Engineering a stable and selective peptide blocker of the Kv1.3 channel in T lymphocytes.

Kv1.3 potassium channels maintain the membrane potential of effector memory (T(EM)) T cells that are important mediators of multiple sclerosis, type 1 diabetes mellitus, and rheumatoid arthritis. The polypeptide ShK-170 (ShK-L5), containing an N-terminal phosphotyrosine extension of the Stichodactyla helianthus ShK toxin, is a potent and selective blocker of these channels. However, a stability study of ShK-170 showed minor pH-related hydrolysis and oxidation byproducts that were exacerbated by increasing temperatures. We therefore engineered a series of analogs to minimize the formation of these byproducts. The analog with the greatest stability, ShK-192, contains a nonhydrolyzable phosphotyrosine surrogate, a methionine isostere, and a C-terminal amide. ShK-192 shows the same overall fold as ShK, and there is no evidence of any interaction between the N-terminal adduct and the rest of the peptide. The docking configuration of ShK-192 in Kv1.3 shows the N-terminal para-phosphonophenylalanine group lying at the junction of two channel monomers to form a salt bridge with Lys(411) of the channel. ShK-192 blocks Kv1.3 with an IC(50) of 140 pM and exhibits greater than 100-fold selectivity over closely related channels. After a single subcutaneous injection of 100 microg/kg, approximately 100 to 200 pM concentrations of active peptide is detectable in the blood of Lewis rats 24, 48, and 72 h after the injection. ShK-192 effectively inhibits the proliferation of T(EM) cells and suppresses delayed type hypersensitivity when administered at 10 or 100 microg/kg by subcutaneous injection once daily. ShK-192 has potential as a therapeutic for autoimmune diseases mediated by T(EM) cells.

Osteoclast formation and bone resorption are inhibited by megakaryocytes.

It has been previously reported that addition of megakaryocytes (MKs) to osteoblasts in vitro results in increased osteoblastic collagen and osteoprotegerin (OPG) production, suggesting a role for MKs in bone formation. To further investigate this role, we have studied the effects of MKs on osteoclast formation and activity. Human osteoclasts were generated from CD14 monocytes isolated from peripheral blood and cultured in the presence of M-CSF and sRANKL on dentine and calcium phosphate substrates. MKs were generated from CD34+ cells isolated from either human peripheral blood or cord blood and cultured in liquid medium for 6 days, after which time maturing MKs (CD61-positive cells) were isolated and added to monocyte cultures. After 6 and 9 days of culture, the number of osteoclasts identified morphologically and by TRAP staining was counted. Cells were removed and the area of resorption was identified by von Kossa staining and quantitatively assessed by image analysis. The addition of MKs to osteoclast cultures at day 0 inhibited the number of osteoclasts formed 1.9-fold (p>0.003), whereas addition at 3 days had no effect on osteoclast number. The presence of MKs inhibited resorption 8.7-fold when co-cultured with osteoclasts from day 0 (p>0.004), but only by 3.1-fold when co-cultured from day 3 (p>0.01). In dose-response experiments, it was found that 1-10% of MKs added to monocyte cultures elicited the greatest inhibition of resorption. Similar osteoclast cultures were treated with CD61-negative cells (non-MKs) to confirm that the inhibition of osteoclast formation and activity was specifically due to MKs. Experiments with a cell-impermeable membrane indicated that both cell to cell contact and release of soluble factor(s) were involved in mediating these effects. These results show that MKs inhibit osteoclast formation and activity. The most pronounced effects were seen when MKs and osteoclasts were co-cultured from day 0, suggesting that MKs act primarily on osteoclast precursors.

Circulating levels of interleukin-6 and its soluble receptor in patients with head injury and fracture.

