Thorough design and pre-trial quality assurance (QA) decrease dosimetric impact of delineation and dose planning variability in the STRICTLUNG and STARLUNG trials for stereotactic body radiotherapy (SBRT) of central and ultra-central lung tumours.

INTRODUCTION: SBRT of central lung tumours implies significant risk of toxicity. We are initiating two phase II trials prescribing 56 Gy/eight fractions to PTV, allowing for dose escalation of GTV. We prioritize organs at risk (OAR) constraints over target coverage, making the treatment plans very sensitive to OAR delineation variations. The aim of this study is to quantify the dosimetric impact of contouring variations and to provide a thorough description of pre-trial quality assurance to be used in upcoming trials to provide consistent clinical care. MATERIALS AND METHODS: Delineation: Seven physicians delineated OAR in three rounds, with evaluations in-between. For each patient case, seven treatment plans, repeatedly using each of the OAR structure sets from the seven physicians, were made and compared to evaluate the dosimetric effect of delineation variability. Treatment planning: Treatment plans for seven cases were made at six departments in two rounds, with discussion in-between. RESULTS: OAR delineation variation between centres resulted in high variabilities in OAR dose for simulated plans and led to potential overdosage of the lobar bronchus (constraint: D0.03cc < 45 Gy), with maximum doses ranging between 58 Gy (first round), and 50 Gy (third round). For mediastinal tissue, the constraint (D0.03cc < 45 Gy) was violated for the majority of the delineations in all three rounds, with maximum doses of 84 Gy (first round), and 72 Gy (third round).For the treatment planning study, the range of the standard deviation for GTV mean dose was 12.8-18.5 Gy (first round) and 2.8-3.5 Gy (second round). CONCLUSIONS: Even small variations in OAR delineation led to high OAR overdosage. The study demonstrates the importance of having extensive QA procedures in place before initiating clinical trials on dose escalation in SBRT.

A giant outburst two years before the core-collapse of a massive star.

The death of massive stars produces a variety of supernovae, which are linked to the structure of the exploding stars. The detection of several precursor stars of type II supernovae has been reported (see, for example, ref. 3), but we do not yet have direct information on the progenitors of the hydrogen-deficient type Ib and Ic supernovae. Here we report that the peculiar type Ib supernova SN 2006jc is spatially coincident with a bright optical transient that occurred in 2004. Spectroscopic and photometric monitoring of the supernova leads us to suggest that the progenitor was a carbon-oxygen Wolf-Rayet star embedded within a helium-rich circumstellar medium. There are different possible explanations for this pre-explosion transient. It appears similar to the giant outbursts of luminous blue variable stars (LBVs) of 60-100 solar masses, but the progenitor of SN 2006jc was helium- and hydrogen-deficient (unlike LBVs). An LBV-like outburst of a Wolf-Rayet star could be invoked, but this would be the first observational evidence of such a phenomenon. Alternatively, a massive binary system composed of an LBV that erupted in 2004, and a Wolf-Rayet star exploding as SN 2006jc, could explain the observations.

Functional and immunohistochemical evaluation of porcine neonatal islet-like cell clusters.

Porcine neonatal islet-like cell clusters (NICCs) may be an attractive source of insulin-producing tissue for xenotransplantation in type I diabetic patients. We examined the functional and immunohistochemical outcome of the islet grafts in vitro during long-term culture and in vivo after transplantation to athymic nude mice. On average we obtained 29,000 NICCs from each pancreas. In a perifusion system, NICCs responded poorly to a glucose challenge alone, but 10 mmol/L arginine elicited a fourfold increase in insulin secretion and 16.7 mmol/L glucose + 10 mmol/L arginine caused a sevenfold increase in insulin section indicating some sensitivity towards glucose. Hormone content as well as the number of hormone-containing cells increased for the first 14 days of culture. When NICCs were stained for hormones, proliferation (Ki67), and duct cells (CK7), some insulin- and glucagon-positive cells co-stained for proliferation. However no co-staining was observed between insulin- and glucagon-positive cells or between hormone-and CK-positive cells. Following transplantation of 2000 NICCs under the renal capsule of diabetic nude mice, BG levels were normalized within an average of 13 weeks. Oral and IP glucose tolerance tests revealed a normal or even faster clearance of a glucose load compared with normal controls. Immunohistochemical examination of the grafts revealed primarily insulin-positive cells. In summary, in vitro, NICCs responded to a challenge including glucose and arginine. There was a potential for expansion of the beta-cell mass of NICCs in vitro as well as in vivo where NICCs eventually may normalize blood glucose of diabetic mice.

