Pseudarthrosis is an exceedingly common, costly, and morbid complication in the treatment of long bone fractures and after spinal fusion surgery. Electrical bone growth stimulation (EBGS) presents a unique approach to accelerate healing and promote fusion success rates. Over the past three decades, increased experience and widespread use of EBGS devices has led to significant improvements in stimulation paradigms and clinical outcomes. In this paper, we comprehensively review the literature and examine the history, scientific evidence, available technology, and clinical applications for EBGS. We summarize indications, limitations, and provide an overview of cost-effectiveness and future directions of EBGS technology. Various models of electrical stimulation have been proposed and marketed as adjuncts for spinal fusions and long bone fractures. Clinical studies show variable safety and efficacy of EBGS under different conditions and clinical scenarios. While the results of clinical trials do not support indiscriminate EBGS utilization for any bone injury, the evidence does suggest that EBGS is desirable and cost efficient for certain orthopedic indications, especially when used in combination with standard, first-line treatments. This review should serve as a reference to inform practicing clinicians of available treatment options, facilitate evidence-based decision making, and provide a platform for further research.

Calmodulin (CaM) is a ubiquitous Ca(2+) receptor protein mediating a large number of signaling processes in all eukaryotic cells. CaM plays a central role in regulating a myriad of cellular functions via interaction with multiple target proteins. This review focuses on the action of CaM and CaM-dependent signaling systems in the control of vertebrate cell proliferation, programmed cell death and autophagy. The significance of CaM and interconnected CaM-regulated systems for the physiology of cancer cells including tumor stem cells, and processes required for tumor progression such as growth, tumor-associated angiogenesis and metastasis are highlighted. Furthermore, the potential targeting of CaM-dependent signaling processes for therapeutic use is discussed.

Calmodulin (CaM) is one of the most well-studied Ca(2+) transducers in eukaryotic cells. It is known to regulate the activity of numerous proteins with diverse cellular functions; however, the functions of apoplastic CaM in plant cells are still poorly understood. By combining pharmacological analysis and microscopic techniques, we investigated the involvement of apoplastic CaM in pollen tube growth of Cedrus deodara (Roxb.) Loud. It was found that the tip-focused calcium gradient was rapidly disturbed as one of the early events after application of pharmacological agents, while the cytoplasmic organization was not significantly affected. The deposition and distribution of acidic pectins and esterified pectins were also dramatically changed, further perturbing the normal modeling of the cell wall. Several protein candidates from different functional categories may be involved in the responses to inhibition of apoplastic CaM. These results revealed that apoplastic CaM functions to maintain the tip-focused calcium gradient and to modulate the distribution/transformation of pectins during pollen tube growth.

Photocurrent therapy with participation of light and electrical stimulations could be an innovative and promising approach in regenerative medicine, especially for skin and nerve regeneration. Photocurrent is generated when light irradiates on a photosensitive device, and with more and more types of photosensitive materials being synthesized, photocurrent could be applied for enhanced regeneration of tissue. Photosensitive scaffolds such as composite poly (3-hexylthiophene)/polycaprolactone (P3HT/PCL) nanofibers are fabricated by electrospinning process in our lab for skin regeneration in presence of applied photocurrent. This review article discuss on the various in vitro, in vivo and clinical studies that utilized the principle of 'electrotherapy' and 'phototherapy' for regenerative medicine and evaluates the potential application of photocurrent in regenerative medicine. We conclude that photocurrent therapy will play an important role in regenerative medicine.

Traditionally, calmodulin (CaM) was thought to be a multi-functional receptor for intracellular Ca(2+) signals. But in the last ten years, it was found that CaM also exists and acts extracellularly in animal and plant cells to regulate many important physiological functions. Laboratory studies by the authors showed that extracellular CaM in plant cells can stimulate the proliferation of suspension cultured cell and protoplast; regulate pollen germination and pollen tube elongation, and stimulate the light-independent gene expression of Rubisco small subunit (rbcS). Furthermore, we defined the trans-membrane and intracellular signal transduction pathways for extracellular CaM by using a pollen system. The components in this pathway include heterotrimeric G-protein, phospholipase C, IP(3), calcium signal and protein phosphorylation etc. Based on our findings, we suggest that extracellular CaM is a polypeptide signal in plants. This idea strongly argues against the traditional concept that there is no intercellular polypeptide signal in plants.

