20 that had any adenylyl cyclase (AC) stimulating activity. In this work we have studied substitutions of Arg-20 with nonencoded amino acids and conformationally constrained analogues with side chains mimicking that of Arg. No analogue had more than 20% of the AC-stimulating ability of the natural Arg-20-bearing peptide. In descending order of activity, the most active analogues had (S)-4-piperidyl-(N-amidino)glycine (PipGly), norleucine (Nle), citrulline (Cit), or ornithine (Orn) at residue 20. Analogues with Arg-20 substituted with L-4-piperidyl-(N-amidino)alanine, Lys, Glu, Ala, Gln, (S)-2-amino-4-[(2-amino)pyrimidinyl]butanoic acid, or L-(4-guanidino)phenylalanine had very low or negligible activity. Low or negligible activities of Lys or Orn analogues suggested ionic interactions play a minor role in the Arg interaction with the receptor. The conformational constraints imposed by the PipGly ring had a negative effect on its ability to substitute for Arg. The side-chain H-bonding potential of the Cit ureimido group was likely an important factor in its mimicry of Arg. The increase in amphiphilicity, as demonstrated by its greater high-performance liquid chromatographic retention, and increased alpha-helix, as shown by circular dichroic spectroscopy, likely contributed to the activity of the Nle-20 analogue. The data demonstrated that specific H-bonding, hydrophobicity of the side chain, stabilization of alpha-helix, and possibly specific cation positioning were all important in the interaction of Arg-20 with receptor groups.

Adenylyl Cyclases↗
PubMed2001

The effect of monocyclic and bicyclic analogs of human parathyroid hormone (hPTH)-(1-31)NH2 on bone formation and mechanical strength in ovariectomized rats.

The [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 lactam is a stronger stimulator of adenylyl cyclase activity and a better stimulator of trabecular bone in the ovariectomized (OVX) rat model of osteopenia than hPTH-(1-31)NH2. This enhanced activity is due in large part to the stabilization of the amphiphilic receptor-binding alpha-helix in the Ser17-Gln29 region. The goal of the present study was to determine whether further cyclization could produce a more active hPTH analog. To this end, we compared the relative bioactivities of the bicyclic hPTH analog [Glu17,Leu27]cyclo(Lys13-Glu17,Glu22-Lys26)-hPTH-(1-31)NH2, made by replacing Ser17 with Glu17 and introducing a second lactam linkage between Lys13 and Glu17. The relative EC50 for adenylyl cyclase stimulation by the bicyclic hPTH analog was similar to the EC50 of the monocyclic [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, but the bicyclic analog was still more active than hPTH-(1-31)NH2. As expected from adenylyl cyclase stimulation being responsible for PTH's anabolic action, the bicyclic hPTH analog [Glu17, Leu27]cyclo(Lys13-Glu17, Glu22-Lys26)-hPTH-(1-31)NH2 was able to increase femoral trabecular volume and thickness and mechanical strength in OVX rats, but it was no more effective than [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 when injected once daily in a dose of 0.8 nmol/100 g body weight. Thus, further constraint of the conformation of hPTH-(1-31)NH2 by introducing two lactam link-ages between Lys13-Glu17 and Glu22-Lys26 did not raise the osteogenicity above that of the monocyclic analog.

Adenylyl Cyclases↗
PubMed2001

Stimulation of protein kinase C activity in cells expressing human parathyroid hormone receptors by C- and N-terminally truncated fragments of parathyroid hormone 1-34.