Cyclopentyladenosine improves cell proliferation, wound healing, and hair growth.

BACKGROUND: N(6)-Cyclopentyladenosine (CPA), a structural analog of adenosine, is a vasodilator with extensive pharmacological effects. However, little is known about the effect of CPA on wound healing and hair growth. METHODS: Cellular responses to CPA were measured in vitro by tetrazolium dye reduction and in vivo by bromodeoxyuridine (BrdU) uptake. The effect of CPA on healing of incisional and excisional wounds on the dorsum of diabetic (db/db, n = 94) and nondiabetic (db/+, n = 20) mice and hair growth along the wound margin was evaluated with wound breaking strength, wound closure rate, and quantitative histology. RESULTS: CPA stimulated proliferation of BALB/3T3 fibroblasts and human dermal microvascular endothelial cells in both quiescent and nonquiescent phases. Wounds treated with CPA at 10 microM showed a significant increase in the number of BrdU-labeled cells, including keratinocytes, fibroblasts, endothelial cells, and cells in sebaceous glands and the outer root sheath of hair follicles, compared with controls (P < 0.05). CPA application (5.1 microg/daily for 12 days) significantly increased the breaking strength of incisional wounds at day 24 postwound (P < 0.05). Excisional wound closure rate in the CPA-treated group (3.4 microg/daily for 15 days) was accelerated starting at day 10 postwound compared with controls (P < 0.01). Tissue sections from CPA-treated wounds showed a sevenfold increase in hair follicle number, compared with controls (P < 0.01). Enhanced hair growth along the wound margin was revealed in CPA-treated groups. CONCLUSION: CPA stimulated proliferation of many cell types in vivo and in vitro and enhanced wound healing and hair growth. Therefore, CPA could be an interesting candidate for clinical application.

Electric cautery lowers the contamination threshold for infection of laparotomies.

BACKGROUND: Interplay between wound resistance factors and bacterial innoculum determines the risk of surgical infection. Since cautery causes more damage than the scalpel, our hypothesis is that lower numbers of bacteria are required to infect wounds made by electric cautery than to infect wounds made with a scalpel. METHODS: Abdominal fascia was incised in 375 rats by cold knife, cutting current, or coagulation current. Wounds were innoculated with increasing numbers of bacteria and histologically scored at 7 days for necrosis, inflammation, and abscess. RESULTS: Coagulation current causes more inflammation, necrosis, and abscesses than the scalpel at all bacterial levels. Electric cutting current is intermediate, causing more damage than the scalpel only after contamination reached 10(5). Above this threshold most wounds were infected in all groups. CONCLUSIONS: Electric coagulation current should be used only when the need for meticulous hemostasis outweighs the considerably increased risk of infection. Electric cutting current is less destructive but also less hemostatic; indications for its use are difficult to identify.

Transfection with aFGF cDNA improves wound healing.

Somatic gene therapy is a potentially useful strategy for the delivery of growth factors or cytokines to enhance wound healing. Experimental excisional and incisional wounds in impaired-healing diabetic mice (db/db) were treated with aFGF and with a plasmid coding for aFGF. A eukaryotic expression plasmid composed of the Hst signal peptide sequence in-frame with the human aFGF sequence was used. Transfection of tissues was accomplished either by direct plasmid uptake or by uptake facilitated with cationic liposomes. The results show that the closure of excisional wounds was significantly accelerated (p < 0.05) by topical application of human recombinant aFGF or by transfection with the aFGF plasmid but not by vehicle or control plasmid not containing the aFGF sequence. In incisional wounds, aFGF or transfection with the plasmid significantly increased the wound-breaking strength compared to their corresponding controls (p < 0.05). Quantitative histology of the plasmid-treated incisional wound sections revealed improved wound quality. The transcription of mRNA from human aFGF cDNA in the incisional wound tissue extracts was confirmed by RT-PCR, and the expressed aFGF was detected by immune dot blot and immunohistochemistry assays. The transfection was a transient process with a peak at 9 d in db/+ (littermates of the diabetic mice) incisional wounds, at 36 d in db/db incisional wounds, and at 27 d in db/db excisional wounds. Cells transfected with human aFGF occupied up to 6.4% of the transectional area in the wound sites. Thus, aFGF gene delivery resulted in both gene expression and a functional improvement in healing.

