Na(+)-Ca2+ exchange, myoplasmic Ca2+ concentration, and contraction of arterial smooth muscle. Na(+)-Ca2+ exchange is proposed to be an important regulator of myoplasmic intracellular Ca2+ concentration ([Ca2+]i) and contraction in vascular smooth muscle. We investigated the role of Na(+)-Ca2+ exchange in regulating [Ca2+]i in swine carotid arterial tissues that were loaded with aequorin to allow simultaneous measurement of [Ca2+]i and force. Reversal of Na(+)-Ca2+ exchange, by reduction of extracellular Na+ concentration ([Na+]o) to 1.2 mM, induced a large increase in aequorin-estimated [Ca2+]i and a low [Ca2+]i sensitivity. The contraction induced by 1.2 mM [Na+]o was partially caused by depolarization and opening of L-type Ca2+ channels because 10 microM diltiazem partially attenuated the 1.2 mM [Na+]o-induced increases in [Ca2+]i. High dose ouabain (10 microM), a putative endogenous Na+,K(+)-ATPase inhibitor, increased both [Ca2+]i and force. However, the increases in [Ca2+]i and force were mostly blocked by 10 microM phentolamine, suggesting the predominant effect of ouabain was to increase norepinephrine release from nerve terminals. In the presence of 10 microM phentolamine, 10 microM ouabain slightly accentuated 1 microM histamine-induced increases in [Ca2+]i and force. The ouabain dose necessary to induce contraction in the absence of phentolamine was significantly less than the ouabain dose necessary to accentuate histamine-induced contractions in the presence of phentolamine. These results suggest that Na(+)-Ca2+ exchange exists in swine arterial smooth muscle. These data also suggest that ouabain (which should increase [Na+]i and inhibit Na(+)-Ca2+ exchange) primarily enhances contractile function in the swine carotid artery by releasing catecholamines from nerve terminals; direct action of Na+,K(+)-ATPase inhibitors on smooth muscle appears to occur only with very high doses.


C-type natriuretic peptide inhibits thrombin- and angiotensin II-stimulated endothelin release via cyclic guanosine 3',5'-monophosphate. We examined the inhibitory effect of porcine C-type natriuretic peptide (CNP) on endothelin-1 secretion stimulated by thrombin and angiotensin II (Ang II) in cultured porcine endothelial cells. The results were compared with the effects of atrial (ANP) and brain (BNP) natriuretic peptides. Thrombin and Ang II produced a concentration-dependent stimulation of immunoreactive endothelin-1 secretion, and porcine CNP-22 potently inhibited this stimulated secretion in a concentration-dependent manner. CNP-22 had a stronger inhibitory effect than either porcine ANP(1-28) or porcine BNP-26. In addition, CNP potently increased the cellular level of cyclic guanosine 3',5'-monophosphate (GMP), with the inhibition of immunoreactive endothelin-1 secretion in response to thrombin and Ang II being paralleled by the increase in the cyclic GMP level. The increase of cyclic GMP produced by CNP was also greater than that due to porcine ANP(1-28) or porcine BNP-26. The immunoreactive endothelin-1 in the culture medium had two components on high-performance liquid chromatography; the major one corresponded to endothelin-1 (1-21) and the minor one to big endothelin-1 (porcine 1-39). Treatment with CNP did not affect this profile. Our results suggest that CNP probably inhibits the endothelin-1 secretion stimulated by thrombin and Ang II through a cyclic GMP-dependent process. The increase of cyclic GMP levels and the inhibition of immunoreactive endothelin-1 secretion produced by CNP appear to be greater than those produced by ANP or BNP.


Pressure natriuresis in rats during blockade of the L-arginine/nitric oxide pathway. The natriuretic response was studied in anesthetized rats during the intravenous infusion of L-arginine analogues to inhibit the production of endothelium-derived nitric oxide. In an initial experimental series, rats were administered saline vehicle or vehicle containing 300 mumol/kg body wt N omega-monomethyl-L-arginine, N omega-nitro-L-arginine methyl ester, N omega-monomethyl-D-arginine, or L-arginine. Infusion of the competitive inhibitors N omega-monomethyl-L-arginine and N omega-nitro-L-arginine methyl ester significantly increased mean arterial pressure to 155 +/- 3 and 145 +/- 5 mm Hg, respectively, compared with a mean arterial pressure of 118 +/- 3 mm Hg determined in the vehicle control group. Sodium excretion averaged 3.27 +/- 1.08 and 2.52 +/- 0.78 mu eq/min in the N omega-monomethyl-L-arginine- and N omega-nitro-L-arginine methyl ester-treated rats, respectively, and each was significantly higher than the basal sodium excretion of 0.20 +/- 0.05 mu eq/min in the vehicle-treated control animals. Plasma renin activity was significantly lower in the N omega-monomethyl-L-arginine- and N omega-nitro-L-arginine methyl ester-treated groups than in the vehicle-treated group. Neither L-arginine nor N omega-monomethyl-D-arginine administration significantly altered any of the measured variables compared with vehicle alone. In a second experimental series, an adjustable snare was placed around the suprarenal aorta for the purpose of controlling renal perfusion pressure independently of increases in the systemic mean arterial pressure initiated by infusion of N omega-nitro-L-arginine methyl ester (75 mumol/kg i.v.).(ABSTRACT TRUNCATED AT 250 WORDS)


On the role of renal alpha-adrenergic receptors in spontaneously hypertensive rats. We tested the hypothesis that a genetically determined increase in renal alpha-adrenergic receptor density might be a pathophysiologically important factor in the spontaneously hypertensive rat model of genetic hypertension. In a first study, we compared renal alpha 1 and alpha 2-adrenergic receptor density with systolic blood pressure in 45 rats of an F2 generation of Wistar-Kyoto x spontaneously hypertensive rat hybrids but were unable to detect significant cosegregation between either receptor density or blood pressure. In a second study, we determined renal alpha 1- and alpha 2-adrenergic receptor density in Wistar-Kyoto and spontaneously hypertensive rat kidneys that were transplanted into an F1 generation of Wistar-Kyoto x spontaneously hypertensive rat hybrids. Although Wistar-Kyoto kidneys lowered blood pressure in these animals and spontaneously hypertensive rat kidneys increased blood pressure, renal alpha-adrenergic receptor densities were similar in membranes from both types of kidneys. Since rat kidney coexpresses alpha 1A- and alpha 1B-adrenergic receptors, we also investigated whether differential regulation of these two subtypes might conceal ongoing alterations. The alpha 1A/alpha 1B-adrenergic receptor ratio, however, was similar in Wistar-Kyoto rats, spontaneously hypertensive rats, and F1 rats transplanted with a kidney from either strain. Taken together these data do not support the hypothesis that genetically determined alterations of renal alpha-adrenergic receptor numbers play an important role in the development of elevated blood pressure in the spontaneously hypertensive rat.


Leukotriene release from neutrophils of patients on hemodialysis with cellulose membranes. The role of cytokines in patients with chronic renal failure is currently under investigation. We therefore studied the release of leukotriene B4 (LTB4) from polymorphonuclear leukocytes (PMN) in stable dialysis patients treated with two different cellulose membranes, Cuprophan and Hemophan, a modified cellulose with less complement activation. Six patients were treated for four weeks with Cuprophan then switched to Hemophan for another four weeks. Before and after the last treatment of each period, PMN were separated from 20 ml heparinized blood by FICOLL density gradient centrifugation. Portions of 5 x 10(6) PMN were resuspended in Hanks' buffer and stimulated for 5 minutes with calcium ionophore A23187 (5 micrograms/ml). LTB4 in cell supernatants was determined by specific radioimmunoassay. PMN from dialysis patients before HD released significantly (p less than 0.01) more LTB4 than healthy donors. No significant difference between pre- and post-dialysis values was observed with Cuprophan or Hemophan dialyzers. Our data suggest that the acute effects of blood membrane interaction with either complement activating or non-activating dialyzers do not lead to changes in post-dialysis leukotriene metabolism, but leukotriene production is enhanced chronically in dialysis patients.


Cellular heterogeneity of DNA/total-protein content in human lung tumors, as determined by flow cytometry. With the aim of distinguishing neoplastic cell sub-populations of different prognostic and diagnostic significance, dual-parameter measurements (DNA/protein) have been simultaneously determined in a (256, 256) channel matrix in lung samples derived from 110 patients affected by neoplastic and non-neoplastic lung diseases. Biparametric analysis demonstrated that cells with abnormally high red fluorescence (i.e., protein content), which is indicative of unbalanced growth, were often observed in malignant tumors as compared with normal lung samples. Furthermore, the dual-parameter analysis allowed recognition of additional aneuploid tumor-cell lines, indicating that the frequency of cytometrically determined diploid tumor is lower than that previously described by DNA monoparametric analysis. The recognition of aneuploid subpopulations by dual-parameter analysis in clinically and histologically negative one-parameter flow-cytometric "diploid" samples assumes important diagnostic value. The results have also shown the presence of multiple protein sub-populations in clones with the same ploidy value, indicating a higher level of cellular heterogeneity than demonstrated by DNA monoparametric measurements.