The parathyroid hormone (PTH) fragment PTH(1-34) stimulates adenylyl cyclase, phospholipase C (PLC), and protein kinase C's (PKCs) in cells that express human, opossum, or rodent type 1 PTH/PTH-related protein (PTHrP) receptors (PTHR1s). Certain carboxyl (C)-terminally truncated fragments of PTH(1-34), such as human PTH(1-31) [hPTH-(1-31)NH2], stimulate adenylyl cyclase but not PKCs in rat osteoblasts or PLC and PKCs in mouse kidney cells. The hPTH(1-31)NH2 peptide does fully stimulate PLC in HKRK B7 porcine renal epithelial cells that express 950,000 transfected hPTHR1s per cell. Amino (N)-terminally truncated fragments, such as bovine PTH(3-34) [bPTH(3-34)], hPTH(3-34)NH2, and hPTH(13-34), stimulate PKCs in Chinese hamster ovary (CHO) cells expressing transfected rat receptors, opossum kidney cells, and rat osteoblasts, but an intact N terminus is needed to stimulate PLC via human PTHR1s in HKRK B7 cells. We now report that the N-terminally truncated analogs bPTH(3-34)NH2 and hPTH(13-34)OH do activate PKC via human PTHR1s in HKRK B7 cells, although less effectively than hPTH(1-34)NH2 and hPTH(1-31)NH2. Moreover, in a homologous human cell system (normal foreskin fibroblasts), these N-terminally truncated fragments stimulate PKC activity as strongly as hPTH(1-34)NH2 and hPTH(1-31)NH2. Thus, it appears that unlike their opossum and rodent equivalents, hPTHR1s can stimulate both PLC and PKCs when activated by C-terminally truncated fragments of PTH(1-34). Furthermore, hPTHR1s, like the PTHR1s in rat osteoblasts, opossum kidney cells, and rat PTHR1-transfected CHO cells also can stimulate PKC activity by a mechanism that is independent of PLC. The efficiency with which the N-terminally truncated PTH peptides stimulate PKC activity depends on the cellular context in which the PTHR1s are expressed.

Amino Acid Sequence↗
PubMed2001

Statins: new drugs for treating osteoporosis?

Statins are widely used lipid-lowering drugs that reduce cholesterol synthesis by inhibiting 3-hydroxy-3-glutaryl-coenzyme A (HMG-CoA) reductase activity. They also strongly stimulate bone formation in rodents. If the drugs' potent bone-building activity results directly from inhibition of HMG-CoA reductase, there should be less bone fracturing in humans who have taken statins to lower their serum cholesterol and prevent heart attacks, but the data gleaned from several databases are contradictory. According to some reports the lipid-lowering doses of oral statins increased bone mineral density and more than halved the risk of fracturing various bones, while according to others, including the very large Women's Health Initiative Observational Study (WHI-OS), the drugs did not significantly affect the fracturing risk. Such contradictory data could be due in part to one of the commonly used statins, pravastatin which only targets hepatocytes, or due to bone growth being stimulated by something other than inhibition of HMG-CoA reductase. Therefore, different doses of statins may be needed to build bone or optimally lower serum cholesterol. To be able to answer the question posed by the title of this editorial, it will be necessary to carry out a controlled trial using designer statins that are less liver-oriented and thus better for assessing the optimal doses needed, specifically for osteogenicity rather than for their cholesterol-lowering ability.

Animals↗
PubMed2001

Leptin: brains and bones.

Leptin, is a 16 kDa protein derived mainly, though not entirely, from white fat cells. It regulates the size of the body's fat load (i.e., energy reserves) and has a growing list of other functions. The latest addition is bone growth. The evidence so far indicates that leptin controls bone growth in two ways. It stimulates the release of an undefined hypothalamic osteoblast-inhibiting factor(s), which limits the amount of bone matrix that osteoblasts can make. Interestingly, it is itself a bone anabolic factor that directly stimulates bone growth by inducing osteoblasts to make IGF-I (insulin-like growth factor-I) and inhibiting osteoclast generation.

Animals↗
PubMed2001

Parathyroid hormone: an anabolic treatment for osteoporosis.

Osteoporosis is a disease characterised by low bone mass, structural deterioration of bone and increased risk of fracture. The prevalence, and cost, of osteoporosis is increasing dramatically with our ageing population and the World Health Organization now considers it to be the second-leading healthcare problem. All currently approved therapies for osteoporosis (eg., estrogen, bisphosphonates, calcitonin and selective estrogen receptor modulators) are anti-resorptive agents that act on osteoclasts to prevent further bone loss. A new class of bone anabolic agent capable of building mechanically strong new bone in patients with established osteoporosis is in development. While the parathyroid hormone (PTH) is classically considered to be a bone catabolic agent, when delivered intermittently at low doses PTH potently stimulates cortical and trabecular bone growth in animals humans. The native hPTH-(1-84) and its osteogenic fragment, hPTH-(1-34), have already entered Phase III clinical trials. Understanding the mechanism of PTH's osteogenic actions has led to the development of smaller PTH analogues which can also build mechanically normal bone in osteopenic rats. These new PTH analogues are promising candidates for treating osteoporosis in humans as they are as efficacious as hPTH-(1-84) and hPTH-(1-34), but there is evidence that they may have considerably less ability to induce hypercalcemia, the major side effect of PTH therapy. In addition to treating osteoporosis, PTHs may be used to promote fracture healing, to restore bone loss in immobilized patients, or following excessive glucocorticoid or prolonged spaceflight, and to treat psoriasis.