Calmodulin-like protein (CaLP) was believed to be involved in the shell formation of pearl oyster. However, no further study of this protein was ever performed. In this study, the in vitro crystallization experiment showed that CaLP can modify the morphology of calcite. In addition, aragonite crystals can be induced in the mixture of CaLP and a nacre protein (at 16 kDa), which was detected and purified from the EDTA-soluble matrix of nacre. These results agreed with that of immunohistological staining in which CaLP was detected not only in the organic layer sandwiched between nacre (aragonite) and the prismatic layer (calcite), but also around the prisms of the prismatic layer. Take together, we concluded that (1) CaLP, as a component of the organic layer, can induce the nucleation of aragonite through binding with the 16-kDa protein, and (2) CaLP may regulate the growth of calcite in the prismatic layer.

Calmodulin, a highly conserved protein family that has long been well known as an intracellular calcium sensor, was identified in the culture medium and cell walls of Arabidopsis thaliana suspension-cultured cells by immunoblotting assay. A promotion effect by applying exogenous purified calmodulin and an inhibition effect by the addition of anti-calmodulin anti-serum or calmodulin antagonist to the medium on proliferation of suspension cells were found by monitoring incorporation of [methyl-3H]thymidine into nuclear DNA. Radioligand binding analysis with 35S-labeled calmodulin indicated the presence of specific, reversible, and saturable calmodulin binding sites on the surface of both A. thaliana suspension-cultured cells and its protoplasts; among them at least one is on the surface of Arabidopsis protoplasts, with the Kd approximately 9.2 nM, and two are on the out-surface of Arabidopsis suspension-cultured cells, with Kd values of approximately 47.5 and 830 nM. Chemical crosslinking of 35S-labeled calmodulin to protoplasts revealed 117- and 41-kDa plasma membrane proteins specifically bound to calmodulin, whereas cross-linking with intact suspension-cultured cells verified more calmodulin binding proteins which might be cell wall-associated in addition to membrane-localized. Taking together, our data provide first evidence for the presence of apoplastic calmodulin receptor-like binding proteins on the cell surface of Arabidopsis suspension-cultured cells, which strongly supports our previous idea that apoplastic calmodulin functions as a peptide signal involved in regulation of cell growth and development.

An important feature of male fertility is the physiological priming of spermatozoa by a multifaceted process collectively referred to as capacitation. The end point of this evasive process is the hyperactivated spermatozoa capable of binding to terminal sugar residues on the egg's extracellular coat, the zona pellucida (ZP), and undergoing acrosomal exocytosis (i.e., induction of the acrosome reaction). The hydrolytic action of acrosomal enzymes released at the site of zona binding, along with the enhanced thrust generated by the hyperactivated beat pattern of the bound spermatozoa, are important factors that regulate the penetration of ZP and fertilization of the egg. Despite many advances in identifying sperm components that promote capacitation, the mechanism underlying the calcium-triggered process remains elusive. The purpose of this review article is to focus on new advances that have enhanced our understanding of in vivo/in vitro capacitation, a prerequisite event resulting from a dramatic modification and reorganization of the sperm membrane molecules. Special emphasis has been laid on accumulating evidence suggesting potential similarities between the sperm capacitation and early phases of calcium-triggered membrane fusion (i.e., tethering and docking) during secretory and endocytotic pathways among eukaryotes.

Calmodulin (CaM) is an essential component of calcium signaling in multicellular organisms. We used null mutations of the Drosophila CaM gene (Cam) in combination with clonal analysis and immunolocalization to examine the effects of loss of Cam function in the ovarian germline and developing embryo. These studies have uncovered unexpected and striking movements of CaM protein within these tissues. In the ovary, evidence for transfer of CaM from an external source, across plasma membranes, into the germline cells was obtained. In late embryogenesis, maternally derived CaM protein relocalizes dramatically within the nervous system of both wildtype and Cam null embryos-a process that may also involve movement across cell membranes. These findings indicate dynamic, unsuspected elements to the in vivo functions of CaM in the whole organism.