Serum oncoproteins and growth factors in asbestosis and silicosis patients. Levels of 9 different oncoproteins and growth factors were assayed by immunoblotting with monoclonal antibodies in 91 serum samples collected between March 1983 and August 1987 from 46 pneumoconiosis patients (36 asbestosis, 10 silicosis) at high risk for the development of cancer. Follow-up of these patients through June 1991 showed that 18 had developed cancer (11 lung, 2 pleural mesothelioma, 2 transitional-cell carcinomas of the urinary bladder, 1 osteosarcoma, 1 non-Hodgkin's lymphoma, 1 adenocarcinoma of the gallbladder). Increased serum levels of ras oncogene-related protein (p21) were found in 7 of the 18 patients who developed cancer (5 lung, 2 pleural mesothelioma) versus 2 of the 28 patients without cancer, a statistically significant difference (p = 0.012). In addition, 6 of the 7 p21-positive cancer cases had positive serum samples prior to clinical diagnosis of disease (average = 16.3 months, range = 3-26 months prior to diagnosis), suggesting that elevated serum p21 levels may be a useful marker for earlier detection in a significant percentage of respiratory malignancies. Finally, elevated serum levels of PDGF-related protein were detected significantly more frequently in advanced pneumoconiosis cases (ILO radiographic classification of 2/1 or greater) than in less advanced cases (80% vs. 41.9%; p = 0.016), and there was a tendency for these PDGF-positive patients to have progression of their disease (68.2% vs. 41.7%; p = 0.065), suggesting that elevated serum PDGF levels may be a marker for the development of severe and progressive pneumoconioses.


Mesothelioma in rats following intrapleural injection of chrysotile and phosphorylated chrysotile (chrysophosphate). Pathological effects of asbestos are probably dependent on the size and surface properties of the fibers. Surface-modified chrysotile fibers were injected into the pleural cavity of rats to investigate the potency of the fiber to induce mesothelioma. Chrysotile fibers were modified by a phosphorylation process, resulting in the presence of phosphorus at the fiber surface. Phosphorylated samples were characterized by enhanced durability and reduced affinity for biological macromolecules. Five samples were tested: 1 untreated and 4 phosphorylated. ChrP1, ChrP2 and ChrP3 corresponded to phosphorylated samples obtained by first, second and third passages through an Alpine classifier; Pm was defibrillated ChrP1. The number of fibers per microgram and the size distribution were determined by transmission electron microscopy and classified in 4 size groups. Groups of 35 rats were inoculated with 20 mg of fibers suspended in 0.9% NaCl solution. No mesothelioma was found in the saline controls. All fiber samples were proficient in producing mesothelioma; the percentages were different between groups and untreated chrysotile but not significantly so. The differences may be explained on the basis of the number of fibers injected which were greater than 8 microns in length and less than 0.25 microns in diameter. The findings of a proficiency of long fibers to produce mesothelioma, previously reported by others for glass fibers, could be applied to chrysotile.


Anti-idiotype vaccine against Hodgkin's lymphoma: induction of B- and T-cell immunity across species barriers against CD30 antigen by murine monoclonal internal image antibodies. Murine monoclonal antibody HRS-4 (Ab1), which defines the cell-bound and soluble CD30 antigen associated with Hodgkin's lymphoma, was used to generate monoclonal anti-idiotypic antibodies (Ab2) in syngeneic BALB/c mice. Murine monoclonal Ab2 14G9 and Ab2 9G10 directed against HRS-4 were shown to be anti-idiotypic Ab2 beta carrying the internal image of the CD30 antigen. These antibodies bound specifically to HRS-4 and effectively inhibited binding of HRS-4 to a CD30 antigen preparation at concentrations as low as 50 ng/ml. KLH-coupled Ab2 beta 14G9 and 9G10 induced in BALB/c mice and New Zealand white rabbits a specific polyclonal humoral response against the 120 kDa band of the CD30 antigen. Moreover, BALB/c mice immunized i.p. with KLH-coupled 14G9 and 9G10 exhibited a statistically significant (p less than 0.01) delayed-type hypersensitivity reaction against CD30 expressing Hodgkin-derived L540-cells. We conclude from these data that Ab2 beta 14G9 and 9G10, mimicking structures of the nominal CD30 antigen, are capable of inducing a CD30-specific T-cell- and B-cell-mediated immune response in mice and even across species barriers in rabbits. These CD30 anti-id antibodies may hold promise for use as vaccines against CD30-antigen-expressing lymphomas.


Differential expression of myogenic regulatory genes, MyoD1 and myogenin, in human rhabdomyosarcoma sublines. A cell line (SCMC-MM-1) was established from a human abdominal tumor that was initially diagnosed as a malignant mesenchymoma by histological, immunohistochemical and clinical criteria. The cell line was composed of 2 morphologically and immunohistochemically distinct cell types, one with a small polygonal phenotype (P-type), characterized by the immunostaining of vimentin and the presence of a few electron-microscopically visible organelles, and the other with a giant tubular phenotype (T-type), characterized by the immunostaining of desmin, alpha-sarcomeric actin and skeletal-muscle myosin, and the presence of thick and thin myofilaments and Z-line materials. The parental cell line was cloned into 2 sublines, a P-type clone (SCMC-MM-1-19P) and a T-type clone (SCMC-MM-1-1T), which shared both 2q37 and 11p15 translocations, the characteristic chromosomal aberrations for rhabdomyosarcoma, with the parental SCMC-MM-1 cell line. Northern-blot analyses of the myogenic regulatory genes, including MyoD1 and myogenin, demonstrated the expression of MyoD1 in both of these sublines. Myogenin was very weakly expressed in the SCMC-MM-1-19P subline, but strongly expressed in the SCMC-MM-1-1T subline. Chromosomal and myogenic-regulatory-gene analyses revealed that both of these sublines were rhabdomyosarcoma cell lines. Furthermore, the regulatory-gene analyses indicated that these 2 sublines represented 2 distinct differentiation stages of myoblasts, and that MyoD1 and myogenin could serve as the lineage marker and the differentiation marker, respectively, of human rhabdomyosarcoma.


Regional hyperthermia combined with radiotherapy in the treatment of lung cancers. Twenty locally advanced lung cancers were treated by hyperthermia in combination with radiotherapy between November 1980 and January 1990. All tumors selected had invaded or were in contact with the chest wall, so that transcutaneous insertion of thermal probes into the tumor was possible. Using an 8 or 13.56 MHZ RF capacitive heating device, hyperthermia was given once or twice a week after irradiation for 30-60 min per session (1-12 sessions in total). Radiotherapy was delivered at dose of 13.6-70 Gy. The thermal parameters analyzed were a) maximum, average, and minimum intratumor temperatures (Tmax, Tav, and Tmin), which were recorded at the termination of each treatment, and b) the percentages of the intratumor points that exceeded 41 C (%T greater than or equal to 41 C). The mean +/- SD for Tmax, Tav, Tmin, and %T greater than or equal to 41 C was 42.9 +/- 1.7 C, 41.6 +/- 1.2 C, 39.7 +/- 1.1 C, and 56.2 +/- 25.8, respectively. Larger tumors showed higher thermal parameters than the smaller tumors. Of the 12 tumors treated by definitive therapy, 2 (17%) achieved CR, 7 (58%) PR, and 3 (25%) NR. Four of 10 tumors that did not achieve CR showed large intratumor low density areas on post-treatment CT, reflecting massive coagulation necrosis. Higher thermal parameters were closely related to the appearance of low-density areas but not to changes in tumor size. Four tumors treated preoperatively were successfully resected 2 weeks after thermoradiotherapy, whereas four palliatively-treated tumors showed no regression. The side effects associated with hyperthermia were pain in 12 patients (60%) and dyspnea in 3 (15%), all of which resolved after termination of treatment. A skin abscess and a pneumothorax attributed to thermal probe insertion were observed in one patient each. These results indicate that regional RF capacitive hyperthermia is clinically feasible for local treatment of selected lung cancers.


Prognostic assessment of tumor regression after external irradiation for cervical cancer. From February 1980 to December 1986, 428 cases of cervical cancer in Stage I through IVA were given curative radiation therapy at Chang Gung Memorial Hospital, Taipei. All of them received external irradiation and intracavitary brachytherapy. The degree of tumor regression was assessed immediately before the first intracavitary brachytherapy treatment. Patients were classified at this time as having (a) no gross residual tumor, or (b) gross residual tumor. Factors found to be associated with tumor regression by logistic regression analysis were stage, age and hemoglobin level. Patients with advanced cancer (Stage III, IVA), young age (less than 40 years), and low hemoglobin level (less than 10 g/dl) had a low incidence of no gross residual tumor. Five-year survival rate was 77% in patients with no gross residual tumor and 31% in patients with gross residual tumor (p less than 0.001). This significant difference held true even when one compared these two groups stage for stage; the difference was 77% versus 41% (p less than 0.001) in Stage II and 72% versus 28% (p less than 0.001) in Stage III. The local relapse rate was 59% in the gross residual tumor groups, significantly greater (p less than 0.001) than the 12% found in the no gross residual tumor group. It was concluded that patients whose tumors did not regress after external pelvic irradiation tended to recur after intracavitary brachytherapy, most often locally. This would justify a more aggressive treatment to improve local tumor control in this subset of high risk patients.