Animals↗
PubMed2000

Structure and activities of constrained analogues of human parathyroid hormone and parathyroid hormone-related peptide: implications for receptor-activating conformations of the hormones.

Parathyroid hormone (PTH) has a helix-bend-helix structure in solution. Part of the C-terminal helix, residues 21-31, is amphiphilic and forms a critical receptor-binding region. Stabilization of this alpha-helix by lactam formation between residues spaced i, i + 4 on the polar face was previously reported to increase adenylyl cyclase-stimulating (AC) activity if between residues 22 and 26 but to diminish it if between residues 26 and 30 [Barbier et al. (1997) J. Med. Chem. 40, 1373-1380]. This work reports the effects of other cyclizations on the polar face, differing in ring size or position, on alpha-helix conformation, as measured by circular dichroism, and on AC-stimulating activity. All analogues cyclized between residues 22 and 26 had at least a 1. 5-fold increase in activity, suggesting an alpha-helical structure between about residues 21 and 26. Cyclization between residues 25 and 29 or residues 26 and 30 diminished activity by 20-30%, despite stabilizing alpha-helix, suggesting that residues 25-31 bind to the receptor in a helical, but not classical alpha-helical, conformation. Analogues cyclized between residues 13 and 17 had slightly increased activity. A bicyclic analogue, with lactams between residues 13 and 17 and residues 22 and 26, had about the same activity as that cyclized only between 22 and 26. Parathyroid hormone-related peptide (PTHrP) may bind in a manner similar to the common receptor, but hydrophobic moment calculations suggest that it must bind as a tighter helix in order to optimally present its hydrophobic residues to the receptor. Both PTHrP analogues cyclized between either residues 22 and 26 or residues 26 and 30 had more stable alpha-helices but reduced AC activities, consistent with this hypothesis.

Adenylyl Cyclases↗
PubMed2000

Increased activity of the protein kinase C-delta holoenzyme in the cytoplasmic particulate fraction precedes the activation of caspases in polyomavirus-transformed pyF111 rat fibroblasts exposed to calphostin C or topoisomerase-II inhibitors.

A caspase-mediated release of the 40-kDa catalytic fragment of the delta isoform (CF-delta) of protein kinase C (PKC-delta) is involved in apoptosis, but its actual role in apoptosis development is still unknown. In an effort to understand this role, we have used polyomavirus-transformed pyF111 rat fibroblasts, which are hypersusceptible to apoptosis as they constitutively hyperexpress PKC-delta, but cannot make the antiapoptotic Bcl-2 and Bcl-X(L) proteins, while making the proapoptotic Bax protein. Calphostin C is reportedly both a specific inhibitor of PKC-delta activity (C. Keenan, N. Goode, and C. Pears, 1997, FEBS Lett. 415, 101-108) and an effective apoptogen (M. Murata et al., 1997, Cell. Mol. Life Sci. 53, 737-743). Exposure of pyF111 cells to calphostin C (75 nM) stimulated the translocation of the PKC-delta holoenzyme (holo-PKC-delta) onto the cytoplasmic particulate (CP) fraction between 15 and 45 min, which was after the release of mitochondrial cytochrome c but before the activation of cytoplasmic DEVD-specific caspases. The CF-delta fragment started accumulating only between 2 and 4 h, while apoptosis occurred mostly within 6 h. Incubating pyF111 cells with the much slower acting, apoptogenic topoisomerase-II inhibitors etoposide (VP-16) and teniposide (VM-26) also caused within 6 h a doubling of the CP-bound holo-PKC-delta-related activity but with no significant translocation of the holoenzyme to the CP fraction. Again this occurred after the release of cytochrome c but before the activation of DEVDases and the accumulation of the CF-delta. However, while calphostin C did not affect the delta-related activity in the nuclear membrane (NM) and nucleoplasmic (NP) fractions, VP-16 and VM-26 caused a prompt, large, and irreversible drop in the delta activity at the NM and a transient surge followed by a fall in the NP-associated activity. Hence, a surge of CP-anchored holo-PKC-delta activity is a common part of the signals given by various apoptogenic drugs to pyF111 cells. On the other hand, inhibition of delta-related activity, first at the NM and then in the NP fraction, is a specific feature only of the signals given by apoptogenic DNA-damaging agents.

Animals↗
PubMed2000

The stimulation of vertebral and tibial bone growth by the parathyroid hormone fragments, hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-30)NH2.