Sperm capacitation in vitro is thought to be correlated with the increased protein tyrosine phosphorylation of a subset of sperm components. Our group recently used a pharmacological approach to demonstrate that calmodulin (CaM), a 17 kDa calcium sensor protein, has a role in sperm capacitation. In the present study, we have used several CaM antagonists in an attempt to characterize further the role of CaM in capacitation-associated protein tyrosine phosphorylation of sperm components. Our data demonstrate, first, that mouse spermatozoa incubated in a medium that favors capacitation undergo increased protein tyrosine phosphorylation in a time-dependent manner. Second, inclusion of six CaM antagonists individually in an in vitro incubation medium prevented sperm capacitation, as demonstrated by their diminished ability to undergo agonist-induced acrosome reaction. Third, half of the CaM antagonists (compound 48/80, W13 and CaM-binding domain) had no effect on protein tyrosine phosphorylation or sperm motility. Fourth, by contrast, three CaM antagonists (W7, ophiobolin A and calmidazolium) significantly inhibited protein tyrosine phosphorylation of sperm components (42, 56, 66, 82 and 95 kDa) and adversely affected their motility without altering viability as assessed by propidium iodine staining. Finally, inclusion of purified CaM in the capacitation medium significantly increased tyrosine phosphorylation of 82 kDa and 95 kDa components. Combined, these data suggest that CaM antagonists prevent capacitation by interfering with multiple regulatory pathways, and do so either with or without adverse effects on sperm motility and protein tyrosine phosphorylation.

The RbcS genes encode the small subunits of rubisco; the expression of these genes is controlled in a light-dependent and independent manner. It has been reported that intracellular calmodulin (CaM) is involved in light-dependent RbcS expression. In this report, the role of extracellular CaM in regulating expression of RbcS in darkness was examined. The time course of expression of RbcS-GUS and that of the secretion of CaM in the suspended transgenic tobacco cells in darkness were very similar. Both showed initial increase followed by decline with maximum CaM secretion preceding maximum GUS expression by 24 h. The concentration of CaM in the culture medium is regulated light independently. Purified CaM alone added to the media enhanced RbcS-GUS expression in darkness. The addition of membrane-impermeable CaM inhibitors, such as anti-CaM antiserum or W7-agarose, repressed the expression of RbcS-GUS in darkness, but this inhibitory effect was completely reversed by adding exogenous purified CaM. These results provide the first evidence that extracellular CaM is involved in the regulation of light-independent RbcS gene expression.

Electrical stimulation is used to treat nonunions and to augment spinal fusions. We studied the biochemical pathways that are activated in signal transduction when various types of electrical stimulation are applied to bone cells.

Cultured MC3T3-E1 bone cells were exposed to capacitive coupling, inductive coupling, or combined electromagnetic fields at appropriate field strengths for thirty minutes and for two, six, and twenty-four hours. The DNA content of each dish was determined. Other cultures of MC3T3-E1 bone cells were exposed to capacitive coupling, inductive coupling, or combined electromagnetic fields for two hours in the presence of various inhibitors of signal transduction, with or without electrical stimulation, and the DNA content of each dish was determined.

All three signals produced a significant increase in DNA content per dish compared with that in the controls at all time-points (p < 0.05), but only exposure to capacitive coupling resulted in a significant, ever-increasing DNA production at each time-period beyond thirty minutes. The use of specific metabolic inhibitors indicated that, with capacitive coupling, signal transduction was by means of influx of Ca(2+) through voltage-gated calcium channels leading to an increase in cytosolic Ca(2+) (blocked by verapamil), cytoskeletal calmodulin (blocked by W-7), and prostaglandin E2 (blocked by indomethacin). With inductive coupling and combined electromagnetic fields, signal transduction was by means of intracellular release of Ca(2+) leading to an increase in cytosolic Ca(2+) (blocked by TMB-8) and an increase in activated cytoskeletal calmodulin (blocked by W-7).

The initial events in signal transduction were found to be different when capacitive coupling was compared with inductive coupling and with combined electromagnetic fields; the initial event with capacitive coupling is Ca(2+) ion translocation through cell-membrane voltage-gated calcium channels, whereas the initial event with inductive coupling and with combined electromagnetic fields is the release of Ca(2+) from intracellular stores. The final pathway, however, is the same for all three signals-that is, there is an increase in cytosolic Ca(2+) and an increase in activated cytoskeletal calmodulin.

Calmodulin (CaM) is the main intracellular Ca2+ sensor protein responsible for mediating Ca2+ triggered processes. Chicken DT40 lymphoma B cells express CaM from the two genes, CaMI and CaMII. Here we report the phenotypes of DT40 cells with the CaMII gene knocked out. The disruption of the CaMII gene causes the intracellular CaM level to decrease by 60%. CaMII-/- cells grow more slowly and die more frequently as compared to wild type (wt) cells but do not exhibit significant differences in their cell cycle profile. Both phenotypes are more pronounced at reduced serum concentrations. Upon stimulation of the B-cell receptor (BCR), the resting Ca2+ levels remain elevated after the initial transient in CaMII-/- cells. Despite higher Ca2+ resting levels, the CaMII-/- cells are partially protected from BCR induced apoptosis indicating that CaM plays a dual role in apoptotic processes.