There is evidence that fractures heal more rapidly in patients with head injury. We measured the circulating level of interleukin-6 (IL-6) and its soluble receptor (sIL-6R) and soluble glycoprotein 130 (sgp130) in serum from patients who had sustained a head injury with and without fracture and compared these with levels found in control subjects. Within 12 hours of injury the serum level of IL-6 was significantly higher in patients with head injury and fracture compared with the control group. Levels of IL-6 were also significantly higher in patients with head injury and fracture compared with fracture only. While there was no significant difference in circulating levels of sIL-6R in the initial samples they were increased one week after surgery in patients with head injury and fracture and with head injury only. In addition, reduced levels of sgp130 in patients with head injury with and without fracture indicated a possible reduction of the inhibitory effect of this protein on the activity of IL-6. Our study suggests that IL-6 may be involved in altered healing of a fracture after head injury.

[Ion channels and demyelination: basis of a treatment of experimental autoimmune encephalomyelitis (EAE) by potassium channel blockers]. / Canaux ioniques et démyélinisation: les fondements d'un traitement de l'encéphalomyélite autoimmune expérimentale (EAE) par des bloqueurs des canaux potassium.

Voltage-gated potassium channels (Kv channels) are ion channels, openings of which provide an outward flow of potassium ions repolarising the cell. In neurons, Kv channels play a crucial role in action potential repolarisation and in shaping neuronal excitability. In non-excitable cells, such as T lymphocytes, Kv channels and calcium-activated K+ channels (KCa channels) determine the driving force for Ca2+ entry. During T cell activation the calcium entry depolarises the cell and increases the cytosolic calcium concentration, which in return activates Kv and KCa channels. K+ channel opening repolarises the cell and drives the membrane potential to a negative voltage. The roles of Kv channels in nervous and immune systems have been investigated here by means of a rat experimental autoimmune disease of the central nervous system, the experimental autoimmune encephalomyelitis (EAE). EAE is characterised clinically by paralysis, and pathologically by inflammatory cell infiltrations into the brain and the spinal cord. Among the inflammatory cells, T lymphocytes play a major role. Hence, EAE can be adoptively transferred into syngenic animals by the injection of T cells reactive to myelin antigens. During adoptive-EAE, somato-sensory evoked potentials recorded along the spinal tracts decrease in amplitude and axonal propagation is disrupted. We have analysed the consequences of Kv channels blockade by peptidyl toxins on central nerve conduction, on T cell activation and on the time course of EAE. In rat optic nerves, Kv channels have been identified up from postnatal day 1. Their blockade by kaliotoxin (a scorpion toxin) or by dendrotoxin-I (a snake toxin) enlarges the compound action potentials, demonstrating the participation of Kv channels to spike repolarisation. This effect disappears at adult age due to the sequestration of Kv channels under the myelin, in the paranodal regions. During acute demyelination by lysophosphatidyl-choline, the surface area of compound action potential decreased probably because conduction block occurred. Demyelination unmasked Kv channels, which are again accessible to toxins. Their blockade by dendrotoxin-I or kaliotoxin favoured a slow delayed conduction suggesting that those Kv channel blockers exert a neurological benefit during demyelinating diseases. In a T-cell line reactive to myelin basic protein antigen, which is used to adoptively transfer experimental autoimmune encephalomyelitis, Kv1.3 channels are constitutively expressed. Their blockade leads to a pronounced reduction of the T cell proliferative response, cytokine production and Ca2+ influx. In the rat, blockade of Kv1.3 inhibits the delayed type hypersensitivity response to myelin basic protein prevents and treats adoptive experimental autoimmune encephalomyelitis. Blockade of Kv channels alone or in combination with KCa channels improves the symptoms of the disease. These results demonstrate that K+ channel blockers displaying high selectivity are potent immunosuppressive agents with beneficial symptomatic effects in experimental autoimmune encephalomyelitis.

Circulating levels of insulin-like growth factor-1 and insulin-like growth factor binding protein-3 in patients with severe head injury.