The native human parathyroid hormone, hPTH-(1-84), and certain carboxyl truncated analogs such as hPTH-(1-34) and even smaller fragments such as hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-30)NH2 stimulate femoral trabecular and cortical bone growth in ovariectomized (OVX) rats. Here we show that when injected once daily for 6 weeks starting 2 weeks after OVX in doses of 1 or 2 nmol/100 g of body weight, hPTH-(1-31)NH2, [Leu27] cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-34)NH2 prevented the loss of trabecular volume in the L5 vertebrae induced by OVX. In fact, by the end of the sixth week of injections (i.e., the eighth week after OVX) the fragments had increased the volume and trabecular thickness significantly above the values in vehicle-injected sham-operated rats. hPTH-(1-30)NH2 can stimulate vertebral bone growth as much as the larger fragments, but 10-25 times more of it was needed to do so. The same daily doses of hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-34)NH2 also raised the trabecular volume and thickness in the L5 vertebrae of rats well above the values in vehicle-treated animals when the injections were started 9 weeks after OVX. This restoration of trabecular bone in the L5 vertebrae in estrogen-deprived animals was accompanied by a significant increase in the bone mineral density (BMD) of the L1-L4 vertebrae and tibias. However, there was no significant drop in the pelvic BMD in the estrogen-deprived animals and the effects of hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-(Lys) hPTH-(1-31)NH2, and hPTH-(1-34)NH2 on the pelvic BMD were equivocal.

Animals↗
PubMed2000

High-yield expression of fully bioactive N-terminal parathyroid hormone analog in Escherichia coli.

A fully active analog of human parathyroid hormone (hPTH) has been produced by recombinant expression in Escherichia coli. Initially, a nucleotide sequence encoding hPTH(1-34)-Asp-Pro was ligated to a proinsulin gene in the plasmid pUC8, for the eventual expression of a fusion protein of 137 amino acids. Unexpectedly, the proinsulin gene and 340 bp downstream were deleted by an unknown mechanism during transformation of the E. coli. This resulted in a new plasmid encoding a small (72-amino acid) fusion product of hPTH(1-34)-Asp35-Pro36-X, where X is a 36-residue "arbitrary" downstream sequence of pUC8. The fusion product was efficiently expressed and the hPTH analog, [Asp35]hPTH-(1-35), was readily released by acid cleavage, with a yield of 100 mg/L. This analog had an effective concentration for half-maximal adenylyl cyclase stimulation (EC50) in rat osteosarcoma cells of 14 nM, which was identical to that for hPTH-(1-34). In the ovariectomized rat model of osteoporosis, [Asp35]hPTH-(1-35) was fully active as a bone anabolic agent.

Adenylyl Cyclases↗
PubMed2000

Lactam formation increases receptor binding, adenylyl cyclase stimulation and bone growth stimulation by human parathyroid hormone (hPTH)(1-28)NH2.

Human parathyroid hormone (1-28)NH2 [hPTH(1-28)NH2] is the smallest of the PTH fragments that can fully stimulate adenylyl cyclase in ROS 17/2 rat osteoblast-like osteosarcoma cells. This fragment has an IC50 of 110 nM for displacing 125I-[Nle8,18,Tyr34]bovine PTH(1-34)NH2 from HKRK B7 porcine kidney cells, which stably express 950,000 human type 1 PTH/PTH-related protein (PTHrP) receptors (PTH1Rs) per cell. It also has an EC50 of 23.9 nM for stimulating adenylyl cyclase in ROS 17/2 cells. Increasing the amphiphilicity of the alpha-helix in the residue 17-28 region by replacing Lys27 with Leu and stabilizing the helix by forming a lactam between Glu22 and Lys26 to produce the [Leu27]cyclo(Glu22-Lys26)hPTH(1-28)NH2 analog dramatically reduced the IC50 for displacing 125I-[Nle8,18,Tyr34]bPTH(1-34)NH2 from hPTH1Rs from 110 to 6 nM and dropped the EC50 for adenylyl cyclase stimulation in ROS 17/2 cells from 23.9 to 9.6 nM. These modifications also increased the osteogenic potency of hPTH(1-28)NH2. Thus, hPTH(1-28)NH2 did not significantly stimulate either femoral or vertebral trabecular bone growth in rats when injected daily at a dose of 5 nmol/100 g body weight for 6 weeks, beginning 2 weeks after ovariectomy (OVX), but it strongly stimulated the growth of trabeculae in the cancellous bone of the distal femurs and L5 vertebrae when injected at 25 nmol/100 g body weight. By contrast [Leu27]cyclo(Glu22-Lys26)hPTH(1-28)NH2 significantly stimulated trabecular bone growth when injected at 5 nmol/100 g of body weight. Thus, these modifications have brought the bone anabolic potency of hPTH(1-28)NH2 considerably closer to the potencies of the larger PTH peptides and analogs.