Chemodectoma of the head and neck: results of treatment in 84 patients. Eighty-four patients with chemodectoma of the head and neck presented to the Royal Marsden Hospital between 1949 and 1985. For tumors arising at the skull base (glomus jugulare and glomus tympanicum) 46 were treated with radiotherapy alone resulting in an actuarial local control rate of 73% at 25 years; 13 were treated with surgery plus radiotherapy with no recurrences during a median follow-up of 9 years; 4 had surgery alone but all recurred by 7 years. For tumors of the soft tissues of the neck (carotid body and glomus vagale) 13 were treated with surgery alone with an actuarial control rate of 54% at 15 years; 4 were treated with radiotherapy which resulted in local control at 1, 2, 8 and 11 years; and one patient who received both surgery and radiotherapy remained controlled at 1 year. Although comparison between radiotherapy and surgery in terms of tumor control is not simple, the case is argued for more frequent use of radiotherapy at all sites. This case is strengthened by minimal morbidity from radiotherapy in doses which appear effective: in the range of 45-50 Gy in 25 daily fractions over 5 weeks.


Radiotherapy for pleomorphic adenoma of the parotid gland. A study was made of 187 patients with parotid pleomorphic adenoma treated by radiotherapy. This followed surgery but with incomplete removal or tumor spillage. In the early years of the study radiotherapy was given by radium needle implant done usually at the time of surgery, but from the late 1960s beam-directed external radiotherapy with a head shell was used most commonly. A 3-field technique or wedge pair was the standard technique. The median age was 46 with nearly half the patients (87/187) aged between 40 and 60, and the ratio of women to men was 1.4:1 (110:77). Median follow-up for all patients was 14 years. One hundred fifteen patients had radiotherapy immediately after their first operation with a recurrence rate of 0.9% (1/115). Of the 115 there were 2 cases of radionecrosis (1 major, 1 minor), 1 case of permanent facial nerve palsy, 1 Frey Syndrome (post-gustatory sweating), and 1 salivary fistula. Seventy-two patients had radiotherapy delayed until one or more recurrences had been surgically treated. Nine (12.5%) of these developed yet further recurrence after radiotherapy. There were 2 cases of radionecrosis (1 major), 4 cases of facial nerve palsy (3 of which were complete), 16 cases (22.2%) of Frey Syndrome, and 1 case of malignant change in a parotid tumor. In addition one squamous cell carcinoma developed at the site of a needle implant 25 years later. Recurrences after radiotherapy continued beyond 20 years of follow-up. Patients having unsatisfactory surgery due to spill at operation or residual tumor left behind should have radiotherapy immediately and not delayed until local recurrence occurs because of the increased morbidity and the higher incidence of yet further recurrence.


Detection of Epstein-Barr virus DNA in a Japanese case of lymphoepithelioma-like thymic carcinoma. Epstein-Barr virus DNA was detected in a case of lymphoepithelioma-like thymic carcinoma. A homogeneous terminal structure of the viral DNA was demonstrated in this case, indicating the presence of the viral genome in clonally expanded tumor cells. Since all of 26 other thymic epithelial tumors (eight non-invasive, 13 invasive thymomas and four non-lymphoepithelioma-like thymic carcinomas) in Japanese were negative by polymerase chain reaction, it is suggested that lymphoepithelioma-like thymic carcinoma may represent a unique pathological entity distinct from Epstein-Barr virus-negative thymic epithelial tumors, which are in the majority in Japan.


Quartromicin, a complex of novel antiviral antibiotics. I. Production, isolation, physico-chemical properties and antiviral activity. A strain of Amycolatopsis orientalis No. Q427-8 (ATCC 53884) was found to produce a complex of new antiviral antibiotics, quartromicin which consisted of at least six components A1, A2, A3, D1, D2 and D3. Structural studies suggested that they are a novel type of molecules unrelated to any known antibiotics. Each component of quartromicin exhibited antiviral activity against herpes simplex virus type 1, influenza virus type A and human immunodeficiency virus.


Protective effects of immunoactive peptide, FK565 against systemic and local infections with herpes simplex virus and murine cytomegalovirus and respiratory tract infection with influenza virus in mice. The protective effects of FK565 against systemic infections with herpes simplex virus (HSV) and murine cytomegalovirus (MCMV), respiratory tract infection with influenza virus and zosteriform rash with HSV investigated in mice. FK565 showed excellent protective activities against systemic infections with both acyclovir (ACV)-sensitive and -resistant HSV at intravenous and subcutaneous doses of 0.1 and 1 mg/kg and oral dose of 1 mg/kg. FK565 showed superior protective activities at subcutaneous doses of 0.01 and 0.1 mg/kg compared to ACV at subcutaneous dose of 15 mg/kg against MCMV infection. In respiratory tract infection with influenza virus, FK565 showed potent protective effects at intravenous, subcutaneous and oral doses of 0.001 to 1 mg/kg. FK565 markedly inhibited zosteriform spread of HSV on the flank skin at an intravenous dose of 0.1 mg/kg and the mice given FK565 survived longer than the control mice. The peritoneal exudate cells from FK565-treated mice suppressed the growth of HSV in mouse embryo fibroblast more strongly than those from the control mice, although FK565 had no direct antiviral activity against HSV. These findings suggest that FK565 enhanced the host defense ability against viral infections by nonspecific activation of macrophages.


Use of gene probes based on the insertion sequence IS986 to differentiate between BCG vaccine strains. Gene probes derived from the insertion sequence IS986, which have previously been shown to differentiate isolates of Mycobacterium tuberculosis for epidemiological analysis, are also capable of distinguishing two groups of BCG vaccine strains. Most BCG strains have a single copy of IS986, at the same chromosomal site, while the Brazilian, Japanese and USSR strains have an additional copy at a different, common location. These results correlate with the results of previous antigenic analysis and may reflect a different clonal origin of the two groups of BCG strains.


Cell density-dependent modulation of the Vibrio fischeri luminescence system in the absence of autoinducer and LuxR protein. Expression of the Vibrio fischeri luminescence genes (luxR and luxICDABEG) in Escherichia coli requires autoinducer (N-3-oxohexanoyl homoserine lactone) and LuxR protein, which activate transcription of luxICDABEG (genes for autoinducer synthase and the luminescence enzymes), and cyclic AMP (cAMP) and cAMP receptor protein (CRP), which activate transcription of the divergently expressed luxR gene. In E. coli and in V. fischeri, the autoinducer-LuxR protein-dependent induction of luxICDABEG transcription (called autoinduction) is delayed by glucose, whereas it is promoted by iron restriction, but the mechanisms for these effects are not clear. To examine in V. fischeri control of lux gene expression by autoinducer, cAMP, glucose, and iron, lux::Mu dI(lacZ) and lux deletion mutants of V. fischeri were constructed by conjugation and gene replacement procedures. beta-Galactosidase synthesis in a luxC::lacZ mutant exhibited autoinduction. In a luxR::lacZ mutant, complementation by the luxR gene was necessary for luminescence, and addition of cAMP increased beta-galactosidase activity four- to sixfold. Furthermore, a luxI::lacZ mutant produced no detectable autoinducer but responded to its addition with induced synthesis of beta-galactosidase. These results confirm in V. fischeri key features of lux gene regulation derived from studies with E. coli. However, beta-galactosidase specific activity in the luxI::lacZ mutant, without added autoinducer, exhibited an eight- to tenfold decrease and rise back during growth, as did beta-galactosidase and luciferase specific activities in the luxR::lacZ mutant and luciferase specific activity in a delta(luxR luxICD) mutant. The presence of glucose delayed the rise back in beta-galactosidase and luciferase specific activities in these strains, whereas iron restriction promoted it. Thus, in addition to transcriptional control by autoinducer and LuxR protein, the V. fischeri lux system exhibits a cell density-dependent modulation of expression that does not require autoinducer, LuxR protein, or known lux regulatory sites. The response of autoinducer-LuxR protein-independent modulation to glucose and iron may account for how these environmental factors control lux gene expressions.


Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions. Helicobacter pylori produces a potent urease that is believed to play a role in the pathogenesis of gastroduodenal diseases. Four genes (ureA, ureB, ureC, and ureD) were previously shown to be able to achieve a urease-positive phenotype when introduced into Campylobacter jejuni, whereas Escherichia coli cells harboring these genes did not express urease activity (A. Labigne, V. Cussac, and P. Courcoux, J. Bacteriol. 173:1920-1931, 1991). Results that demonstrate that H. pylori urease genes could be expressed in E. coli are presented in this article. This expression was found to be dependent on the presence of accessory urease genes hitherto undescribed. Subcloning of the recombinant cosmid pILL585, followed by restriction analyses, resulted in the cloning of an 11.2-kb fragment (pILL753) which allowed the detection of urease activity (0.83 +/- 0.39 mumol of urea hydrolyzed per min/mg of protein) in E. coli cells grown under nitrogen-limiting conditions. Transposon mutagenesis of pILL753 with mini-Tn3-Km permitted the identification of a 3.3-kb DNA region that, in addition to the 4.2-kb region previously identified, was essential for urease activity in E. coli. Sequencing of the 3.3-kb DNA fragment revealed the presence of five open reading frames encoding polypeptides with predicted molecular weights of 20,701 (UreE), 28,530 (UreF), 21,744 (UreG), 29,650 (UreH), and 19,819 (UreI). Of the nine urease genes identified, ureA, ureB, ureF, ureG, and ureH were shown to be required for urease expression in E. coli, as mutations in each of these genes led to negative phenotypes. The ureC, ureD, and ureI genes are not essential for urease expression in E. coli, although they belong to the urease gene cluster. The predicted UreE and UreG polypeptides exhibit some degree of similarity with the respective polypeptides encoded by the accessory genes of the Klebsiella aerogenes urease operon (33 and 92% similarity, respectively, taking into account conservative amino acid changes), whereas this homology was restricted to a domain of the UreF polypeptide (44% similarity for the last 73 amino acids of the K. aerogenes UreF polypeptide). With the exception of the two UreA and UreB structural polypeptides of the enzyme, no role can as yet be assigned to the nine proteins encoded by the H. pylori urease gene cluster.


