Zoledronic acid inhibits the function of Toll-like receptor 4 ligand activated monocyte-derived dendritic cells.

Zoledronic acid (ZA) is a nitrogen-containing bisphosphonate with antitumor activity used to treat patients with malignant diseases. ZA treatment induces, as a side effect, inflammatory responses, which are accompanied by expansion of gammadelta T cells. In our study, we analyzed the function and differentiation of monocyte-derived immature and lipopolysaccharide (LPS)-stimulated dendritic cells (moDCs) treated with different ZA concentrations, which are achieved in patients. We found that moDC activation with TLR4 ligand LPS is modulated by ZA. The expression of maturation markers was diminished with increasing ZA levels upon LPS activation. The migratory capacity, interleukin-12 secretion and generation of cytotoxic- T-cell responses were reduced at higher ZA levels. Increasing ZA concentrations downregulated nuclear factor-kappaB family members and interferon-regulatory factor (IRF)-3. Surprisingly, in immature moDCs, low ZA concentrations caused upregulation of RelB, c-Rel, IRF-3 and IRF-8. We conclude that ZA concentrations used to treat patients have inhibitory effects on DC activation. This might lead to immunosuppression or result in infectious complications.

Capacitance-torsion balance for measuring small stresses and strains.

A versatile and inexpensive capacitance-torsion balance has been developed to measure, simultaneously and continuously, small forces and displacements in remotely located samples. It can also be used as a sensitive dilatometer or as a microbalance. The present instrument provides an output voltage change of 1 V for a load change of 3 g and a displacement of 5x10(-5) m. These load and displacement ranges can be independently chosen by adjusting the geometrical configuration of the balance, whereas the sensitivity is independent of the range.

[The occurrence and significance of Chlamydia psittaci and Coxiella burnetii in dogs and cats. A study of the literature]. / Vorkommen und Bedeutung von Chlamydia psittaci und Coxiella burnetii bei Hund und Katze. Eine Literaturstudie.

With this review of the literature it is demonstrated that not only infections with Chl. psittaci and C. burnetii do occur in dogs and cats in the Federal Republic of Germany but also clinical diseases caused by these agents may occur. Especially in diseases with uncharacteristic symptoms both bacteria have to be taken into consideration as causative agents. Since both agents cause zoonotic infections a correct diagnosis is of special importance, especially if the owner of a diseased dog or cat or members of his family show signs of disease also. In cases of unidentified disease or of a rickettsial zoonosis identification of the causative agent must be tackled by cultural or serological investigation. Direct or cultural identification is often not possible so that serological techniques have to be applied. Besides established serological techniques sensitive enzyme immunoassays are useful tools for securing a diagnosis.

[Experiences with a mobile laboratory during Operation "Kurdenhilfe" in Iran in 1991]. / Erfahrungen mit einem mobilen Labor während der Operation "Kurdenhilfe" im Iran 1991.

During the operation "Kurdenhilfe" from 9th of May to 14th of June in 1991 the German army managed a field-hospital in the province Bakhtaran of Iran. Part of this field-hospital was a mobile field-laboratory in which microbiological/serological and parasitological examinations could be done. It is reported about equipment, methods and results.

Cardiac calmodulin-stimulated protein phosphatase: purification and identification of specific sarcolemmal substrates.

A calmodulin-stimulated protein phosphatase has been purified from bovine myocardium. The purification procedure involves sequential DEAE-Sephacel ion exchange chromatography, calmodulin-Sepharose affinity chromatography, and high performance liquid chromatography using a Spherogel TSK DEAE 5PW column. By SDS polyacrylamide gel electrophoresis, the purified cardiac phosphatase consists of two subunits of Mr 61,000 and 19,000, similar to the brain enzyme, calcineurin. Protein phosphatase activity of the cardiac enzyme is stimulated by Ca2+-calmodulin and inhibited by the calmodulin antagonist drug, calmidazolium. Effects of a series of divalent cations on catalytic activity of the cardiac calmodulin-stimulated protein phosphatase are similar to those observed with calcineurin, when the two enzymes are assayed under identical conditions. Highly enriched preparations of bovine cardiac sarcolemma contain substrates of cAMP-dependent protein kinase of Mr 166 K, 133 K, 108 K, 79 K, 39 K, and 14 K, which are specifically dephosphorylated by the calmodulin-stimulated phosphatase with pseudofirst-order rate constants of 0.23, 0.46, 0.69, 0.35, 0.69, and 0.115 min-1, respectively. These substrates are not present in purified preparations of cardiac sarcoplasmic reticulum. These results support a role of the calmodulin-stimulated phosphatase in the Ca2+-regulation of specific sarcolemmal processes by protein dephosphorylation.