Cellular responses to surgical trauma, hemorrhage, and resuscitation with diaspirin cross-linked hemoglobin in rats.

BACKGROUND: Resuscitation with acellular oxygen carrier solutions offers the potential advantage of improved oxygen delivery compared with crystalloid solutions, but the detailed consequences of improved resuscitation have not been fully evaluated. This study evaluated local and systemic cellular effects of trauma, hemorrhage, and resuscitation in a model of hemorrhage and surgical trauma. METHODS: Rats with a 10 cm full-thickness incisional wound and a 15 mL/kg hemorrhage were either not resuscitated or resuscitated with blood or diaspirin cross-linked hemoglobin (DCLHb). Cellular proliferative responses were evaluated at 1.5, 6, 24, and 48 hours after wounding by labeling in vivo with 5-bromo-2'-deoxyuridine. Plasma levels of interleukin-6, tumor necrosis factor-alpha, and interferon-gamma were measured by bioassay or enzyme-linked immunosorbent assay (ELISA). Bacterial translocation was measured by culturing liver homogenates. RESULTS: Trauma inhibited keratinocyte and hepatocyte proliferation at 1.5 and 6 hours, and stimulated subsequent proliferation of keratinocytes and liver nonparenchymal cells. DCLHb stimulated wound keratinocyte proliferation, attenuated the inhibition of hepatocyte proliferation, eliminated bacterial translocation to the liver, protected the intestine from ischemic damage, and induced a rapid increase of interleukin-6 during the early phase of injury. CONCLUSIONS: Surgical trauma alone, or in combination with hemorrhage, modulated cell proliferation both in the wound and in the remote organs of intestine and liver. DCLHb enhanced wound healing and cell proliferation as well as, or better than, freshly drawn blood, which may be beneficial for trauma care.

Systemic bacteraemia following haemorrhagic shock in mice: alleviation with oral Interleukin 6.

A murine model of haemorrhagic shock was used to investigate bacterial translocation from the gut and subsequent systemic immunoreduction. Anaesthetized mice were bled from the femoral artery, and held at a mean arterial blood pressure of 35 mm Hg for one hour then resuscitated with shed blood and two-fold volume lactated Ringer's solution. Upon awakening, they were given cytokines or control media orally. Bacteriological cultures of livers, spleens and mesenteric lymph nodes from haemorrhaged mice given cytokine had significantly fewer bacteria/gm of tissue than those given media. Recombinant IL-6 mimicked the effects seen with crude cytokines. Reduction of proliferation among spleen cells from haemorrhaged mice was observed and could be partially returned to normal by cytokine feeding. Mixing experiments in which cells from haemorrhaged mice were added to those of normal mice in an MLR showed no suppressor activity. Flow cytometry analysis revealed a reduction in CD 3+ cells at 16 hours post-haemorrhage in mice fed control media or cytokines, suggesting that reduced proliferative capacity may be due to loss of function rather than active suppression. Histological examination of the intestines of haemorrhaged mice fed cytokines or media revealed restoration of intestinal mucosal integrity by cytokine administration. These results suggest that oral administration of IL-6 may be an important treatment for the prevention of systemic sepsis following haemorrhage.

A new wound healing agent--sphingosylphosphorylcholine.