Adenylyl Cyclases↗
PubMed1999

Ca(2+) and extracellular acidification rate responses to parathyroid hormone fragments in rat ROS 17/2 and human SaOS-2 cells.

To examine the importance of the N- or C-termini of PTH(1-34) the effects of truncated fragments of PTH on human receptors in osteoblast-like SaOS-2 cells and rat receptors in rats ROS 17/2 cells were examined. Fura-2-loaded cells were used to monitor cytosolic free Ca(2+) concentration ([Ca2+]i), and the Cytosensor microphysiometer was used to monitor extracellular acidification rate (ECAR). C-terminally truncated fragments (1-31) and (1-28) of hPTH(1-34)NH(2) stimulated an increase in [Ca(2+)](i) and ECAR in both cell lines. hPTH(3-34)NH(2) and other N-terminally truncated fragments did not stimulate [Ca(2+)](i) or ECAR in either cell type. The signal transduction pathway of PTH-induced ECAR in ROS 17/2 cells was investigated to compare with previous results in SaOS-2 cells. Potentiation by IBMX, attenuation by 8Br-cAMP and lack of effect of the PKC inhibitor chelerythrine chloride support a cAMP/PKA-mediated signal transduction pathway in ROS 17/2, while the protein kinase C pathway was predominant in SaOS-2 cells. We conclude that the intact N-terminus of PTH is essential in PTH signaling mediated via either the cAMP/PKA or inositol lipid/Ca(2+)/PKC pathways in osteoblast-like cells.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Changes in nuclear protein kinase C-delta holoenzyme, its catalytic fragments, and its activity in polyomavirus-transformed pyF111 rat fibroblasts while proliferating and following exposure to apoptogenic topoisomerase-II inhibitors.

Protein kinase C-delta (PKC-delta) appears to be variously involved in proliferation and apoptosis. To compare the changes of this enzyme in these two processes, we have determined the levels and activities of the 79-kDa PKC-delta holoenzyme and its catalytically active 47- and 40-kDa C-terminal fragments in the nuclei of proliferating untreated polyomavirus-transformed pyF111 rat fibroblasts and pyF111 cells treated with the apoptogenic topoisomerase-II inhibitors VP-16 (etoposide), VM-26 (teniposide), and doxorubicin. PyF111 cells were chosen because they hyperexpress PKC-delta and they are hypersusceptible to apoptosis because they do not express the antiapoptotic proteins Bcl-2 and Bcl-XL. The highest PKC-delta activity in cells before they started proliferating or were exposed to one of the inhibitors was in the NM (nuclear envelope-containing) fraction, which contained the holoenzyme and both C-terminal fragments, while only the two fragments were in the nucleoplasmic (NP) fraction where they were tightly associated with chromatin. When the cells began proliferating the amounts of the PKC-delta holoenzyme and the two fragments increased in the NM and the NP fractions and the already high PKC-delta activity either increased or stayed the same in these fractions until the end of the 72-h incubation. And there was no leakage of cytochrome c from the mitochondria into the cytoplasm. VP-16 exposure caused a prompt release of cytochrome c from the mitochondria into the cytosol and at the same time triggered a sharp drop (35% by 3 h and 60% by 6 h) in the PKC-delta activity in the NM fraction without changing the actual amounts of the holoenzyme or its fragments. This prompt inactivation of PKC-delta and its fragments during the first 6 h of exposure to the drug was not due to their dephosphorylation and could not be reversed by phosphatidylserine and/or 12-O-tetradecanoylphorbol 13-acetate (TPA). Between 6 and 24 h the PKC-delta activity in the NM fraction dropped a further 20%, the kinase's activity transiently surged in the NP fraction, and cytoplasmic CPP-32-like (DEVD-specific caspase) activity increased without an increase in the proteolysis of nuclear PKC-delta or PARP. Between 24 and 72 h nuclear CPP-32-like activity increased along with a massive proteolysis of PKC-delta, an accumulation of various PKC-delta fragments, and the cleavage of PARP. But despite this proteolysis, the cells were still able to maintain or even increase the amounts of holoenzyme and 40- and 47-kDa fragments in the NM and NP fractions before dying. VM-26 and doxorubicin caused the same prompt release of cytochrome c from the mitochondria and dramatic drop of NM PKC-delta activity as did VP-16. Thus, high levels of activity of nuclear PKC-delta, particularly PKC-delta in the nuclear membrane, might have a role driving the cell cycle of pyF111 cells. On the other hand, the prompt and sustained large drop in the activity of PKC-delta at this site that precedes the onset of the caspase-mediated proteolysis of the isoform may be involved in starting and driving apoptogenesis in pyF111 fibroblasts exposed to topoisomerase-II inhibitors.