Vinculin phosphorylation in response to calcium and phorbol esters in intact cells.

Vinculin phosphorylation in both chick embryo fibroblasts and Swiss 3T3 cells was increased by either calcium or biologically active phorbol esters. Increased phosphorylation of vinculin was noted as early as 10 min following phorbol 12-myristate 13-acetate treatment and was maximal at about 1 h. Maximal increases in phosphorylation were noted at approximately 100 nM phorbol 12-myristate 13-acetate. Phorbol 12,13-dibutyrate (80 nM), a less potent phorbol ester, resulted in smaller increases in vinculin phosphorylation than phorbol 12-myristate 13-acetate at equimolar concentrations. Phorbol, dibutyryl cAMP, and dibutyryl cGMP had no significant effect on phosphorylation. No correlation was found between vinculin phosphorylation and the morphological changes induced by phorbol esters. Tryptic peptide analysis of vinculin revealed multisite phosphorylation. Phosphorylation of only three of the peptides was significantly increased following phorbol 12-myristate 13-acetate treatment. Phosphoamino acid analysis revealed increases at both serine and threonine residues. The low level of phosphotyrosine present in control cells was not significantly increased by phorbol 12-myristate 13-acetate treatment. These findings combined with studies of vinculin phosphorylation by purified protein kinase C (Werth, D. K., Niedel, J. E., and Pastan I. (1983) J. Biol. Chem. 258, 11423-11426) suggest the hypothesis that protein kinase C may be involved in regulation of phosphorylation of vinculin, a cytoskeletal protein.

Serodiagnosis of Q fever by enzyme-linked immunosorbent assay (ELISA).

Indirect enzyme-linked immunosorbent assays (ELISAs) specific for IgG and IgM antibodies against Coxiella burnetii were applied to test 208 serum samples collected within 1983 to 1986 from 128 individuals suspected of having Q fever, and from 1611 serum samples of normal blood donors. Among them were 2 patients with Q fever endocarditis, one patient with myocarditis, one patient with chronic hepatitis, 3 patients with pneumonia, one woman who had aborted a monstrous child, 38 state veterinarians, 26 farms workers, 21 persons employed in veterinary medicine, and 4 laboratory workers. Comparison with the complement fixation test (CF) revealed 46 (38%) subjects seropositive by CF and 77 (60%) seropositive by IgG and/or IgM ELISA. Among the normal blood donors 22% had antibodies to C. burnetii by ELISA. With exception of two CF titers of 1:2 and 1:8, all positive results detected by CF were confirmed by ELISA. Early stages of C. burnetii infections could be diagnosed in four cases with a single serum sample through demonstration of specific IgM by ELISA before appearance of CF antibodies. In 9 patients with acute Q fever and rising CF titers or IgG levels, diagnosis was already possible with the first serum sample by demonstration of high IgM levels by ELISA. In the two cases of endocarditis investigated, high CF titers against phase I antigen of C. burnetii confirmed the diagnosis "chronic Q fever".

Mechanisms of enhanced phosphorylase activation in the hyperthyroid rat heart.