The role of heterotrimeric G proteins in pollen germination, tube growth, and signal transduction of extracellular calmodulin (CaM) was examined in lily pollen. Two kinds of antibodies raised against animal Gzalpha, one against an internal sequence and the other against its N terminus, cross-reacted with the same 41-kD protein from lily pollen plasma membrane. This 41-kD protein was also specifically ADP ribosylated by pertussis toxin. Microinjection of the membrane-impermeable G protein agonist GTP-gamma-S into a pollen tube increased its growth rate, whereas microinjection of the membrane-impermeable G protein antagonist GDP-beta-S and the anti-Galpha antibody decreased pollen tube growth. The membrane-permeable G protein agonist cholera toxin stimulated pollen germination and tube growth. Anti-CaM antiserum inhibited pollen germination and tube growth, and this inhibitory effect was completely reversed by cholera toxin. The membrane-permeable heterotrimeric G protein antagonist pertussis toxin completely stopped pollen germination and tube growth. Purified CaM, when added directly to the medium of plasma membrane vesicles, significantly activated GTPase activity in plasma membrane vesicles, and this increase in GTPase activity was completely inhibited by pertussis toxin and the nonhydrolyzable GTP analogs GTP-gamma-S and guanylyl-5'-imidodiphosphate. The GTPase activity in plasma membrane vesicles was also stimulated by cholera toxin. These data suggest that heterotrimeric G proteins may be present in the pollen system where they may be involved in the signal transduction of extracellular CaM and in pollen germination and tube growth.

Calmodulin, an EF-hand protein, inhibited the fusion between CD4+ human cells and cells stably expressing HIV-1 envelope proteins. Fusion was also inhibited when HIV-1, HIV-2 or SIV envelope glycoproteins were expressed by vaccinia virus (VV) recombinants, but calmodulin did not inhibit syncytia formation induced by measles virus glycoproteins. Calmodulin also inhibited fusion induced by vPE17, a VV-recombinant expressing a truncated form of HIV-1gp160 which lacks the two known calmodulin-binding sites located in the cytoplasmic domain of gp41. The inhibitory activity was specific to calmodulin among the EF-hand proteins. These observations may be important in understanding the mechanism of retroviral envelope glycoprotein-mediated cell fusion. Several possible mechanisms of action are discussed.

Cultured human umbilical vein endothelial cells inhibited tumor necrosis factor-alpha release from whole blood or isolated mononuclear cells exposed to endotoxin. In contrast, the endothelial cells augmented neutrophil elastase release in the same blood. A protein with these functional properties was isolated from endothelial cell-conditioned media and, surprisingly, was identified as calmodulin. Authentic calmodulin mimicked the effect of endothelium. 125I-Calmodulin bound to a high affinity site on monocytic cell lines (Kd approximately 30 nM, in agreement with its functional activity). Cross-linking of 125I-calmodulin to monocytic cells identified a candidate calmodulin receptor. We conclude that calmodulin possesses an extracellular signaling role in addition to its intracellular regulatory functions. Calmodulin released at sites of tissue injury or possibly by specific mechanisms in the endothelium can bind to receptors, modulating the activities of inflammatory cells.

A 21-kDa calmodulin (CaM)-binding protein and a 19-kDa calmodulin-binding protein were detected in 0.1 M CaCl₂ extracts of Angelica dahurica L. suspension-cultured cells and carrot (Daucus carota L.) suspension-cultured cells, respectively, using a biotinylated cauliflower CaM gel-overlay technique in the presence of 1 mM Ca²⁺. No bands, or very weak bands, were shown on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels overlayed with biotinylated cauliflower CaM when 1 mM Ca²⁺ was replaced by 5 mM EGTA, indicating that the binding of these two CaM-binding proteins to CaM was dependent on Ca²⁺. Less 21-kDa CaM-binding protein was found in culture medium of Angelica dahurica suspension cells; however, a 21-kDa protein was abundant in the cell wall. We believe that the 21-kDa CaM-binding protein is mainly in the cell wall of Angelica dahurica. Based on its reaction with periodic acid-Schiff (PAS) reagent, this 21-kDa protein would appear to be a glycoprotein. The 21-kDa CaM-binding protein was purified by a procedure including Sephadex G-100 gel filtration and CM-Sepharose cation-exchange column chromatography. The purity reached 91% according to gel scanning. The purified 21-kDa CaM-binding protein inhibited the activity of CaM-dependent NAD kinase and the degree of inhibition increased with augmentation of the 21-kDa protein, which appeared to be the typical characteristic of CaM-binding protein.