Ty insertions upstream and downstream of native DUR1,2 promoter elements generate different patterns of DUR1,2 expression in Saccharomyces cerevisiae. Expression of allantoin pathway genes is subject to induction and nitrogen catabolite repression. Two classes of cis-dominant mutations (DUR80 and DUR1,2-Oh) result in overproduction of DUR1,2 mRNA. In DUR80 mutants, DUR1,2 expression remained inducible, nitrogen catabolite repression sensitive, and unresponsive to cell ploidy, i.e., overproduction was superimposed on normal gene regulation. DUR1,2-Oh mutations, in contrast, generated a pattern of DUR1,2 expression similar to that often reported when a Ty element inserts upstream of a gene, the ROAM phenotype. We analyzed four independent DUR80 and DUR1,2-Oh alleles. The DUR1,2-Oh mutation was, as expected, a Ty insertion at -445 3' of the native DUR1,2 upstream activation sequences (UASs). All three DUR80 alleles were also Ty insertions between -644 and -653 immediately 5' of the native DUR1,2 USASs. We suggest that the difference in DUR1,2-Oh and DUR80 phenotypes depends on whether the native cis-acting elements and transcription factors associated with them can operate. If they can, enhancement of normally regulated DUR1,2 expression is observed. This is a novel phenotype for Ty insertions. If the native DUR1,2 cis-acting elements are not present, the case when Ty insertion occurs 3' of them, a ROAM phenotype is generated. Nitrogen-regulated upstream activation sequence (UASNTR)-homologous sequences present in the Ty delta elements rather than cis-acting elements required for Ty transcription are the most likely candidates to serve as the cis-acting elements mediating the DUR80 phenotype.


Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris. Nonpathogenic mutants of Xanthomonas campestris pv. campestris, generated from transposon mutagenesis, accumulated extracellular polygalacturonate lyase, alpha-amylase, and endoglucanase in the periplasm. The transposon Tn5 was introduced by a mobilizable, suicidal plasmid, pSUP2021 or pEYDG1. Genomic banks of wild-type X. campestris pv. campestris, constructed on the broad-host-range, mobilizable cosmid pLAFR1 or pLAFR3, were conjugated with one of the mutants, designated XC1708. Recombinant plasmids isolated by their ability to complement XC1708 can be classified into two categories. One, represented by pLASC3, can complement some mutants, whereas the other, represented by a single plasmid, pLAHH2, can complement all of the other mutants. Restriction mapping showed that the two recombinant plasmids shared an EcoRI fragment of 8.9 kb. Results from subcloning, deletion mapping, and mini-Mu insertional mutation of the 8.9-kb EcoRI fragment suggested that a 4.2-kb fragment was sufficient to complement the mutant XC1708. Sequence analysis of this 4.2-kb fragment revealed three consecutive open reading frames (ORFs), ORF1, ORF2, and ORF3. Hybridization experiments showed that Tn5 in the genome of XC1708 and other mutants complemented by pLASC3 was located in ORF3, which could code for a protein of 83.5 kDa. A signal peptidase II processing site was identified at the N terminus of the predicted amino acid sequence. Sequence homology of 51% was observed between the amino acid sequences predicted from ORF3 and the pulD gene of Klebsiella species.


Detection of lipid radicals by electron paramagnetic resonance spin trapping using intact cells enriched with polyunsaturated fatty acid. Electron paramagnetic resonance (EPR) spin trapping was used to detect lipid-derived free radicals generated by iron-induced oxidative stress in intact cells. Using the spin trap alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), carbon-centered radical adducts were detected. These lipid-derived free radicals were formed during incubation of ferrous iron with U937 cells that were enriched with docosahexaenoic acid (22:6n-3). The EPR spectra exhibited apparent hyperfine splittings characteristic of a POBN/alkyl radical, aN = 15.63 +/- 0.06 G and aH = 2.66 +/- 0.03 G, generated as a result of beta-scission of alkoxyl radicals. Spin adduct formation depended on the FeSO4 content of the incubation medium and the number of 22:6-enriched cells present; when the cells were enriched with oleic acid (18:1n-9), spin adducts were not detected. This is the first direct demonstration, using EPR, of a lipid-derived radical formed in intact cells in response to oxidant stress.


Identification of cysteine-rich domains of the type 1 tumor necrosis factor receptor involved in ligand binding. The extracellular portion of the type 1 (p55) and type 2 (p75) tumor necrosis factor (TNF) receptors contains a repetitive amino acid sequence pattern of four cysteine-rich domains (CRDs). This pattern is found also in several other cell surface proteins, including the p75 nerve growth factor receptor and the CD40, 4-1BB, OX40, Fas, and CD27 antigens. To investigate whether CRDs play a role in TNF binding, we have constructed soluble variants of the extracellular portion of human type 1 TNF receptor (sTNFR1), in which CRD1 (N-terminal) or CRD4 (C-terminal) was deleted by mutagenesis. These variants or a wild type sTNFR1 were linked in their C terminus to the hinge and Fc portion of IgG1 heavy chain to create sTNFR1-IgG chimeras (immunoadhesins). Deletion of either CRD1 or -4 did not cause any major perturbations in the structure of the sTNFR1 variants, as evidenced by their efficient expression and secretion from transfected cells, and by their binding to conformation-dependent monoclonal antibodies that recognize diverse epitopes on sTNFR1. The wild type sTNFR1 immunoadhesin exhibited high affinity binding to TNF alpha (Kd = 65 pM) and TNF beta (Kd = 640 pM). Deletion of CRD4 resulted in about a 10-fold reduction in affinity for TNF alpha (Kd = 660 pM) and for TNF beta (Kd = 5.7 nM). In contrast, deletion of CRD1 resulted in a complete loss of binding to TNF alpha and to TNF beta. These results indicate that CRD4 is important but not necessary for TNF binding, while CRD1 is required. In addition, the results suggest some overlap between the TNFR1 binding sites for TNF alpha and TNF beta, despite low amino acid sequence homology between these cytokines.


Phospholipid base exchange activity in rat liver plasma membranes. Evidence for regulation by G-protein and P2y-purinergic receptor. Phospholipid base exchange activity using choline as substrate was detected in plasma membranes (PM) and other subcellular fractions of rat liver, with microsomes (MS) showing the highest specific activity. In contrast, phospholipase D activity was only detected in PM. In PM, choline exchanged for phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), whereas ethanolamine exchanged for PE and PS, and serine exchanged for PS. Ca2+ (10 microM or higher) stimulated choline incorporation into PC in MS and PM, whereas Mg2+ (10 microM or higher) stimulated it only in PM. Ethanolamine and serine incorporation into PM phospholipids was also stimulated by Ca2+, and inositol incorporation by Mn2+. Phospholipase D activity was substantial in the presence of EGTA and was slightly stimulated by Ca2+ concentrations less than 500 microM. It was undetectable without Mg2+. Low concentrations of oleate (1 mM or less) stimulated phospholipase D activity. These concentrations inhibited choline base exchange activity, whereas higher concentrations (3-8 mM) were stimulatory. Comparison of the subcellular distribution and Ca2+, Mg2+, and oleate effects on choline base exchange and phospholipase D activities supports the view that they are catalyzed by different enzymes. The incorporation of choline, but not ethanolamine or serine, into the phospholipids of PM, but not MS, was stimulated by micromolar concentrations of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) and other slowly hydrolyzable analogues of GTP. GDP, GMP, and other nucleoside triphosphates and their analogues were ineffective. GTP gamma S stimulation of base exchange activity was dependent upon Mg2+ and was inhibited by high concentrations of guanosine 5'-O-2-(thio)diphosphate. In the presence of low concentrations of GTP gamma S, ATP and its slowly hydrolyzable analogues stimulated base exchange activity. Dose-response curves for these nucleotides revealed a potency order consistent with mediation by purinergic receptors of the P2Y type. Base exchange activity stimulated by ATP plus GTP gamma S or GTP gamma S alone was not altered by treatment with pertussis or cholera toxins. These results suggest that the choline base exchange activity of liver PM is regulated by a pertussis toxin-insensitive G-protein linked to P2Y purinergic receptors.


rab15, a novel low molecular weight GTP-binding protein specifically expressed in rat brain. rab3A is a low molecular weight (LMW) GTP-binding protein specifically expressed in brain and localized to synaptic vesicles. rab3A has been proposed to play a role in neurotransmitter release by regulating membrane flow in the nerve terminal. In an attempt to define other LMW GTP-binding proteins that may regulate neurotransmitter release, seven cDNA clones encoding new members of the rab family of LMW GTP-binding proteins were isolated from a rat brain cDNA library. The rab proteins contain the four conserved structural domains essential for GTP binding in addition to domains required for membrane localization and effector protein interactions. One protein, rab16, is closely related to members of the rab3 subfamily, whereas two others are assigned as the rat homologs of canine rab8 and rab10. Four additional clones, rab12, rab13, rab14, and rab15, revealed unique sequences and are new members of the rab family of LMW GTP-binding proteins. The patterns of expression of rab15 and rab3A closely overlap but differ from that observed for all other known LMW GTP-binding proteins. This data suggests that rab15 may act in concert with rab3A in regulating aspects of synaptic vesicle membrane flow within the nerve terminal.