Enhanced phosphorylase activation in hearts from hyperthyroid animals has been well documented. To elucidate the mechanisms responsible for the enhanced phosphorylase a formation, hearts from euthyroid and hyperthyroid rats were perfused by the Langendorff method with calcium (3.75 mM), isoproterenol, dibutryl cAMP and trifluoperazine, an inhibitor of calcium-calmodulin dependent enzymes. Comparative biochemical analyses revealed increased phosphorylase a formation in hearts from both euthyroid and hyperthyroid animals following exposure to calcium, dibutryl cAMP and isoproterenol. Hearts from hyperthyroid rats had an increased sensitivity to threshold concentrations of isoproterenol for both cAMP formation and phosphorylase b to a conversion. At higher concentrations of isoproterenol (10(-8) M and 3 x 10(-8) M), no significant differences in cAMP formation were noted between euthyroid and hyperthyroid animals in spite of persistently increased phosphorylase a levels in the hyperthyroid state. Trifluroperazine had no effect on basal phosphorylase a levels but significantly inhibited phosphorylase a formation in both groups following calcium or isoproterenol stimulation. However, enhanced phosphorylase a formation was still present in the hearts from hyperthyroid rats following trifluoperazine preperfusion. Determinations of phosphorylase kinase activity revealed a specific activity in the hyperthyroid animals twice that of the euthyroid controls. At least two mechanisms, an increased sensitivity to beta-adrenergic agents and increased cardiac phosphorylase kinase activity, may mediate the enhanced phosphorylase a formation found in hearts from hyperthyroid rats.

Vinculin, a cytoskeletal substrate of protein kinase C.

Vinculin, a cytoskeletal protein localized at adhesion plaques, is a phosphoprotein containing phosphoserine, phosphothreonine, and phosphotyrosine. Vinculin has been previously shown to be a substrate for pp60src, a phosphotyrosine protein kinase, but the kinase(s) responsible for phosphorylation of the other amino acid residues is unknown. The present report examines the phosphorylation of vinculin by various serine- and threonine-specific protein kinases. Only protein kinase C, the calcium-activated phospholipid-dependent protein kinase, phosphorylates vinculin at a significant rate (24 nmol/min/mg) and displays marked specificity for vinculin. Both calcium and phosphatidylserine were required for vinculin phosphorylation by protein kinase C. In addition, both phorbol 12,13-dibutyrate (10 nM) and phorbol 12-myristate 13-acetate (10 nM) stimulated vinculin phosphorylation by protein kinase C at a limiting calcium concentration (10(-6) M). Tryptic peptide analysis revealed two major sites of phosphorylation. One site contained phosphoserine and the other contained phosphothreonine. When compared with tryptic maps of vinculin phosphorylated by src kinase, no overlapping phosphorylated peptides were found. The present findings coupled with the plasma membrane location of both these proteins suggest that vinculin may be a physiologic substrate for protein kinase C.

Purification of a myosin phosphatase from bovine aortic smooth muscle.

A myosin phosphatase has been purified to homogeneity from bovine aortic smooth muscle. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme eluted from nondenaturing gels revealed two subunits (Mr = 67,000 and 38,000). Densitometric scans of the subunits indicated a molar ratio of 1:1. Several phosphoproteins were substrates for the phosphatase including histone II-A, isolated 20,000-dalton smooth muscle myosin light chains, phosphorylase a, and smooth muscle myosin. In the presence of 0.25 M NaCl and a substrate concentration of 2 microM, myosin was preferentially dephosphorylated. The specific activity of the phosphatase for myosin at a concentration of 10 microM was found to be 5 mumol/mg/min. The phosphatase required Mn2+ or Co2+ ions for activity. Mg2+, Ca2+, or Mg-ATP would not substitute for Mn2+ or Co2+ at equimolar concentrations. This phosphatase may play an important role in regulating actin-myosin interaction in smooth muscle by serving to dephosphorylate myosin.

Limited autolysis reduces the Ca2+ requirement of a smooth muscle Ca2+-activated protease.