Tape stripping induces transient increase in keratinocyte proliferation in vivo. The effects of tacalcitol (1,24-(R)-dihydroxyvitamin D3) ointment on the cell kinetics of pig epidermis after the tape stripping were investigated. The tacalcitol ointment (2 micrograms/g) was applied once to the back of pigs immediately after the tape stripping. The pig epidermal cell kinetics were analyzed at various times following the treatment. Tape stripping transiently increased thymidine incorporation of keratinocytes; the maximal effect was observed at 24 h. Tape stripping-induced increase in thymidine incorporation was markedly augmented by tacalcitol treatment. At 24 h following the tape stripping DNA-flow cytometry revealed an accelerated transition from G0/1 to S phase of cell cycle in tacalcitol treated epidermis. There was no significant difference, however, in mitotic counts and G2/M phase fractions between tape stripping-treated and tape stripping plus tacalcitol ointment-treated epidermis. We also measured calmodulin content of pig epidermis following the treatments. Although tape stripping slightly increased calmodulin content of pig epidermis, this was statistically not significant. Tape stripping plus tacalcitol ointment treatment resulted in a significant increase in calmodulin content at 24 h following the treatment. There was no significant difference in calmodulin content between tape stripping treated- and tape stripping plus tacalcitol-treated epidermis.

Although calmodulin (CaM) is commonly considered to be an intracellular protein, it has been suggested lately that it is released and exerts functions extracellularly. In the present investigation this was studied in in vitro regenerating adult frog (Rana temporaria) sciatic nerves. Using a multi-compartment incubation chamber, the non-neuronal cells in the outgrowth region of such nerves were radiolabelled with amino acid precursors. Based on immunological criteria, these cells were shown to release CaM. When the nerves were treated with CaM, both the outgrowth of sensory axons and the injury-induced proliferation of non-neuronal cells were partially inhibited. The inhibitory effects occurred even when the incubation medium contained as little as 30 pM CaM. Furthermore, treatment with anti-CaM antibodies resulted in reduced outgrowth, which suggested that during normal conditions extracellular CaM is kept at an optimal concentration. Finally, conditioned medium was found to contain several CaM-binding proteins. The present findings may reflect an earlier unknown function of extracellular CaM in controlling various growth mechanisms in integrated tissues.

Calmodulin like protein has been identified for the first time in dermatophyte--M. gypseum (by specific radioimmunoassay). Maximum amount of this protein was present in the early and mid log phase of growth and was mainly localized in the cytosolic fraction. Cells treated or grown with calmodulin antagonists (phenothiazine and trifluoperazine) exhibited lower uptake of [14C]acetate or labelled phosphate into phospholipids. This is probably due to lower levels of calmodulin seen in these cells. Our results suggest the relationship between calmodulin levels and phospholipid synthesis in Microsporum gypseum.

The anti-depressive drug trifluoperazine (TFP) was studied on in vitro immune responses. TFP proved to be an inhibitor of lymphokine-activated killer (LAK) cells in its generative step, as well as in its effector phase. Natural killer (NK) activity and interleukin-2 (IL-2) or mitogen-induced lymphocyte proliferation were just as sensitive to the drug effects, whereas the division of tumor cells was more resistant. The mechanism through which TFP suppresses these lymphocytic systems remains unclear. It does not, however, affect an early stage of cellular activation as the addition of the drug as late as 24 h after the start of the culture was still inhibitory for lymphocyte mitogenesis. Neither the expression of CD25, nor that of CD56 was affected by TFP, and exogenous IL-2 was unable to overcome the suppression of proliferation. In relation to cell-mediated cytotoxicity, TFP partially interfered with the effector/target binding. However, addition of lectin to the assay did not overcome the inhibition of lysis produced by the drug. Although further work remains to be done, the effect of TFP on immune responses must be taken into consideration when treating immunosuppressed patients.