Spingosylphosphorylcholine (lysosphingomyelin or SPC) is an effective and broad spectrum cell growth promoting agent and a candidate for evaluation on wound healing. The effect of SPC on full-thickness excision and incision wounds in genetically healing-impaired diabetic (db/db) mice was evaluated by measurement of wound area, skin strength, and tissue histology. The effect on cell proliferation was measured in vivo by incorporation of bromo-deoxyuridine and in vitro by [3H] thymidine incorporation. SPC increased the rate of wound closure, with a statistically significant improvement in measured wound areas (p < 0.02, compared with vehicle controls). The optimum concentration was 2-3 microM. SPC, alone and in combination with insulin, stimulated DNA synthesis in cells known to participate in wound healing, including microvascular endothelial cells. In vivo, SPC stimulated proliferation of keratinocytes, fibroblasts, endothelial cells, and cells around sebaceous glands and hair follicles at day 2-4 postwound, resulting in a complete re- epithelialization and profound granulation tissue formation in excisional and incisional wound sites of db.db and db/+ mice. Quantitative assessment of wound tissue section morphology indicated that SPC induced up to a 3-fold increase in the numbers of mitotic cells, resulted in smaller cross-sectional scar area, and led to more normalized tissue in the wound sites. SPC had no deleterious effect on wound skin strength. In conclusion, the acceleration of dermal wound healing animal models suggests that SPC could be an interesting candidate for clinical application.

Multiple responses to administration of liposome-encapsulated hemoglobin (LEH): Effects on hematopoiesis and serum IL-6 levels.

Liposome-encapsulated hemoglobin (LEH) has been tested in animals as an oxygen-carrying red cell substitute and has been shown to be beneficial in the treatment of hemorrhagic shock. The effects of LEH on immune responses have not been studied thoroughly in any well-controlled model. Using a murine model, we evaluated nephrotoxicity and hepatotoxicity as well as immune function parameters following LEH administration. Following intravenous administration of LEH, 1) a serum spike of interleukin-6 (IL-6) occurred in mice at 4-8 hours, with no elevation of IL-1, tumor necrosis factor (TNF), or interferon-gamma (IFN-gamma); 2) the serum liver function enzymes SGOT (AST, aspartate aminotransferase) and SGPT (ALT, alanine aminotransferase) were elevated at 48 hours; 3) only a slight increase in serum antibody to bovine hemoglobin was observed; and 4) increased hematopoietic activity was observed in the spleen and bone marrow. The finding that only IL-6 but not the associated TNF, IL-1, or IFN-gamma is secreted in vivo following LEH administration is novel and may have significance in defining the mechanisms underlying specific adverse responses observed with LEH administration in animals.

Biochemical properties of cloned lipases from the Pseudomonas family.

Three Pseudomonas lipases, representing three subfamilies, were analysed for pH optima, destabilization by EGTA and surfactants, phospholipase and cholesterolesterase side activities. All the Pseudomonas lipases tested showed alkaline pH optima. The Pseudomonas cepacia and the P. pseudoalcaligenes lipases were totally inhibited by EGTA at pH 9, and the latter was also fully inhibited at pH 7. The lipase from P. mendocina was not inhibited by EGTA at any of the pH values tested. These findings indicate that a calcium binding site exists in some of the Pseudomonas lipases. The P. pseudoalcaligenes, P. cepacia and P. mendocina lipases were inhibited by the anionic surfactant SDS at concentrations between 0.01-0.5 mg/ml. The P. pseudoalcaligenes and P. cepacia lipases were not inhibited by the nonionic surfactant Brij35 in concentration up to 1 mg/ml, whereas the lipase from P. mendocina was inhibited at 0.1 mg/ml. The P. pseudoalcaligenes and P. cepacia lipases were found to possess high cholesterol esterase activity. P. pseudoalcaligenes lipase was further found to have high phospholipase activity. Ten Pseudomonas lipase sequences were compared by automatic sequence alignment. On the basis of sequence identity we have classified Pseudomonas lipases into five subfamilies.

Spatially controlled adhesion, spreading, and differentiation of endothelial cells on self-assembled molecular monolayers.

Chemically modified glass substrates were used to demonstrate differential adhesion, growth, and differentiation of endothelial cells. Endothelial cells were examined for adhesion and growth on glass, glass treated with N-(2-aminoethyl)-3-aminopropyl trimethoxysilane (EDA), or EDA with a subsequent treatment with physically adsorbed extracellular matrix components human fibronectin and heparin sulfate. EDA and EDA/human fibronectin showed similar abilities to support adhesion, spreading, and proliferation of endothelial cells. In contrast, heparin sulfate inhibited endothelial cell adhesion to EDA. Differentiation of endothelial cells resulting in precapillary cord formation was triggered by addition of basic fibroblast growth factor (bFGF). On EDA and EDA/human fibronectin bFGF causes confluent endothelial cell monolayers to differentiate and form cords, which resulted in a large-scale spatial redistribution of cells on the surface. Formation of organized neovascular assemblies was demonstrated on coplanar molecular patterns of EDA and a nonadhesive perfluorinated alkylsilane (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-dimethylchloros ilane (13F). Endothelial cells preferentially adhered to the EDA lines and after 24-48 hr, microfilaments aligned with the long axes of the patterned EDA region. Finally, endothelial cells that became confluent within the confines of the EDA region (bound by the nonadhesive, 13F domains) were observed to differentiate into neovascular cords in long-term culture (7-10 days) with bFGF.