Chicken gizzard smooth muscle contains large amounts of Ca2+-activated protease activity. Approximately 15 mg of purified enzyme can be obtained from 1 kg of fresh muscle. The enzyme consists of two subunits (Mr = 80,000 and 30,000) present in a 1:1 molar ratio. In the presence of CaCl2, the 80,000/30,000-dalton heterodimer (form I) is rapidly converted by limited autolysis to a 76,000/18,000-dalton species (form II). Both the 80,000- and 30,000-dalton subunits are degraded simultaneously. Moreover, the Ca2+ dependence for autolysis (K0.5 = 300 microM) is identical for both subunits. Neither the time course nor the Ca2+ dependence of the autolytic conversion reaction is altered by 10- and 20-fold molar excesses of substrate. Limited autolysis markedly reduces the Ca2+ requirement for substrate degradation. Using N-[ethyl-2-3H]maleimide-labeled 27,000-dalton cardiac myosin light chains as substrate, the Ca2+ requirement of form I was found to be quite high (K0.5 = 150 microM). Under similar conditions, the Ca2+ requirement of form II was 30-fold lower (K0.5 = 5 microM). Limited autolysis did not alter the specific activity of the enzyme. Our results demonstrate that smooth muscle contains an abundant amount of Ca2+-activated protease. Moreover, autolysis of this enzyme may play an important regulatory role by converting the native form to a species that is fully active at physiological levels of intracellular calcium ion.

Regulation of phosphorylase kinase in rat ventricular myocardium. Role of calmodulin.

Conversion of phosphorylase b to a which is catalyzed by the enzyme phosphorylase kinase is known to require Ca++. Trifluoperazine, an inhibitor of calmodulin-dependent enzymes, was utilized in the present study to clarify the role in vivo of calcium-calmodulin regulation of phosphorylase kinase. Twenty-minute preperfusion of isolated rat ventricles with 10(-5) M trifluoperazine had no effect on basal levels of phosphorylase a but significantly attenuated phosphorylase activation induced by either calcium (3.75 mM) or isoproterenol (3 x 10(-9) M, 3 x 10(-8) M). The positive inotropic effect of both agents and cyclic adenosine 3',5'-monophosphate (cAMP) levels were not altered by trifluoperazine in the perfused hearts. In addition, no effects of 10(-5) M trifluoperazine were noted on beta-adrenergic receptor binding of [3H](+/-)carazolol or on adenylate cyclase activity. In vitro studies with partially purified rat cardiac phosphorylase kinase demonstrated 1.5- to 3-fold stimulation by exogenous calmodulin. The addition of 10(-5) M trifluoperazine prevented calmodulin stimulation but had little effect on activity in the absence of exogenous calmodulin. The present results suggest that reversible binding of calcium-calmodulin may represent a physiological means for regulating phosphorylase kinase activity in rat cardiac muscle.

Primary neoplasms of the ureter.

The role of tobacco as a carcinogenic agent should be considered in ureteral carcinoma. In our study 77 per cent of the patients had a history of smoking, with an average of 50 packs a year consumption. Various studies have shown that the prognosis for ureteral carcinoma is related primarily to the grade and stage of the disease at the time of treatment. Therefore, it is imperative that the clinician maintain a high index of suspicion. This index of suspicion should be heightened in any patient with lower tract urothelial tumors. In addition to endoscopic followup excretory urography should be done on an annual basis, with retrograde studies when necessary. Ureteral brush biopsies or tissue obtained with the Dormia basket may be diagnostic. A Dormia stone basket was used in 3 patients in our study and sufficient tissue for diagnosis was obtained in each case. No complications were encountered. The results obtained in treating a primary leiomyosarcoma of the ureter are noteworthy in that complete remission of pulmonary metastases was obtained after the patient received a course of doxorubicin hydrochloride and dimethyl-triazeno imidazole carboxamide.

Functional studies on the EGF receptor with an antibody that recognizes the intracellular portion of the receptor.