There is evidence to suggest that fractures heal more rapidly in patients with a head injury as a result of systemic factors released from the site of this injury. We have measured the circulating level of insulin-like growth factor-1 (IGF-1) and IGF binding protein-3 (IGFBP-3) in serum because of their known involvement in the stimulation of the activity of osteoblasts and the healing of fractures. The serum level of IGF-1 was significantly lower in patients with both head injury and fracture and fracture only compared with that in healthy volunteers (p < 0.01 and p < 0.02, respectively). The level of IGFBP-3 was also significantly lower in patients with both head injury and fracture (p < 0.01). Our findings showed, however, that the level of IGF-1 and IGFBP-3 varied from week to week in both the patients and healthy control subjects. These results indicate that the levels of circulating IGF-1 and IGFBP-3 are unlikely to be responsible for the altered healing of fractures seen in conjunction with head injury.

Selective blockade of T lymphocyte K(+) channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis.

Adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE), a disease resembling multiple sclerosis, is induced in rats by myelin basic protein (MBP)-activated CD4(+) T lymphocytes. By patch-clamp analysis, encephalitogenic rat T cells stimulated repeatedly in vitro expressed a unique channel phenotype ("chronically activated") with large numbers of Kv1.3 voltage-gated channels (approximately 1500 per cell) and small numbers of IKCa1 Ca(2+)-activated K(+) channels (approximately 50-120 per cell). In contrast, resting T cells displayed 0-10 Kv1.3 and 10-20 IKCa1 channels per cell ("quiescent" phenotype), whereas T cells stimulated once or twice expressed approximately 200 Kv1.3 and approximately 350 IKCa1 channels per cell ("acutely activated" phenotype). Consistent with their channel phenotype, [(3)H]thymidine incorporation by MBP-stimulated chronically activated T cells was suppressed by the peptide ShK, a blocker of Kv1.3 and IKCa1, and by an analog (ShK-Dap(22)) engineered to be highly specific for Kv1.3, but not by a selective IKCa1 blocker (TRAM-34). The combination of ShK-Dap(22) and TRAM-34 enhanced the suppression of MBP-stimulated T cell proliferation. Based on these in vitro results, we assessed the efficacy of K(+) channel blockers in AT-EAE. Specific and simultaneous blockade of the T cell channels by ShK or by a combination of ShK-Dap(22) plus TRAM-34 prevented lethal AT-EAE. Blockade of Kv1.3 alone with ShK-Dap(22), but not of IKCa1 with TRAM-34, was also effective. When administered after the onset of symptoms, ShK or the combination of ShK-Dap(22) plus TRAM-34 greatly ameliorated the clinical course of both moderate and severe AT-EAE. We conclude that selective targeting of Kv1.3, alone or with IKCa1, may provide an effective new mode of therapy for multiple sclerosis.

Early joint erosions and serum levels of matrix metalloproteinase 1, matrix metalloproteinase 3, and tissue inhibitor of metalloproteinases 1 in rheumatoid arthritis.

OBJECTIVE: To further evaluate the roles of matrix metalloproteinase 1 (MMP-1), MMP-3, and tissue inhibitor of metalloproteinases 1 (TIMP-1) in the pathogenesis of joint inflammation and articular erosions in early inflammatory arthritis. METHODS: Untreated patients with joint symptoms for <2 years were evaluated at presentation and followed up prospectively for 18 months. Swollen joint count and serum levels of C-reactive protein (CRP) were determined every 6 months. Serum levels of MMP-1, MMP-3, and TIMP-1 were measured by double-antibody sandwich enzyme-linked immunosorbent assay at the same time intervals. The number of joint erosions in serial radiographs of the hands and feet was also recorded. Analysis of synovial fluid levels of MMPs and TIMP-1 at presentation was completed in some patients. RESULTS: Of 175 patients evaluated at baseline, 85 had rheumatoid arthritis (RA), 39 had seronegative spondylarthropathy, 38 had undifferentiated arthritis, and 13 had self-limiting arthritis. Of 164 patients with available radiographs of the hands and feet at presentation, 33 (20.1%) had joint erosions. Baseline levels of MMP-1, MMP-3, and TIMP-1 were significantly higher (P = 0.0001, P = 0.013, and P = 0.0001, respectively) and ratios of TIMP-1:MMP-1 and TIMP-1:MMP-3 were significantly lower (P = 0.0001 and P = 0.013, respectively) in RA versus non-RA patients. In RA patients, serum levels of CRP correlated with MMP-3 and TIMP-1 levels, but not with MMP-1 levels. The number of erosions at presentation correlated with baseline levels of both MMP-1 and MMP-3, but not with levels of TIMP-1. One hundred one patients were followed up for the next 18 months. The number of patients with erosions and the number of erosions per patient increased significantly during this period. Area under the curve (AUC) measurements of MMP-1 and TIMP-1 levels, but not of MMP-3 levels, yielded significantly higher values in RA than in non-RA patients. In RA patients, only the AUC level of MMP-3 correlated with the AUC CRP level (r = 0.67, P = 0.0001), while only the AUC level of MMP-1 correlated with the number of new joint erosions (r = 0.28, P = 0.034). CONCLUSION: These data suggest an uncoupling of the pathophysiologic mechanisms associated with joint inflammation and articular erosion. Treatments that inhibit the production and activity of MMP-1 may preferentially limit the formation of new joint erosions and improve the long-term functional outcome of some patients with inflammatory arthritis.