Examination of the functional roles of 5 highly conserved residues in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides. The cytochrome b subunit of the bc1 complexes contains two cytochrome components (bL and bH) and is the locus of both a quinol-oxidizing site (Qo or Qz) and a quinone-reducing site (Qi or Qc). Sequence alignments of this subunit from over 20 eukaryotic and prokaryotic species have revealed a remarkable degree of conservation, including approximately 20 totally conserved residues. In this paper, site-directed mutagenesis has been used to examine the structural or functional roles of 5 of these highly conserved residues, Gly48, Gln58, Ser102, Phe104, and Pro202, all predicted to be within transmembrane alpha-helical segments. The mutants were made in the bc1 complex of Rhodobacter sphaeroides, a photosynthetic bacterium. The ability to use spectroscopic, electrochemical, and flash-induced kinetic methods allows the mutants to be analyzed for influences both on cytochrome spectra and thermodynamic properties and on the kinetics of specific electron transfer reactions. The results show that none of the 5 residues is absolutely essential. Substitution of aspartate or valine for Gly48 results in the loss of photosynthetic growth. The G48V mutant assembles a bc1 complex, but with modified cytochromes bH and bL, and a dysfunctional quinone reductase (Qc) site; an alanine is tolerated at this position. Possibly, a small residue is important here for heme packing. Gln58 and Ser102 are the only highly conserved polar residues predicted to be within the transmembrane spans, apart from the histidines which are heme axial ligands. Neither Gln58 nor Ser102 is essential for assembly or function of the bc1 complex, although substitution of other amino acids in these positions does cause subtle, but measurable changes. Phe104 lies midway between the axial ligands to cytochromes bL and bH and can be modeled to project in the space separating the two hemes. Replacement of this highly conserved aromatic residue by isoleucine has no measurable influence on the rate of electron transfer through the cytochrome b chain containing the two hemes. Finally, Pro202 is a totally conserved proline which is in the middle of transmembrane helix D, in between the 2 histidines which provide ligands to the hemes. No major inhibition of electron transfer resulted from replacing this proline by a leucine, although subtle changes in spectra of the b cytochromes and their electrochemical properties were noted.


The effect of histidine modification on the activity of myo-inositol monophosphatase from bovine brain. The pH dependence of myo-inositol monophosphatase may indicate a role for histidine residues in the catalytic mechanism (Ganzhorn, A. J., and Chanal, M.-C. (1990) Biochemistry 29, 6065-6071). This possibility was investigated by chemical modification. At pH 6.0 and 25 degrees C, the enzyme was inactivated by diethylpyrocarbonate in a pseudo-first order reaction with a bimolecular rate constant of 0.37 M-1 s-1. Two histidines were modified rapidly with no effect on enzyme activity, while 3 residues were modified at a slower rate corresponding to the rate of inactivation. No noticeable changes in the secondary structure of the enzyme were observed by comparison of circular dichroic spectra before and after modification. Treatment of myo-inositol monophosphatase with diethylpyrocarbonate in the presence of inositol 1-phosphate, Mg2+, and Li+ protected 2 residues from modification and decreased the inactivation rate by about 5-fold. Spectrophotometric analysis, the restoration of enzyme activity by hydroxylamine, and the lack of any inhibitory effect with alkylating agents suggest that inactivation is due solely to modification of histidine. We conclude that a histidine residue is essential for activity and may act as a base catalyst during hydrolysis of the substrate.


Calcium-induced inhibition of phosphoserine phosphatase in insulin target cells is mediated by the phosphorylation and activation of inhibitor 1. In this study, we examined the mechanism of inhibition of phosphoserine phosphatase (PSPase) activity by elevated [Ca2+]i in insulin target cells. In in vitro studies, isolated rat adipocytes were incubated with either 40 mM K+ or parathyroid hormone (PTH) (20 ng/ml) for 1 h. In in vivo studies, rats were injected with PTH (three hourly injections of 40 micrograms intraperitoneally) prior to isolation of either adipocytes or skeletal muscle. Under these conditions, intracellular [Ca2+]i changed from 100 +/- 8.7 to 263 +/- 10.5 nM. There was a concomitant 30% decrease in adipocyte PSPase activity and a 35% decrease in skeletal muscle PSPase activity, assayed using 32P-labeled phosphorylase "a" as a substrate. The inhibition of PSPase was accompanied by a 60% increase in adipocytes (p less than 0.05) and a 118% increase (p less than 0.01) in skeletal muscle inhibitor 1 (I1) activities, respectively. Since I1 is active only in the phosphorylated state, we studied the effect of [Ca2+]i on I1 phosphorylation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of heat treated extracts immunoprecipitated with I1 antibody revealed significant increase in 32P incorporation (45-60%, p less than 0.05) into I1 protein in cells with elevated [Ca2+]i. Nitrendipine, a calcium channel blocker, completely prevented increases in I1 phosphorylation and activity in cells exposed to K+ but was only partially effective in the PTH-treated cells. In contrast, a cyclic AMP antagonist, RpcAMP, prevented both the K(+)-and the PTH-induced increases in I1 phosphorylation and activity, even though it failed to block the elevations in [Ca2+]i in these cells. We conclude that [Ca2+]i-induced and cAMP-mediated phosphorylation and activation of I1 results in inhibition of PSPase activity in insulin target cells. The inhibition of PSPases may cause inappropriate serine dephosphorylation of substrates of insulin action resulting in insulin resistance.


Salivary statherin. Dependence on sequence, charge, hydrogen bonding potency, and helical conformation for adsorption to hydroxyapatite and inhibition of mineralization. The structural domains of salivary statherin that are partly responsible for the protection and recalcification of tooth enamel were examined with respect to charge, sequence, hydrophobicity, hydrogen bonding potential, and conformation. Several fragments of statherin, 1-15 (SN15), 5-15 (SN11), 15-29 (SM15), 29-43 (SC15), 19-43 (SC25), and analogs of the N-terminal 15-residue sequence, where phosphoserines at positions 2 and 3 have been replaced by Ser (SNS15) and Asp (SNA15), respectively, were synthesized. The abilities of these fragments to adsorb at hydroxyapatite (HAP) surfaces and to inhibit its mineralization in supersaturated solutions were determined and compared with those of the whole statherin molecule, reported previously. The conformational preferences of the fragments both in aqueous and nonaqueous solutions were examined by circular dichroism. The highly charged N-terminal SN15 fragment has the greatest adsorption to HAP as compared with statherin and all other fragments. Its mineralization inhibitory activity is significantly greater than those of other fragments and comparable with that of the whole molecule. The dephosphorylated N-terminal fragment SNS15 shows a decreased tendency to adhere to and inhibit the formation of HAP, as compared with SN15. However, the substitution of Asp residues in place of phosphoserines (SNA15), restores the binding affinity and crystal growth inhibition properties, suggesting that the negative charge density at the N-terminal rather than any specific interaction of the phosphate group is important for HAP surface interactions. The C-terminal SC15 and SC25 fragments elicit a much higher affinity for HAP surface than that of the middle sequence (SM15), indicating that hydrogen bonding potential of the C-terminal sequence also contributes to the interaction of statherin with HAP. CD studies provide evidence that the N-terminal SN15 fragment has a strong tendency to adopt an ordered helical conformation, whereas the shorter N-terminal sequence, middle, and C-terminal fragments are structurally flexible and prefer to adopt scattered turn structures or unordered random conformations in organic and aqueous solutions. Collectively, the data indicate that the negative charge density, sequence (1-15), and helical conformation at the N-terminal region of statherin are important for its surface interaction with HAP.


Peptide sequences from the hypervariable regions of two monoclonal anti-idiotypic antibodies against the thyrotropin (TSH) receptor are similar to TSH and inhibit TSH-increased cAMP production in FRTL-5 thyroid cells. Monoclonal antibodies, D2 and 4G11, selected by the autoantiidiotypic approach following injection of thyrotropin (TSH) into mice, mimic TSH in binding to receptors on thyroid membranes. Based on TSH receptor transfection studies, D2 and 4G11 show unequivocal specificity for the TSH receptor. To see if the complementary determining regions (CDRs) of these antibodies share any primary sequence similarities to regions of TSH critical for receptor binding, we deduced the primary structure of the variable regions of D2 and 4G11 by sequencing the immunoglobulin mRNA. We found that CDR1 of 4G11K and CDR2 of D2 mu show sequence similarity to regions of TSH alpha and TSH beta that had been previously implicated in the interaction of the hormone with its receptor. We tested the inhibitory effects of synthetic peptides from D2 mu-CDR2 and 4G11K-CDR1 on the binding of the corresponding antibodies to rat thyroid FRTL-5 cells and found an EC50 of 0.1 and 1 microM, respectively. TSH-derived peptides with similarity to D2 mu-CDR2 and 4G11K-CDR1 showed a significant but lesser effect on the binding of 4G11 or D2 to thyroid cells. Additionally, we tested the effects of the CDR peptides and TSH-derived peptides on TSH-stimulated cAMP production in FRTL-5 cells and found that D2 mu-CDR2 and 4G11K-CDR1 inhibited this activity, D2 mu-CDR2 most strongly (EC50 10 microM). Thus, linear sequences from the CDRs of these autoantiidiotypic antibodies with similarity to sequences from both subunits of TSH appear to interact with the TSH receptor. These data support previous studies indicating the complexity of the interaction between TSH and its receptor and advance earlier findings that such immunologic approaches are useful in dissecting receptor-ligand interactions.