Animals↗
PubMed1999

Stimulation of femoral trabecular bone growth in ovariectomized rats by human parathyroid hormone (hPTH)-(1-30)NH(2).

It has been proposed that intermittent bursts of adenylyl cyclase and the surges of cyclic AMP (cAMP) they produce can trigger PTH's bone anabolic action without the activation of phospholipase-C (PLC). This was based on the osteogenic action in ovariectomized (OVX) rats of hPTH-(1-31)NH(2), which can stimulate adenylyl cyclase but not PLC in ROS 17/2 rat osteosarcoma cells, and the osteogenic impotence of fragments such as 1-desamino-hPTH-(1-34) and hPTH-(8-84) which strongly stimulate PLC but not adenylyl cyclase. But this seems to have been disproven by the inability of hPTH-(1-30)NH(2) to stimulate bone growth despite its having hPTH-(1-31)NH(2)'s ability to strongly stimulate adenylyl cyclase but not PLC in cells with rat type1 PTH/PTHrP receptors. Because of the importance of hPTH-(1-30)NH(2)'s apparent osteogenic impotence for knowing how PTH triggers bone growth, we have reinvestigated the fragment's ability to stimulate trabecular bone growth in the femurs of young OVX rats and have found it to be strongly osteogenic at doses 2-10 times higher than the highest dose used previously. Thus, 6 weeks of once-daily subcutaneous injections of 10-50 nmol of hPTH-(1-30)NH(2)/100 g of body weight into young rats starting 2 weeks after OVX significantly increased the femoral trabecular volume and mean thickness of individual trabeculae above those in sham-operated control rats. In OVX rats treated with 50 nmol of hPTH-(1-30)NH(2)/100 g of body weight, the trabecular volume was 2.6 times higher and the mean trabecular thickness nearly 4 times higher than in the sham-operated control rats. This very large increase in the mean trabecular thickness was as much as the increase induced by 2 nmol/100 g of body weight of hPTH-(1-31)NH(2), [Leu(27)]cyclo(Glu(22)-Lys(26))-hPTH-(1-31)NH(2), hPTH-(1-34)NH(2) and [Leu(27)]cyclo(Glu(22)-Lys(26))-hPTH-(1-34)NH(2). These results have removed a major objection to the proposal that PTH's osteogenic action in rats can be triggered solely by intermittent surges of cAMP and the bursts of cAMP-dependent protein kinase activity they cause.

Animals↗
PubMed1999

Stimulation of membrane-associated protein kinase-C activity in spleen lymphocytes by hPTH-(1-31)NH2, its lactam derivative, [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, and hPTH-(1-30)NH2.

Human parathyroid hormone, hPTH-(1-34), stimulates adenylyl cyclase and phosphatidylinositol-bisphosphate-specific phospholipase-C (PIP2-PLC), as indicated by increased membrane-associated protein kinase C (PKC) activity in ROS 17/2 rat osteosarcoma cells. The C-terminally truncated hPTH-(1-31)NH2 stimulates adenylyl cyclase as strongly as hPTH-(1-34) in these cells, but it does not stimulate PKC activity. Even [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, a 6-fold stronger adenylyl cyclase stimulator than hPTH-(1-34), cannot stimulate PKC activity in ROS cells. Therefore PTH required its 32-34 region to stimulate PIP2-PLC/PKCs in this osteosarcoma line. In contrast, hPTH-(1-31)NH2 [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 and even hPTH-(1-30)NH2 can stimulate PKC activity in freshly isolated rat spleen lymphocytes as strongly as hPTH-(1-34)NH2. The difference in the ability of membrane-associated PKC activity in spleen lymphocytes, but not in ROS cells, to be stimulated by C-terminally truncated PTH fragments might be due to different receptor densities or to the lymphocyte's atypical PTH/PTHrP receptor.

Animals↗