Serum TIMP-1, TIMP-2, and MMP-1 in patients with systemic sclerosis, primary Raynaud's phenomenon, and in normal controls.

BACKGROUND: Excess tissue matrix accumulates in systemic sclerosis (SSc), accounting for both visceral and dermal fibrosis. It is suggested that decreased serum levels of matrix metalloproteinases (MMPs) or increased levels of tissue inhibitors of matrix metalloproteinases (TIMPs) may account for this matrix accumulation. OBJECTIVE: To measure serum levels of tissue inhibitors of metalloproteinases, TIMP-1, TIMP-2, and collagenase-1 (MMP-1), in patients with diffuse cutaneous systemic sclerosis (dcSSc), limited cutaneous systemic sclerosis (lcSSc), primary Raynaud's phenomenon (RP), and in normal controls. METHODS: Serum samples from patients with dcSSc (n=83), lcSSc (n=87), RP (n=80), and normal controls (n=98) were analysed using enzyme linked immunosorbent assays (ELISAs) for total TIMP-1, TIMP-2, and MMP-1. Results from each assay were analysed by the Kruskal-Wallis test. Dunn's multiple comparison post-test was then applied between groups. RESULTS: TIMP-1 levels were significantly raised in dcSSc and lcSSc groups compared with the RP group and normal controls (p<0.01 to p<0.001). In the dcSSc group, TIMP-1 levels were significantly higher in early disease (<2 years) than in late stage disease (>4 years) (p<0.05). This was not found for the lcSSc group. Serum TIMP-2 and MMP-1 levels in dcSSc and lcSSc did not differ significantly from those in normal controls. Increased levels of TIMPs were not convincingly associated with organ disease. No assay result correlated with autoantibody status (anti-topoisomerase 1 (anti-Scl-70), anticentromere antibody, or anti-RNA polymerase). No significant differences in serum TIMP-1, TIMP-2, or MMP-1 levels were shown in the RP group compared with normal controls. CONCLUSIONS: Raised TIMP-1 levels in the SSc groups support the hypothesis that matrix accumulation occurs in SSc at least in part owing to decreased degradation. Moreover, the variation in TIMP-1 levels between the early and late disease stages of dcSSc seems to reflect the early progressive course of dermal fibrosis seen clinically. The expected reduction in serum MMP-1 levels in the SSc groups was not found. This suggests that tissue matrix accumulation is due to increased inhibitors rather than to decreased MMPs.

Selective blocking of voltage-gated K+ channels improves experimental autoimmune encephalomyelitis and inhibits T cell activation.