An antibody against the human epidermal growth factor receptor (EGF), capable of activating its tyrosine kinase has been produced. Antibody 2913 recognizes only the cytoplasmic portion of the EGF receptor in A431 carcinoma cells, in normal human fibroblasts, and in a variety of other human tumor cell lines (Xu, Y.-A., Richert, N., Ito, S., Merlino, G. T., and Pastan, I. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7308-7313). Indirect immunofluorescence and electron microscopy show that the antibody binds to intact cells only after membrane permeabilization. Moreover the antibody immunoprecipitates the v-erb-B gene product in avian myeloblastosis virus-infected cells but does not recognize the secreted form (105 kDa) of the A431 cell EGF receptor which lacks the cytoplasmic domain. Antibody 2913 activates the EGF receptor kinase in solubilized A431 membranes causing autophosphorylation on tyrosine residues only. Tryptic peptide maps suggest that antibody 2913 and EGF stimulate phosphorylation of the same amino acid residues. By electron microscopy, the cytoplasmic portion of the receptor was followed throughout its endocytotic pathway. The results show that the kinase domain is rapidly degraded in lysosomes with no accumulation in the cytoplasm or in the nucleus.

src kinase catalyzes the phosphorylation and activation of the insulin receptor kinase.

When a partially purified insulin receptor preparation immobilized on insulin-agarose is incubated with [gamma-32P]ATP, Mn2+, and Mg2+ ions, the receptor beta subunit becomes 32P-labeled. The 32P-labeling of the insulin receptor beta subunit is increased by 2-3-fold when src kinase is included in the phosphorylation reaction. In addition, the presence of src kinase results in the phosphorylation of a Mr = 125,000 species. The Mr = 93,000 receptor beta subunit and the Mr = 125,000 32P-labeled bands are absent when an insulin receptor-deficient sample, prepared by the inclusion of excess free insulin to inhibit the adsorption of the receptor to the insulin-agarose, is phosphorylated in the presence of the src kinase. These results indicate that the insulin receptor alpha and beta subunits are phosphorylated by the src kinase. The src kinase-catalyzed phosphorylation of the insulin receptor is not due to the activation of receptor autophosphorylation because a N-ethylmaleimide-treated receptor preparation devoid of receptor kinase activity is also phosphorylated by the src kinase. Conversely, the insulin receptor kinase does not catalyze phosphorylation of the active or N-ethylmaleimide-inactivated src kinase. Subsequent to src kinase-mediated tyrosine phosphorylation, the insulin receptor, either immobilized on insulin-agarose or in detergent extracts, exhibits a 2-fold increase in associated kinase activity using histone as substrate. src kinase mediates phosphorylation of predominantly tyrosine residues on both alpha and beta subunits of the insulin receptor. Tryptic peptide mapping of the 32P-labeled receptor alpha and beta subunits by high pressure liquid chromatography reveals that the src kinase-mediated phosphorylation sites on both receptor subunits exhibit elution profiles identical with those phosphorylated by the receptor kinase. Furthermore, the HPLC elution profile of the receptor auto- or src kinase-catalyzed phosphorylation sites on the receptor alpha subunit are also identical with that on the receptor beta subunit. These results indicate that: the src kinase catalyzes tyrosine phosphorylation of the insulin receptor alpha and beta subunits; and src kinase-catalyzed phosphorylation of insulin receptor can mimic the action of autophosphorylation to activate the insulin receptor kinase in vitro, although whether this occurs in intact cells remains to be determined.

Vinculin phosphorylation by the src kinase. Interaction of vinculin with phospholipid vesicles.

Vinculin phosphorylation by pp60src is stimulated by anionic phospholipids (Ito, S., Richert, N., and Pastan, I. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 4628-4631). We have examined whether vinculin interacts with phospholipids, the specificity of the interactions, and a possible mechanism for the enhancement of vinculin phosphorylation by these phospholipids. 3H-labeled vinculin binds to phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. No binding to phosphatidylcholine or phosphatidylethanolamine was observed. The phospholipid binding specificity correlated with the ability of these phospholipids to enhance vinculin phosphorylation by the src kinase. Chlorpromazine (0.1 and 0.3 mM) inhibited both vinculin binding to phosphatidylinositol and the enhanced phosphorylation of vinculin by pp60src in the presence of phosphatidylinositol. Tryptic peptide maps of vinculin phosphorylated in the absence of phospholipid revealed three phosphorylated peptides. The same three peptides were phosphorylated in the presence of phospholipid. However, phosphorylation at one site was markedly increased. In the presence of phospholipid proteolysis of vinculin with both chymotrypsin and V8 protease was markedly enhanced and different peptide maps of vinculin were generated. Microheterogeneity of vinculin was observed with isoelectric focusing. All the isoforms (pI 5.45-5.8) were found to bind phospholipids and undergo phosphorylation by the src kinase. These results suggest that one way anionic phospholipids can enhance vinculin phosphorylation is by binding to vinculin and inducing a conformational change in the vinculin molecule.