Peripheral blood hematopoietic progenitor/stem cells proliferate to form colonies in liquid culture but require contact with vascular endothelial cells and GM-CSF.

To test the hypothesis whether peripheral blood hematopoietic progenitor/stem cells (PBSCs) interact with vascular endothelial cells during events leading to extramedullary hematopoiesis, we cocultured T-cell depleted, peripheral blood mononuclear cells obtained from cytokine treated primates in liquid culture containing a monolayer of porcine aortic endothelial cells (PAECs) for 7 days. Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) added to cocultures of PBSC-PAEC stimulated colony formation, while only a few clusters were observed in cultures without GM-CSF. In contrast, colony formation was not stimulated when either interleukin 1 (IL-1) or IL-3 were added to the cultures. Colony and cluster formation in response to GM-CSF was dose dependent; 20 +/- 5 colonies/5,000 cells were formed at 3 U/ml, and optimal colony formation of 42 +/- 11/5,000 cells occurred at 100 U/ml. Colonies formed in the presence of GM-CSF were large, and most contained greater than 200 cells. Morphological and phenotypical characterization of cells from isolated colonies suggested that the majority of cells were predominantly immature myeloid elements. However, there was also a low but consistent frequency of megakaryocytic lineage cells. Thus, PBSCs interact with non-bone marrow--derived vascular endothelial cells and proliferate, but only in the presence of GM-CSF, suggesting that PBSC interaction with vascular endothelial cells in vivo could lead to extramedullary hematopoiesis.

Retroviral transformation of cerebral microvascular endothelial cells: macrophage-like and microvascular endothelial cell properties.

We report that L-cell-conditioned medium (LCM) transforms porcine cerebral microvascular (PCMV) endothelial cells into cells with macrophage-like properties. LCM is known to contain both cytokine(s) and the L-cell virus, a murine retrovirus found in the L929 cell and LCM. Our evidence suggests that both LCM cytokine(s) and the L-cell virus are involved in this PCMV endothelial cell transformation. Criteria for transformation include focus formation, decreased serum requirements for growth, changes in morphology including nonadherence, propagation in suspension culture, and a decreased growth response to stimulation with a known endothelial cell mitogen. Macrophage-like characteristics of this transformed cell, designated as RVTE, include pinocytosis of low-density lipoprotein, Fc receptor-mediated phagocytosis, phagocytosis of bacteria and zymosan, the expression of macrophage enzyme markers, and constitutive production of colony-stimulating factor 1. However, the transformed cell retains several properties of the nontransformed cell including the expression of FVIII:RAg and in vitro self-organization into capillary-like structures. Cloning of RVTE cells clearly shows that both macrophage-like and cerebral microvascular endothelial cell properties are present in the same cell. During self-organization, nontransformed cells express morphologic and functional characteristics classically associated with the macrophage. These findings suggest that some brain capillary pathophysiologies could involve macrophage-like cerebral microvascular endothelial cells. Furthermore, the "reticuloendothelial" phenotypic repertoire expressed by this transformed cerebral microvascular endothelial cell may show that the cerebral capillary endothelial cell in vivo is derived from a hematopoietic and/or phagocytic precursor.

Morphologic plasticity and periodicity: porcine cerebral microvascular cells in culture.