Kaliotoxin (KTX), a blocker of voltage-gated potassium channels (Kv), is highly selective for Kv1.1 and Kv1.3. First, Kv1.3 is expressed by T lymphocytes. Blockers of Kv1.3 inhibit T lymphocyte activation. Second, Kv1.1 is found in paranodal regions of axons in the central nervous system. Kv blockers improve the impaired neuronal conduction of demyelinated axons in vitro and potentiate the synaptic transmission. Therefore, we investigated the therapeutic properties of KTX via its immunosuppressive and symptomatic neurological effects, using experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. The T line cells used to induce adoptive EAE were myelin basic protein (MBP)-specific, constitutively contained mRNA for Kv1.3. and expressed Kv1.3. These channels were shown to be blocked by KTX. Activation is a crucial step for MBP T cells to become encephalitogenic. The addition of KTX during Ag-T cell activation led to a great reduction in the MBP T cell proliferative response, in the production of IL-2 and TNF, and in Ca(2+) influx. Furthermore, the addition of KTX during T cell activation in vitro led a decreased encephalitogenicity of MBP T cells. Moreover, KTX injected into Lewis rats impaired T cell function such as the delayed-type hypersensitivity. Lastly, the administration of this blocker of neuronal and lymphocyte channels to Lewis rats improved the symptoms of EAE. We conclude that KTX is a potent immunosuppressive agent with beneficial effects on the neurological symptoms of EAE.

Comparison of the kinetic properties of the lipid- and protein-kinase activities of the p110alpha and p110beta catalytic subunits of class-Ia phosphoinositide 3-kinases.

Growth factors regulate a wide range of cellular processes via activation of the class-Ia phosphoinositide 3-kinases (PI 3-kinases). We directly compared kinetic properties of lipid- and protein-kinase activities of the widely expressed p110alpha and p110beta isoforms. The lipid-kinase activity did not display Michaelis-Menten kinetics but modelling the kinetic data demonstrated that p110alpha has a higher V(max) and a 25-fold higher K(m) for PtdIns than p110beta. A similar situation occurs with PtdIns(4,5)P(2), because at low concentration of PtdIns(4,5)P(2) p110beta is a better PtdIns(4,5)P(2) kinase than p110alpha, although this is reversed at high concentrations. These differences suggest different functional roles and we hypothesize that p110beta functions better in areas of membranes containing low levels of substrate whereas p110alpha would work best in areas of high substrate density such as membrane lipid rafts. We also compared protein-kinase activities. We found that p110beta phosphorylated p85 to a lower degree than did p110alpha. We used a novel peptide-based assay to compare the kinetics of the protein-kinase activities of p110alpha and p110beta. These studies revealed that, like the lipid-kinase activity, the protein-kinase activity of p110alpha has a higher K(m) (550 microM) than p110beta (K(m) 8 microgM). Similarly, the relative V(max) towards peptide substrate of p110alpha was three times higher than that of p110beta. This implies differences in the rates of regulatory autophosphorylation in vivo, which are likely to mean differential regulation of the lipid-kinase activities of p110alpha and p110beta in vivo.

Natural variants of human p85 alpha phosphoinositide 3-kinase in severe insulin resistance: a novel variant with impaired insulin-stimulated lipid kinase activity.