Platelet growth factors (e.g. PDGF and TGF-beta) are thought to be pathogenetically important in the stromal reaction characteristic of idiopathic myelofibrosis (IM). We have investigated a possible pathogenetic role for a further platelet mitogen, calmodulin. Platelets are rich in calmodulin, of which 30-40% is releasable with a time course that differs from alpha-granule proteins. In IM urinary calmodulin concentrations were 3-fold those of the normals controls. We suggest that an abnormal release of calmodulin may occur from platelets/megakaryocytes in patients with IM, and that calmodulin should be considered, along with other growth factors, in the pathogenesis of marrow fibrosis.

This study investigated the importance of extracellular calmodulin to the proliferation of the keratinocyte. Normal keratinocytes in culture produced a calmodulin-like protein in their culture media, the level of which increased abruptly and transiently during their growth. This protein was calmodulin-like, in that it specifically bound to a calmodulin affinity column, exhibited calmodulin-like immunoreactivity in both an ELISA and on immunoblots when immunostained with a monoclonal antibody against calmodulin, had an apparent M(r) between 18,000 and 20,000, and stimulated activity in a calmodulin-dependent phosphodiesterase enzyme assay. Addition of exogenous pure calmodulin was of no further mitogenic benefit to the keratinocytes, and slightly reduced proliferation under the culture conditions used. However, addition of either a neutralizing antibody to calmodulin, or W7-agarose, to the culture media of proliferating cells markedly inhibited their proliferation. Accordingly, a calmodulin-like protein was found to satisfy all but one of the criteria for its action as an autocrine growth factor for the keratinocyte. We propose that the lack of mitogenic response to calmodulin in vitro is due to the cell meeting its own requirement for extracellular calmodulin.

Calcium ions (Ca2+) have been identified as mediators of proliferative and morphogenetic processes in many eukaryotic cells. The effects of these ions on the cellular and macromolecular processes that accompany the dimorphic transition from the yeast-to-mycelial form of Sporothrix schenckii have been studied. Ca2+ were found to stimulate germ tube formation and growth in these cells at an optimal concentration of 1.0 mM. Studies concerning the effects of this cation on the molecular processes that precede germ tube formation revealed that the earliest molecular event which was stimulated by 1.0 mM Ca2+ was RNA synthesis. An increased incorporation of radioactivity into RNA in the presence of 1.0 mM Ca2+ was first observed at 0-3 h, and subsequently at all other times tested, following inoculation. A stimulation in rRNA and tRNA synthesis was detected in the presence of 1.0 mM Ca2+. The incorporation of radioactivity into proteins was stimulated 3-5 h following induction in the presence of Ca2+ suggesting a specific effect of Ca2+ on protein synthesis. This increased incorporation takes place prior to the start of DNA synthesis. Incorporation of radioactivity into DNA was also stimulated in the presence of Ca2+, 6 and 9 h after inoculation. This stimulation resulted in nuclear division taking place with a shorter lag period and proceeding with increased kinetics. The results reported here are evidence that Ca2+ plays a role in the control of the early molecular and cellular processes that accompany the yeast-to-mycelium transition in S. schenckii and offer an explanation of how Ca2+ can control the expression of the dimorphic potential of this fungus.

Normal human umbilical vein endothelial cells cultured on gelatin-coated plastic dishes were found to produce a protein in their media which had calmodulin-like immunoreactivity and biological activity. Further identification of the protein was achieved by examining the incorporation of 14C leucine into protein found in the conditioned medium. Cells produced 14C labelled protein in their medium which specifically bound to an affinity column for calmodulin. This latter material stimulated calmodulin dependent phosphodiesterase activity in vitro and this stimulation was inhibited by the addition of the calmodulin antagonist W7. The presence of calmodulin-like activity and immunoreactivity in the media varied as the cells grew from low to high density, a peak of extracellular calmodulin-like activity preceding an increase in cell number. Extracellular calmodulin-like activity did not correlate with the presence of lactate dehydrogenase in the medium. The addition of pure pig brain calmodulin affected the rate of cell proliferation; significant proliferation to pure calmodulin was only seen in cells at low density, at higher density calmodulin either had no effect or inhibited proliferation. Inhibition of extracellular calmodulin activity by a calmodulin antagonist immobilized on agarose beads, or by an antibody to calmodulin significantly decreased proliferation in all dividing cultures. Taken together this data suggests that, in vitro, calmodulin, or a very closely related protein, influences endothelial cell proliferation through an autocrine mechanism.