Porcine cerebral microvascular (PCMV) endothelial cell cultures and pericyte-endothelial cell cocultures were established and the self-organizational properties of the cells were examined in various culture conditions. Cultured PCMV endothelial cells were characterized by the capacity to produce prostacyclin in response to bradykinin. Cultured PCMV pericytes were identified with a smooth muscle actin-specific stain. PCMV endothelial cells organized into cord structures when left in culture for several weeks without passage. Lumina were observed in cross sections of these cords and appeared to form through a process of cell-selective autolysis. PCMV endothelial cells required three dimensions for self-organization, forming suspended cords in planes that either intersected or paralleled the culture vessel floor. After formation, suspended cords continued to exhibit a morphologic plasticity punctuated by the coordinated migrations of PCMV endothelial cells en masse. Sequential propagation of PCMV endothelial cell monolayers and development of suspended capillarylike cords recurred cyclically when cells were left in culture without passage for several weeks. Cord development was also observed in PCMV pericyte-endothelial cell cocultures with large proportions of pericytes. However, pericytes were not located in cross sections of suspended cords formed in coculture. Apparently, in some conditions of PCMV coculture, populations of PCMV endothelial cells and pericytes segregate. Retina-derived growth factor (RDGF) promoted this cell-type segregation and the subsequent formation of suspended cords in PCMV cocultures, although its exact mode of action is unclear. These results indicate that cultured cerebral microvascular endothelial cells and pericytes have capacities for complex, temporal self-organization that varies according to culture conditions.

Effects of phorbol esters on endothelial cell microfilaments: laser scanning confocal microscopy and quantitative morphometry of dose dependent changes.

Phorbol esters are known to alter microfilaments but it is not clear if the changes correspond to modulation of the phosphoinositide turnover/protein kinase C system. The novel technique of laser scanning confocal epifluorescence was used to study fiber orientation in phorbol ester treated cells. We treated endothelial cells with control agents and agents known to stimulate protein kinase C: 4 alpha-phorbol, phorbol 12-myristate 13-acetate (PMA), phorbol dibutyrate (PDB), or lipopolysaccharide. After incubation with the test agents, the endothelial cell microfilaments were stained with rhodamine pholloidin and viewed by conventional epifluorescence and by laser scanning confocal epifluorescence microscopy. The images obtained by the confocal microscopy corresponded to a thin optical section through the cells, 300 nm or more in thickness. The microfilaments extended predominantly in the plane of focus. After exposure of the cells to phorbol esters, the stress fibers became more nearly parallel in arrangement or were shortened, but remained in the plane of focus. The modification of microfilaments in response to phorbol esters was quantitated by a single blind analysis. In order to compare the morphological changes with a biochemical action of the phorbol esters, we measured phosphoinositide turnover. The dose-dependence of morphological changes was compared and contrasted to the dose-dependent effect of phorbol esters on bradykinin-stimulated phosphoinositide turnover. PMA had about the same EC50 (1-5 nM) for both biochemical and morphological processes. PDB was less potent in inducing the disruption of microfilament structure than in inhibiting phosphoinositide turnover. Lipopolysaccharide was ineffective in inducing a morphological change under these conditions. A simple activation of protein kinase C is insufficient to explain the dose-dependent effects of phorbol esters. Thus a morphometric analysis can help distinguish the potency of cytoskeleton modulators.

Role of cellular Ca++ in phosphorylation of 21 K and 19 K polypeptides in cultured thyroid cells: effects of phorbol ester, trifluoperazine, and 8-diethylamino-octyl-3,4,5-trimethoxybenzoate hydrochloride.

Cultured dog thyroid cells contain 21 and 19 kilodalton (K) phosphoproteins which by several criteria have been identified as light chains of myosin (MLC). TSH causes a reduction in the phosphorylation state of the 21 K-19 K proteins, at least in part through activating adenylate cyclase and increasing cAMP levels. We now report that 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also decreases the 21 K-19 K protein phosphorylation state, but in contrast to that due to TSH, the TPA-induced decrease is not associated with elevated cAMP levels. The effect of TPA was not additive to that of TSH. Because Ca++ is a major factor regulating MLC kinase and TPA-stimulated protein kinase C in other systems, the role of Ca++ in the phosphorylation of the 21 and 19 K polypeptides in dog thyroid was examined. In intact cells, both (8-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) (1 X 10(-4) M) and trifluoperazine (TFP) (4 X 10(-5) M) increase basal 21 K-19 K protein phosphorylation and inhibit the decrease in phosphorylation caused by TSH and TPA without affecting cAMP levels. Ionophore A23187 (5 X 10(-6) M) counteracts TMB-8- and TFP-stimulated phosphorylation as well as TMB-8 and TFP inhibition of TSH- and TPA-reduced 21 K-19 K phosphorylation. Incubation of 32PO4-labeled dog thyroid cells in the absence of extracellular Ca++ or with verapamil does not significantly affect basally phosphorylated 21 K-19 K proteins or the decreased 21 K-19 K phosphorylation state caused by TSH. These results strongly suggest that the phosphorylation state of the 21 and 19 K proteins is affected more significantly by intracellular Ca++ pools than by extracellular Ca++, and implicate a kinase(s) other than Ca++-calmodulin-dependent MLC kinase in the phosphorylation of MLC in the dog thyroid.