Homologous desensitization of HEL cell thrombin receptors. Distinguishable roles for proteolysis and phosphorylation. Loss of sensitivity to thrombin following an initial response is characteristic of a number of cell types, including platelets. It has recently been proposed that thrombin receptors resemble other G protein-coupled receptors, but that activation involves a novel mechanism in which thrombin cleaves the receptor, exposing a new N terminus that serves as the ligand for the receptor. Based upon this model, we have examined the mechanism of thrombin receptor desensitization by comparing the effects of thrombin with those of a peptide corresponding to the N-terminal sequence of the receptor following proteolysis by thrombin: SFLLRNPNDKYEPF or TRP42/55. Like thrombin, TRP42/55 stimulated pertussis toxin-sensitive inositol 1,4,5-trisphosphate formation, raised cytosolic Ca2+, and inhibited cAMP formation in the megakaryoblastic HEL cell line. Exposure to either thrombin or TRP42/55 desensitized the cells to both, but not to a third agonist, neuropeptide Y. The rate of recovery after desensitization depended upon the order of agonist addition. Resensitization of the cell to thrombin following a brief exposure to thrombin required up to 24 h and could be inhibited with cycloheximide. Resensitization to TRP42/55 after exposure to thrombin, or to thrombin after exposure to TRP42/55, on the other hand, was detectable within 30 min and could be inhibited by serine/threonine phosphatase inhibitors, but not by cycloheximide. Loss of responsiveness to thrombin and TRP42/55 was also observed following addition of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). However, while the protein kinase inhibitor staurosporine completely prevented the desensitization caused by TPA, it had only a limited effect on the desensitization caused by TRP42/55. These results demonstrate that the G protein-mediated effects of thrombin can be reproduced by a receptor-derived peptide and suggest that desensitization occurs by at least two mechanisms. The first, which is seen with thrombin, but not TRP42/55, involves proteolysis and requires protein synthesis for recovery. The second, which occurs with TRP42/55 and TPA, as well as with thrombin, involves phosphorylation, possibly of the receptor itself. Although protien kinase C is activated by thrombin and is presumably responsible for the desensitization caused by TPA, it does not appear to play a major role in receptor desensitization caused by thrombin and TRP42/55. This suggests that other kinases, such as those which inactivate adrenergic receptors and rhodopsin, are involved in the down-regulation of thrombin receptor function.


Four different DNA helicases from calf thymus. Using a strand displacement assay we have followed DNA helicase activities during the simultaneous isolation of several enzymes from calf thymus such as DNA polymerases alpha, delta, and epsilon, proliferating cell nuclear antigen, and replication factor A. Thus we were able to discriminate and isolate four different DNA helicases called A, B, C, and D. DNA helicase A is identical with the enzyme described earlier (Thömmes, P., and Hübscher, U. (1990) J. Biol. Chem. 265, 14347-14354). The four enzymes can be distinguished by (i) their putative molecular weights after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (ii) glycerol gradient sedimentation under low and high salt conditions, (iii) sensitivity to salt, (iv) binding to DNA, (v) nucleoside- and deoxynucleoside 5'-triphosphate requirements, and (vi) by their direction of movement. DNA helicase A unwinds in the 3'----5' direction on the DNA it was bound to, while DNA helicases B, C, and D do so in the 5'----3' direction. DNA helicase D, and to some extent DNA helicases B and C, are able to unwind long substrates of more than 400 nucleotides. Replication factor A, a single-stranded heterotrimeric DNA binding protein involved in cellular DNA replication and DNA repair stimulates the DNA helicases. The stimulatory effect is most pronounced on DNA helicase A, where replication factor A enables this helicase to unwind longer substrates. DNA helicases B, C, and D are also stimulated by replication factor A. The effect of replication factor A appears to be specific since corresponding single-stranded DNA binding proteins from Escherichia coli and bacteriophage T4 have no or even a negative effect on the four DNA helicases. Heterologous human replication factor A has no stimulatory effect on any of the four DNA helicases suggesting a species specificity of these interactions. Thus it appears that mammalian cells possess, as does E. coli, a variety of different enzymes that can transiently abolish the double helical DNA structure in the cell.


Stimulation by growth hormone of MAP kinase activity in 3T3-F442A fibroblasts. We have previously demonstrated that growth hormone (GH) promotes an increase in tyrosine kinase activity associated with the GH receptor. To gain insight into the role of GH-dependent tyrosine kinase activity in signaling by GH, we investigated the possibility that GH might stimulate MAP kinase, a serine/threonine/tyrosine kinase thought to be a common element in tyrosine kinase-initiated response cascades. Treatment of 3T3-F442A fibroblasts with 100 ng/ml GH results in a 3-6-fold increase in the ability of cell-free extracts to phosphorylate MAP-2 and myelin basic protein. GH-stimulated kinase activity is unaffected by heparin, H7, or cAMP-dependent protein kinase inhibitor peptide, partially reduced by staurosporin and inhibited by fluoride and calcium ions, indicating that the kinase is not protein kinase C or A, casein kinase, or a calcium/calmodulin-dependent protein kinase. Based on gel permeation chromatography, the molecular mass of the GH-stimulated MAP kinase is approximately kDa. Furthermore, anti-phosphotyrosine antibodies revealed the GH-dependent appearance of two phosphotyrosine-containing proteins in cell-free lysates of GH-treated cells that co-migrate with proteins recognized by anti-MAP kinase antibodies. The GH-dependent increase in MAP kinase activity displays a biphasic time course and is dependent on the concentration of GH applied to the cells. GH-dependent MAP kinase activity, partially purified by Mono-Q chromatography, is inactivated by treatment with alkaline phosphatase. Addition of H7 to the cells prior to the addition of GH has no effect, whereas addition of H8 increases MAP kinase activity in control cells with no effect in GH-treated cells, indicating that protein kinase C is unlikely to be an intermediary in the GH-dependent stimulation of MAP kinase activity. These findings indicate that signaling by GH in 3T3-F443A cells may, at least in part, utilize a kinase cascade similar to those that have been proposed for other membrane receptors with associated tyrosine kinase activity.


Structure-function relationships in the activation of platelet thrombin receptors by receptor-derived peptides. According to present models, thrombin activates platelets by cleaving its receptors after Arg41, creating a new N terminus which acts as a tethered ligand. In support of this model, a peptide (SFLLRNPNDKYEPF or TRP42/55) corresponding to residues 42-55 has been shown to activate the receptor. In the present studies, the structural basis for thrombin receptor activation was examined using fragments of this peptide, as well as variants of the peptide with selected amino acid substitutions. The results show that the features of SFLLRNPNDKYEPF required to mimic the effects of thrombin reside within the first 6 residues, SFLLRN. A hexapeptide comprised of these residues was approximately 5 times more potent than the parent peptide in assays of platelet aggregation and, in addition, caused tyrosine phosphorylation, inhibition of cAMP formation, and an increase in cytosolic Ca2+. Omission of either the Ser residue or the Arg and Asn residues greatly diminished peptide activity, as did the substitution of Ala for Phe or Arg. Substitution of Ala for Ser or the initial Leu, on the other hand, had little adverse effect. The inactive peptides SALLRN and NPNDKYEPF had no effect on platelet activation initiated by SFLLRN, but FLLRN inhibited platelet aggregation in response to both SFLLRN and thrombin. These results suggest that within SFLLRN the Phe and Arg residues are particularly important and that Phe must be preceded by another amino acid, the identity of which is not tightly constrained. This observation and comparisons with the homologous domains of proteins whose tertiary structure is known were used to predict the conformation of the SFLLR sequence. The model which emerged suggests that the SFLLR domain may be part of an extended beta structure in the intact receptor and that cleavage by thrombin causes it to contract and assume a modified helical configuration. In this predicted conformation the side chains of Phe and Arg point in the same direction, potentially into a pocket formed by the remainder of the receptor.


Functional domains of membrane-bound human thrombomodulin. EGF-like domains four to six and the serine/threonine-rich domain are required for cofactor activity. Thrombomodulin is an endothelial cell thrombin receptor that serves as a cofactor for thrombin-catalyzed activation of protein C. Structural requirements for thrombin binding and cofactor activity were studied by mutagenesis of recombinant human thrombomodulin expressed on COS-7 and CV-1 cells. Deletion of the fourth epidermal growth factor (EGF)-like domain abolished cofactor activity but did not affect thrombin binding. Deletion of either the fifth or the sixth EGF-like domain markedly reduced both thrombin binding affinity and cofactor activity. Thrombin binding sequences were also localized by assaying the ability of synthetic peptides derived from thrombomodulin to compete with diisopropyl fluorophosphate-inactivated 125I-thrombin binding to thrombomodulin. The two most active peptides corresponded to (a) the entire third loop of the fifth EGF-like domain (Kp = 85 +/- 6 microM) and (b) parts of the second and third loops of the sixth EGF-like domain (Kp = 117 +/- 9 microM). These data suggest that thrombin interacts with two discrete elements in thrombomodulin. Deletion of the Ser/Thr-rich domain dramatically decreased both thrombin binding affinity and cofactor activity and also prevented the formation of a high molecular weight thrombomodulin species containing chondroitin sulfate. Substitutions of this domain with polypeptide segments of decreasing length and devoid of glycosylation sites progressively decreased both cofactor activity and thrombin binding affinity. This correlation suggests that increased proximity of the membrane surface to the thrombin binding site may hinder efficient thrombin binding and the subsequent activation of protein C. Membrane-bound thrombomodulin therefore requires the Ser/Thr-rich domain as an important spacer, in addition to EGF-like domains 4-6, for efficient protein C activation.