Isolated newborn rat calvarial bone cells grown in monolayer on polyurethane membranes in specially constructed culture chambers and subjected to a cyclical biaxial mechanical strain of 0.17% at a frequency of 1 Hz for 30 min demonstrated a 16% increase in DNA synthesis during the subsequent 24 h. The metabolites of the inositol phosphate pathway, shown to be an important second messenger in many cell types, were shown to be elevated using high-performance liquid chromatography to separate and quantitate the various inositol polyphosphates. Inositol 1,4,5-trisphosphate, inositol 1,4-bisphosphate, and inositol 1,3,4,5-tetrakisphosphate reached peak accumulations after 20 s of mechanical strain. Inositol 1,3,4-trisphosphate reached a peak accumulation after 2 min, and inositol 1,2,3,4,5,6 phosphate reached a peak accumulation after 60 min of mechanical strain. Neomycin, an inhibitor of phospholipase C, a membrane-bound enzyme that hydrolyzes phosphatidyl inositol 4,5-bisphosphate to start the inositol phosphate cascade, completely inhibited accumulation of the above inositol phosphates during mechanical straining of the bone cells. Neomycin also completely abolished the increase in DNA synthesis that was seen after a mechanical strain of 0.17%. It is concluded from this study that the inositol phosphate pathway is activated by mechanical strain in bone cells and that this pathway is an important and primary mediator in the transduction of mechanical strain into cellular proliferation in these cells.

Calmodulin affinity chromatography and chromatofocusing were used to purify calmodulin-binding proteins of 32-40-kDa from homogenates of Trypanosoma brucei clone YTat1.1. The trypanosome proteins associated with calmodulins from different sources and reversibly inhibited calmodulin-dependent bovine brain phosphodiesterase. Purified 32-kDa protein bound to calmodulin with an approximate Kd of 1.3 nM. Polyclonal antibodies directed against purified 32-kDa protein and monoclonal antibody ECA6 recognized each of the 32-40-kDa proteins. Immunoprecipitation with biotinylated monoclonal antibody ECA6 (Bio-ECA6) or biotinylated calmodulin (Bio-CaM) identified the 32-40-kDa proteins in phenylmethylsulfonyl fluoride-treated lysates of slender forms of YTat1.1, but not procyclic forms of YTat1.1 or slender forms of EATRO110. In the presence of leupeptin, lysates of slender YTat1.1 contained a single protein of 58 kDa that immunoprecipitated with Bio-ECA6. The 58-kDa protein was exposed to the extracellular space as demonstrated by immunolocalization and sensitivity to pronase treatment in intact cells. The protein was identified as variant surface glycoprotein (VSG) based upon immunolocalization, pattern of expression and cross-reactivity of ECA6 with authentic VSG. The amino-terminal 17 residues of 32-kDa protein were identical with the amino-terminus of YTat1.1 VSG. Putative calmodulin-binding domains were identified in other VSGs by computer modeling. The model was tested with CNBr fragments of VSG 117. The fragments reversibly inhibited calmodulin-dependent activation of phosphodiesterase with approximate Kd of 11 nM. We conclude that endogenously generated proteolytic fragments of VSG from clone YTat1.1, and CNBr fragments of VSG 117 bind with high affinity to calmodulin.

Although calmodulin has been suggested as an important regulator of keratinocyte proliferation, its precise role remains unknown. We employed a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), to examine the role of calmodulin on keratinocyte proliferation. N-(6 aminohexyl-1-naphthalenesulfonamide (W-5), a chlorine-deficient analogue of W-7 with little anti-calmodulin activity, was used as the control. W-7 markedly inhibited thymidine incorporation of pig epidermis at concentrations close to its anti-calmodulin activity; W-5 had no effect on the thymidine incorporation. The inhibitory effect of W-7 was reversible; the removal of W-7 from the incubation medium resulted in the reinitiation of the thymidine incorporation, suggesting that W-7 is not a cytotoxic agent. These results are consistent with the view that calmodulin is an essential regulator of keratinocyte proliferation. The epidermal beta-adrenergic response, which is decreased in various hyperproliferative epidermal abnormalities, was increased in W-7-treated hypoproliferative epidermis. The epidermal SOD activity, which is also decreased in the hyperproliferative epidermis, however, was not affected by the W-7 treatment.