Spare receptors, partial agonists, and ternary complex model of drug action.

The occurrence of spare receptors and partial agonists for smooth muscle contractions mediated by alpha 1-adrenergic receptors can be accounted for with a ternary complex model of drug action presented here. In this model, receptor-ligand complexes are assumed to be inactive until the complex binds to an activating protein. Contractile responses are assumed to be proportional to the concentration of ternary complex (receptor-ligand-activator) regardless of the ligand involved. Antagonists are unable to form the ternary complex. Spare receptors are present as the inactive receptor-ligand complex. Such a model is shown to fit already published data on membrane binding of alpha 1-adrenergic agonists as well as contractile responses to the agonist. Schild plots are expected to resemble those of a single-site model of drug action. The double-reciprocal plots of receptor-inactivation studies will display only a slight curvature as may be seen in previously published articles. Partial agonists may have 50% response doses lowe or higher than full agonists. The hypothesis that ternary complexes are formed with alpha 1-receptors could be tested more critically with receptor-inactivation studies using both antagonists and agonists. Partial inactivation of receptor and activator protein should reduce the binding of antagonist without altering the concentration needed to bind to 50% of the receptors. On the other hand, the concentration of agonist required to displace 50% of a bound antagonist is expected to increase. The proposal that contractile responses are proportional to the ternary complex concentration could be tested by fitting the ternary complex model to the data from studies of contractions induced by partial agonists as well as full agonists.

Association of calmodulin with lysosomes.

We examined the subcellular localization of calmodulin in several cultured cells (primary thyroid follicular cells, thyroid C-cell tumour cells (TT), kidney cells (PtK2-L23) and peritoneal macrophages) by indirect immunofluorescence using affinity-purified antibody to calmodulin. When cells were fixed with 3% formaldehyde for 15 min prior to lysis with 0.5% Triton X-100, spindle fibres in mitotic cells were fluorescent and a diffuse cytoplasmic localization of calmodulin was observed in resting cells. However, when cells were lysed with 0.5% Triton X-100 for 90s prior to fixation for 30 min with 3% formaldehyde, three effects were observed. One: there was little diffuse cytoplasmic staining. Two: discrete vesicles were stained. Three: spindle fibres in mitotic cells were fluorescent. The stained vesicles were phase-dense and ranged from 0.1 to 0.5 micron in primary thyroid follicular cells but were smaller in PtK2 and TT cells. The thyroid follicular cells retained vesicular staining after exposure to thyrotropin and isobutylmethylxanthine, but the number of labelled vesicles decreased by almost 80%. Phase-dense vesicles were identified as lysosomes or other acidic vesicles by vital staining with Acridine Orange. After differential centrifugation of thyroid homogenates, calmodulin was measured by radioimmunoassay (RIA) and found in both cytosolic (89%) and membrane vesicle fractions (11%). The vesicular calmodulin was not eluted by washing with 5 mM-EGTA. The thyroid fractions were subjected to SDS-polyacrylamide gel electrophoresis and the gels incubated with 125I-labelled calmodulin to reveal calmodulin acceptor proteins (CAPs). The vesicle fraction contained quantitatively major CAPs with Mr of 200,000, 140,000, 89,000, 38,000 and 34,000, and minor CAPs of 60,000 and 50,000. Washing the pellet with 5 mM-EGTA did not reduce the content of CAPs. Thus, calmodulin and CAPs are present both in the cytoplasm and in a membrane vesicle fraction. The lysosomal locale of calmodulin and the effect of thyrotropin on vesicle number suggest a role for calcium in the regulation of lysosome function.