Inhibition of membrane fusion in vitro via cyclin B but not cyclin A. It is now clear that complexes of cdc2 kinase with "mitotic" cyclins regulate the transition between the G2 phase of the cell cycle and mitosis and that membrane traffic in mammalian cells is arrested during mitosis. Using a cell-free assay, we have previously reported that the fusion of early endosomes is, in fact, inhibited via the cdc2 kinase (Tuomikoski, T., Felix, M.-A., Dorée, M., and Gruenberg, J. (1989) Nature 342, 942-945). In the present paper, we show that this in vitro inhibition occurs efficiently only when the kinase activity is specifically evoked by a cyclin of the B-type but not by cyclins of the A-type. In addition, high resolution two-dimensional gel analysis revealed that the kinases associated with A- and B-type cyclins exhibit different substrate preferences. These data suggest that the complexes of the cdc2 kinase with different cyclins may control specific events of the cell cycle.


Demonstration of a specific clearance receptor for tissue-type plasminogen activator on rat Novikoff hepatoma cells. The binding, internalization, and degradation of tissue-type plasminogen activator (t-PA) were studied in a rat hepatoma (Novikoff) cell line. Binding of t-PA to specific saturable high affinity binding sites (Kd = 12 nM, 54,000 sites/cell) was followed by internalization and degradation and did not require a functional active site. The catabolism of t-PA was not inhibited by an excess of urokinase-type plasminogen activator (u-PA), and t-PA bound to Novikoff membranes was not complexed to PAI-1, suggesting a mechanism independent of PAI-1. Additionally, a mannose receptor is not involved since t-PA binding was not influenced by an excess of mannose, galactose, ovalbumin, or EDTA. Furthermore, the degradation of t-PA was not influenced by 10 mM 6-aminohexanoic acid, a lysine analogue. The t-PA receptor binds to and can be eluted from wheat germ agglutinin-Sepharose. Cross-linking of t-PA with partially purified receptor and ligand blot analysis, suggest that t-PA binds to two proteins, a principal one of 55 kDa and a minor one of 43 kDa. Novikoff cells are able also to bind (Kd = 1.4 nM, 25,000 sites/cell) and degrade u-PA. The binding was inhibited by pro-u-PA and the amino-terminal fragment of u-PA, but not by an excess of t-PA. The u-PA receptor, but not the t-PA receptor, was removed by treatment with phosphatidylinositol-specific phospholipase C. Our results show that the clearance receptor for t-PA on Novikoff cells is different from the mannose receptor and the PAI-1-dependent receptor described in other cells. The rat hepatoma cells are thus a good model to study the PAI-1 independent hepatocyte-specific clearance of t-PA.


Study of the cytochrome c2-reaction center interaction by site-directed mutagenesis. Photooxidation of Rhodobacter capsulatus cytochrome c2 and four site-directed mutants by detergent solubilized Rhodobacter sphaeroides reaction centers was studied as a function of ionic strength at pH 8.0. Mutants of cytochrome c2 included K12D (lysine 12 substituted by aspartate), K14E (lysine 14 substituted by glutamate), K32E (lysine 32 substituted by glutamate), and K14E/K32E (lysines 14 and 32 substituted by glutamates). With respect to the wild-type, the mutants exhibited decreased second-order rate constants, indicating perturbation of their electrostatic interaction with the reaction center. In the transient complex, the interaction domain charges of the reaction center and wild-type cytochrome c2 were estimated to be -4.8 and +4.8, respectively. In contrast, the interaction domain charges of mutants K12D, K14E, K32E, and K14E/K32E were estimated to be +2.8, +3.7, +3.6 and +1.3, respectively. At infinite ionic strength, the second-order rate constant of the wild-type cytochrome c2 photooxidation (k infinity) was estimated to be 8.7 x 10(6) M-1 s-1. In the case of K32E, k infinity was not changed significantly (8.2 x 10(6) m-1 s-1), suggesting that the electrostatic perturbation of this mutant was largely overcome at high ionic strength. In contrast, the k infinity for K12D, K14E, and K14E/K32E were estimated to be decreased 2-7-fold. Consequently, mutations to R. capsulatus lysines 12 and 14 appear to perturb the distance and/or orientation of the cytochrome c2 relative to the reaction center in the reactive complex, as well as alter electrostatic interactions. Based upon the kinetic results presented here, the cytochrome c2-reaction center transient complex has been modeled.


Ligand-induced polyubiquitination of the platelet-derived growth factor beta-receptor. We have analyzed the nature of ligand-induced shift to higher molecular weight forms of the beta-receptor for platelet-derived growth factor expressed in porcine aortic endothelial cells. The modification of the beta-receptor was found to be due to polyubiquitination, as judged by immunoblotting using an anti-ubiquitin antiserum. A mutant beta-receptor made kinase negative by a point mutation (K634A mutant) did not undergo ubiquitination in response to ligand stimulation. A mutant in which carboxyl-terminal 98 amino acids were deleted (CT98 mutant) and which retained kinase activity was likewise not ubiquitinated. These data suggest that the kinase activity, as well as the carboxyl-terminal 98 amino acids, is required for ubiquitination of the beta-receptor. Ligand-induced degradation of the receptor-bound ligand, as well as of the receptor itself, was partially impaired in the CT98-receptor-expressing cells, suggesting that the ubiquitination is of importance for efficient degradation of the ligand-receptor complex.


Effects of antagonists of protein phosphatases on superoxide release by neutrophils. Neutrophils stimulated with 4 beta-phorbol 12-myristate 13-acetate (PMA) release large quantities of superoxide (O2-) and exhibit phosphorylation of two proteins with molecular masses of 47(p47) and 49 kDa (p49). Addition of inhibitors of protein kinases (e.g. 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7)) to these cells after stimulation with PMA results in the loss of 32P from these proteins and a rapid cessation of O2- release (e.g. Heyworth, P. G., and Badwey, J. A. (1990) Biochim. Biophys. Acta 1052, 299-305). In this paper we report that antagonists of type 1 and 2A protein phosphatases (okadaic acid, calyculin A) prevented both the loss of 32P from p47 and the termination of O2- release in stimulated neutrophils treated with H-7. Calyculin A also caused a remarkable hyperphosphorylation of a number of proteins in neutrophils and increased O2- release from these cells in response to a suboptimal amount of PMA. Enzymes present in both the soluble and particulate fractions of neutrophils catalyzed the near complete dephosphorylation of 32P-labeled p47 and p49 bound to Immobilon-P membranes. Dephosphorylation of these blotted phosphoproteins occurred at physiological rates and was inhibited by okadaic acid and calyculin A. These data strongly suggest that p47 undergoes a continual cycle of phosphorylation and dephosphorylation throughout the period of O2- release when PMA is the stimulus. Moreover, we show that antagonists of type 1 and 2A protein phosphatases block dephosphorylation of p47 both in vivo and in vitro, indicating that these enzymes may modulate O2- release under certain circumstances.


The Caenorhabditis elegans unc-93 gene encodes a putative transmembrane protein that regulates muscle contraction. unc-93 is one of a set of five interacting genes involved in the regulation or coordination of muscle contraction in Caenorhabditis elegans. Rare altered-function alleles of unc-93 result in sluggish movement and a characteristic "rubber band" uncoordinated phenotype. By contrast, null alleles cause no visibly abnormal phenotype, presumably as a consequence of the functional redundancy of unc-93. To understand better the role of unc-93 in regulating muscle contraction, we have cloned and molecularly characterized this gene. We isolated transposon-insertion alleles and used them to identify the region of DNA encoding the unc-93 protein. Two unc-93 proteins differing at their NH2 termini are potentially encoded by transcripts that differ at their 5' ends. The putative unc-93 proteins are 700 and 705 amino acids in length and have two distinct regions: the NH2 terminal portion of 240 or 245 amino acids is extremely hydrophilic, whereas the rest of the protein has multiple potential membrane-spanning domains. The unc-93 transcripts are low in abundance and the unc-93 gene displays weak codon usage bias, suggesting that the unc-93 protein is relatively rare. The unc-93 protein has no sequence similarity to proteins listed in current data-bases. Thus, unc-93 is likely to encode a novel membrane-associated muscle protein. We discuss possible roles for the unc-93 protein either as a component of an ion transport system involved in excitation-contraction coupling in muscle or in coordinating muscle contraction between muscle cells by affecting the functioning of gap junctions.