Enrichment, solubilization, and partial characterization of digitonin-solubilized muscarinic receptors derived from canine ventricular myocardium.

The subcellular distribution of cardiac muscarinic receptors was defined in canine ventricular myocardium, and receptors were solubilized from subcellular fractions enriched in muscarinic receptor content. The subcellular location of muscarinic receptors in cardiac tissue was determined by measurement of the distribution of [3H](+/-)quinuclidinyl benzilate-binding activity in particulate fractions isolated from canine ventricular myocardium. Based upon excellent correlation between [3H](+/-)quinuclidinyl benzilate binding and activity of the sarcolemmal Na+,K+-ATPase throughout the subcellular fractions, muscarinic receptors appeared to be localized to sarcolemma in canine ventricular myocardium. Therefore, membrane fractions enriched in sarcolemma were used as a source of cardiac muscarinic receptors for solubilization. Treatment of membrane vesicle fractions with digitonin (0.6%) resulted in solubilization of [3H](+/-)quinuclidinyl benzilate-binding activity with an extraction yield of 25-35%. Criteria of pharmacological specificity and stereospecificity established the identity of the solubilized binding activity of muscarinic receptors. Solubilization of muscarinic receptors was documented by demonstration of hydrodynamic behavior consistent with molecularly dispersed material. Upon glycerol gradient centrifugation, digitonin-solubilized muscarinic receptors from cardiac tissue sedimented with an apparent sedimentation coefficient of 9S. Pharmacological characterization of the digitonin-solubilized receptors revealed 8- to 39-fold reductions in affinities for muscarinic antagonists compared to the affinities exhibited by receptors in the membrane-bound state. Substantially greater reductions in agonist affinities (reduction of at least 700-fold for all agonists studied) suggested selective loss of ability of the digitonin-solubilized receptors to exhibit high affinity agonist interactions. In contrast to membrane-bound receptors, digitonin-solubilized receptors also demonstrated a loss of guanine nucleotide regulation, as well as steep agonist:radioligand competition curves with slope factors of 1.0, suggesting a homogeneous population of agonist-binding sites. Interpreted within the context of a model of state interconversion for membrane-bound receptors, the results suggested that either muscarinic receptors of a single state were selectively solubilized, or that solubilization induced conversion of all receptors to a single low affinity state, possibly by removal of constituents necessary for assumption of a high affinity agonist conformation.

Phosphorylation of the solubilized insulin receptor by the gene product of the Rous sarcoma virus, pp60src.

Both the insulin receptor and the gene product of the Rous sarcoma virus, pp60src, are protein kinases which phosphorylate themselves and other proteins on tyrosine residues. Addition of the solubilized insulin receptor to purified pp60src increased the phosphorylation of the beta-subunit of the insulin receptor. Phosphorylation of the insulin receptor by pp60src occurred both in the absence and presence of insulin but did not alter the insulin dose response for autophosphorylation of the receptor. Increasing concentrations of pp60src increased the phosphorylation of the receptor and at high concentrations equaled the maximal effect produced by insulin. Our observations suggest a possible mechanism by which the metabolically regulated insulin receptor tyrosine kinase could be altered by other tyrosine kinases such as that associated with pp60src. Further studies will be required to determine if the insulin receptor is phosphorylated by pp60src in Rous sarcoma virus-infected cells.