AIMS/HYPOTHESIS: Phosphoinositide 3-kinase (PI 3K) plays a central part in the mediation of insulin-stimulated glucose disposal. No genetic studies of this enzyme in human syndromes of severe insulin resistance have been previously reported. METHODS: Phosphoinositide 3-kinase p85 alpha regulatory subunit cDNA was examined in 20 subjects with syndromes of severe insulin resistance by single strand conformational polymorphism and restriction fragment length polymorphism analyses. Insulin-stimulated phosphoinositide 3-kinase activity and recruitment into phosphotyrosine complexes of variants of p85 alpha were studied in transiently transfected HEK293 cells. Phosphopeptide binding characteristics of wild-type and mutant p85 alpha-GST fusion proteins were examined by surface plasmon resonance. RESULTS: The common p85 alpha variant, Met326I1e, was identified in 9 of the 20 subjects. Functional studies of the Met326Ile variant showed it to have equivalent insulin-stimulated lipid kinase activity and phosphotyrosine recruitment as wild-type p85 alpha. A novel heterozygous mutation, Arg409Gln, was detected in one subject. Within the proband's family, carriers of the mutation had a higher median fasting plasma insulin (218 pmol/l) compared with wild-type relatives (72 mol/l) (n = 8 subjects, p = 0.06). The Arg409Gln p85 alpha subunit was associated with lower insulin-stimulated phosphoinositide 3-kinase activity compared with wild-type (mean reduction 15%, p < 0.05, n = 5). The recruitment of Arg409Gln p85 alpha into phosphotyrosine complexes was not significantly impaired. GST fusion proteins of wild-type and mutant p85 alpha showed identical binding to phosphopeptides in surface plasmon resonance studies. CONCLUSION/INTERPRETATION: Mutations in p85 alpha are uncommon in subjects with syndromes of severe insulin resistance. The Met326Ile p85 alpha variant appears to have no functional effect on the insulin-stimulated phosphoinositide 3-kinase activity. The impaired phosphoinositide 3-kinase activity of the Arg409Gln mutant suggests that it could contribute to the insulin resistance seen in this family.

The SH3 and BH domains of the p85alpha adapter subunit play a critical role in regulating class Ia phosphoinositide 3-kinase function.

We have investigated the role of the SH3 and BH domains in the function of the p85alpha adapter/regulatory subunit of PI 3-kinase. In these studies epitope-tagged adapter subunit constructs containing wild-type p85alpha, p85alpha lacking the SH3 domain (deltaSH3-p85alpha), or p85alpha lacking the Rac-GAP/BCR homology (BH) domain (deltaBH-p85alpha) were coexpressed with either the p110alpha or p110beta PI 3-kinase catalytic subunit in HEK293 cells. The deletion of either BH or SH3 domains had no effect on the intrinsic activity of the PI 3-kinase heterodimers. However, the ability of activated Rac to stimulate PI 3-kinase activity was only observed in heterodimers containing the p85alpha and deltaSH3-p85alpha, indicating that rac binding to the BH domain is responsible for rac-induced stimulation of class Ia PI 3-kinase. We also investigated the effect of SH3 and BH domain deletion on the ability of insulin to induce recruitment of these constructs into phosphotyrosine-containing signaling complexes. We find that p85alpha expressed alone is poorly recruited into such signaling complexes. However, when coexpressed with catalytic subunit, the p85alpha adapter subunit is recruited to an extent similar to that of endogenous p85alpha. Maximal insulin stimulation caused a similar level of recruitment of p85alpha, deltaSH3-p85alpha, and deltaBH-p85alpha to signaling complexes when these adapter subunits were coexpressed with catalytic subunit. However, there was a higher level of basal association of the deltaSH3-p85alpha and deltaBH-p85alpha with tyrosine-phosphorylated proteins, meaning that the insulin-induced fold increase in recruitment was lower for these forms of the adapter. These results indicate that the N-terminal domains of p85alpha play a critical role in the way the adapter subunit responds to growth factor stimulation.

Immunolocalisation of vascular endothelial growth factor (VEGF) in human neonatal growth plate cartilage.

Angiogenesis is essential for the replacement of cartilage by bone during growth and repair. In order to obtain a better understanding of the mechanisms regulating vascular invasion at sites of endochondral ossification we have investigated the expression of the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), by chondrocytes in human neonatal growth plates. VEGF was absent from chondrocytes in the resting zone and only weakly expressed by occasional chondrocytes in the proliferating region. In the hypertrophic zone the number of chondrocytes stained and the intensity of staining for VEGF increased with chondrocyte hypertrophy, maximum expression of VEGF being observed in chondrocytes in the lower hypertrophic and mineralised regions of the cartilage. These observations provide the first demonstration of the presence of VEGF in situ in developing human bone and are consistent with in vitro observations demonstrating the upregulation of proangiogenic growth factor production with increasing chondrocyte hypertrophy. The presence of numerous small blood vessels and vascular structures in the subchondral region where VEGF expression was maximal indicates that VEGF produced by hypertrophic chondrocytes may play a key role in the regulation of vascular invasion of the growth plate.

Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) distribution in normal and pathological human bone.

Degradation of skeletal connective tissue is regulated, at least in part, by the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinase (TIMPs), their natural inhibitors. The balance between MMPs and TIMPs may therefore be a determinant of normal bone turnover, and imbalance could thus lead to reduced organization of bone structure. To test this hypothesis, the cellular expression of MMPs and TIMP-1 was investigated by immunohistochemistry in human neonatal rib and osteophytic and heterotopic bone; these differ in their structure, with heterotopic bone showing the least and normal rib the most organized development. In all samples, high levels of MMPs were expressed. Collagenase and stromelysin-2 were detected in chondrocytes, osteoblasts, and osteoclasts, whereas gelatinase-B was confined to osteoclasts and mononuclear cells. Matrix-associated stromelysin-1 was present in fibrous tissue and osteoid. In contrast, the expression of TIMP-1 varied markedly between the three types of bone. In heterotopic bone only occasional low level TIMP-1 expression was detected in chondrocytes and osteoblasts. Osteophytic bone showed varying levels of TIMP-1, which was matrix-bound in fibrous tissue and cell-associated in osteoblasts, chondrocytes, and occasional mononuclear cells. In both types of bone, expression of TIMP-1 by osteoclasts was absent despite large numbers of these cells. Neonatal rib bone showed consistent expression of TIMP-1, particularly in chondrocytes, osteoblasts, and lining cells. In contrast to pathological bone, many osteoclasts were TIMP-1 positive. These results suggest that, in heterotopic and osteophytic bone, the low levels of TIMP-1, and in particular its absence in osteoclasts, may partly explain the more poorly organized bone formation in these pathological bone samples. Furthermore, TIMP-1 may play a role in the regulation of bone modeling and remodeling in normal developing human bone.

Insulin-like growth factors require phosphatidylinositol 3-kinase to signal myogenesis: dominant negative p85 expression blocks differentiation of L6E9 muscle cells.

Phosphatidylinositol 3 (PI 3)-kinases are potently inhibited by two structurally unrelated membrane-permeant reagents: wortmannin and LY294002. By using these two inhibitors we first suggested the involvement of a PI 3-kinase activity in muscle cell differentiation. However, several reports have described that these compounds are not as selective for PI 3-kinase activity as assumed. Here we show that LY294002 blocks the myogenic pathway elicited by insulin-like growth factors (IGFs), and we confirm the specific involvement of PI 3-kinase in IGF-induced myogenesis by overexpressing in L6E9 myoblasts a dominant negative p85 PI 3-kinase-regulatory subunit (L6E9-delta p85). IGF-I, des(1-3)IGF-I, or IGF-II induced L6E9 skeletal muscle cell differentiation as measured by myotube formation, myogenin gene expression, and GLUT4 glucose carrier induction. The addition of LY294002 to the differentiation medium totally inhibited these IGF-induced myogenic events without altering the expression of a non-muscle-specific protein, beta1-integrin. Independent clones of L6E9 myoblasts expressing a dominant negative mutant of the p85-regulatory subunit (delta p85) showed markedly impaired glucose transport activity and formation of p85/p110 complexes in response to insulin, consistent with the inhibition of PI 3-kinase activity. IGF-induced myogenic parameters in L6E9-delta p85 cells, ie. cell fusion and myogenin gene and GLUT4 expression, were severely impaired compared with parental cells or L6E9 cells expressing wild-type p85. In all, data presented here indicate that PI 3-kinase is essential for IGF-induced muscle differentiation and that the specific PI 3-kinase subclass involved in myogenesis is the heterodimeric p85-p110 enzyme.

Plasma morphine levels produced by continuous infusion in children.

Blood samples were taken from six children aged between 10 months and 15 years, at intervals over a period of 40 hours while they were receiving continuous morphine infusions. The plasma morphine values obtained showed similar and consistent levels 15-30 minutes after starting the infusions.