The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation. Detergent extracts of canine pancreas rough microsomal membranes were depleted of either the signal recognition particle receptor (SR), which mediates the signal recognition particle (SRP)-dependent targeting of the ribosome/nascent chain complex to the membrane, or the signal sequence receptor (SSR), which has been proposed to function as a membrane bound receptor for the newly targeted nascent chain and/or as a component of a multi-protein translocation complex responsible for transfer of the nascent chain across the membrane. Depletion of the two components was performed by chromatography of detergent extracts on immunoaffinity supports. Detergent extracts lacking either SR or SSR were reconstituted and assayed for activity with respect to SR dependent elongation arrest release, nascent chain targeting, ribosome binding, secretory precursor translocation, and membrane protein integration. Depletion of SR resulted in the loss of elongation arrest release activity, nascent chain targeting, secretory protein translocation, and membrane protein integration, although ribosome binding was unaffected. Full activity was restored by addition of immunoaffinity purified SR before reconstitution of the detergent extract. Surprisingly, depletion of SSR was without effect on any of the assayed activities, indicating that SSR is either not required for translocation or is one of a family of functionally redundant components.


Glycerol shock treatment facilitates purification of herpes simplex virus. We describe an improved method for purification of herpes simplex virus. The primary improvement is the use of glycerol shock treatment to lyse infected cells. This is a rapid and simple technique that efficiently extracts the cytoplasm from the nucleus, while preserving nuclear integrity. Nearly 100% cell lysis is achieved by this method, and cytoplasmic virus yield is optimal. Other improvements to increase virus yield and purity and to economize on equipment are described.


Low density lipoprotein receptor and cation-independent mannose 6-phosphate receptor are transported from the cell surface to the Golgi apparatus at equal rates in PC12 cells. Efficient transport of cell surface glycoproteins to the Golgi apparatus has been previously demonstrated for a limited number of proteins, and has been proposed to require selective sorting in the endocytic pathway after internalization. We have studied the endocytic fate of several glycoproteins that accumulate in different organelles in a variant clone of PC12, a regulated secretory cell line. The cation-independent mannose 6-phosphate receptor and the low density lipoprotein receptor, both rapidly internalized from the cell surface, and the synaptic vesicle membrane protein synaptophysin, were transported to the Golgi apparatus with equivalent, nonlinear kinetics. Transport to the Golgi apparatus (t1/2 = 2.5-3.0 h) was several times faster than turnover of these proteins (t1/2 greater than or equal to 20 h), indicating that transport of these proteins to the Golgi apparatus occurred on average several times for each protein. In contrast, Thy-1, a protein anchored in the membrane by a glycosylphosphoinositide group, was internalized and transported to the Golgi apparatus more slowly than the three transmembrane proteins. Since each of the transmembrane proteins studied showed the same t1/2 for transport to the Golgi apparatus, we conclude that transport of these proteins from the cell surface to the Golgi apparatus does not require sorting information specific to any one of these proteins. These results suggest that one of the functions of late endosomes is constitutive recycling of cell surface receptors through the Golgi apparatus if they fail to recycle to the cell surface directly from early endosomes, and that the late endosome recycling pathway is followed frequently by many rapidly internalized proteins.


Expression of myosin heavy and light chains and phosphorylation of the phosphorylatable myosin light chain in the heart ventricle of the European hamster during hibernation and in summer. We investigated the expression of myosin subunits (myosin heavy chains) as well as light chains and the in vivo phosphorylation of the phosphorylatable myosin light chain in the heart ventricle of the adult male European hamster (Cricetus cricetus L.). Two myosin heavy chain isoenzymes could be detected under native and denaturing electrophoretic conditions having high (alpha-myosin heavy chain) and low (beta-myosin heavy chain) enzymatic activity. Enzymatic activity of alpha- and beta-myosin heavy chain revealed a different temperature dependency. When temperature increased ATPase activity of the alpha-myosin heavy chain isoenzyme increased relatively more than ATPase activity of the beta-myosin heavy chain isoenzyme. Summer animals expressed predominantly the beta-myosin heavy chain (79% of total myosin) while during hibernation the alpha-myosin heavy chain expression increased to 53% of total myosin. Winter-active hamsters kept at 22 degrees C and 12 h day/night rhythm showed the same myosin heavy chain isoenzyme pattern as summer-active animals. Two myosin light chain forms were expressed in the ventricle of all animal groups. The in vivo phosphorylation level of the phosphorylatable myosin light chain decreased from 45% in summer-active hamster to 23% during hibernation.


Relaxant effect of phalloidin on Triton-skinned microvascular and other smooth muscle preparations. Guinea pig mesenteric microarteries (diameter 60-100 microns), the main branch of the mesenteric artery and taenia coli were skinned with 1% Triton X-100 for 4 h at 4 degrees C. Microarteries, mounted for circumferential force measurement, developed maximal active force in response to elevation of the free Ca2+ (pCa = 4.52, in EGTA buffer) in the presence of ATP (7.5 mM) and calmodulin (0.1-0.3 microM). In these preparations, addition of phalloidin (1-100 microM) slowly (greater than 1 h) relaxed submaximal contractions (pCa greater than 4.52) in a dose-dependent manner. Relaxation was irreversible as, after phalloidin wash-out, subsequent active force to pCa = 4.52 was also reduced. By contrast, phalloidin preincubation and wash-out under relaxed conditions (pCa greater than 8) only reduced subsequent force to pCa = 4.52 on prolonged stimulation. The extent of phalloidin-induced relaxation was not dependent on free Ca2+ between pCa 6.40 and 4.52. Phalloidin-induced relaxation did not occur during rigor contractions (i.e. absence of ATP and Ca2+). These mechanical effects of phalloidin were accompanied by a decreased leak of actin out of the skinned preparations and by the prevention of guanidine extraction of actin from these preparations. Phalloidin did not inhibit the myosin light chain kinase or phosphatase activity isolated from these preparations. In addition, the relaxant effects were also noted in taenia coli and the main branch of the mesenteric artery but not in skinned porcine ventricular heart muscle. These experiments suggest the possible participation of actin filament dynamics on the maintenance of active force in Triton-skinned smooth muscle.


Isoform-specific regulation of platelet-derived growth factor activity and binding in osteoblast-enriched cultures from fetal rat bone. In osteoblast-enriched cultures from fetal rat bone, the A-chain homodimer of platelet-derived growth factor (PDGF-AA) is less potent than the PDGF isoforms containing B chain subunits (PDGF-AB and PDGF-BB), but normal osteoblasts appear to synthesize only PDGF-A subunit mRNA and polypeptide. However, other agents may regulate PDGF-AA activity in skeletal tissue. Pretreatment of osteoblast-enriched cultures with interleukin 1 alpha (IL-1 alpha) or tumor necrosis factor-alpha (TNF-alpha) synergistically enhanced the mitogenic effect of PDGF-AA coincident with increased binding site occupancy, but neither factor augmented PDGF-BB activity or binding. Polyacrylamide gel analysis showed 125I-PDGF-AA binding complexes predominantly at greater than 200 kD and faint labeling at 185 kD. After IL-1 alpha or TNF-alpha pretreatment, PDGF-AA binding increased at both sites, but this effect was more striking at 185 kD, which co-migrated with 125I-PDGF-BB-labeled complexes. PDGF-AA binding sites were rapidly lost by comparison to those for PDGF-BB in cycloheximide-treated cultures, but they remained relatively enhanced by IL-1 alpha and TNF-alpha pretreatment. These studies indicate that IL-alpha and TNF-alpha increase PDGF-AA binding and activity for osteoblasts by mechanisms that are at least in part independent of new receptor synthesis, and suggest regulatory events that could control how PDGF binding sites specifically recognize different ligands.


Direct contact between sympathetic neurons and rat cardiac myocytes in vitro increases expression of functional calcium channels. To test the hypothesis that direct contact between sympathetic neurons and myocytes regulates expression and function of cardiac Ca channels, we prepared cultures of neonatal rat ventricular myocytes with and without sympathetic ganglia. Contractile properties of myocytes were assessed by an optical-video system. Contractility-pCa curves showed a 60% greater increase in contractility for innervated myocytes compared with control cells at 6.3 mM [Ca]0 (n = 8, P less than 0.05). Cells grown in medium conditioned by growth of ganglia and myocytes were indistinguishable physiologically from control cells. [Bay K 8644]-contractility curves revealed a 60 +/- 10% enhancement of the contractility response at 10(-6) M for innervated cells compared with control cells. The increased response to Bay K 8644 was not blocked by alpha- or beta-adrenergic antagonists. Moreover, increased efficacy of Bay K 8644 was maintained for at least 24 h after denervation produced by removal of ganglia from the culture. Dihydropyridine binding sites were assessed with the L channel-specific radioligand 3[H]PN200-110. PN200-110 binding sites were increased by innervation (51 +/- 5 to 108 +/- 20 fmol/mg protein, P less than 0.01), with no change in KD. Peak current-voltage curves were determined by whole-cell voltage clamp techniques for myocytes contacted by a neuron, control myocytes, and myocytes grown in conditioned medium. Current density of L-type Ca channels was significantly higher in innervated myocytes (10.5 +/- 0.4 pA/pF, n = 5) than in control myocytes (5.9 +/- 0.3 pA/pF, n = 8, P less than 0.01) or myocytes grown in conditioned medium (6.2 +/- 0.2 pA/pF, n = 10, P less than 0.01). Thus, physical contact between a sympathetic neuron and previously uninnervated neonatal rat ventricular myocytes increases expression of functional L-type calcium channels as judged by contractile responses to Ca0 and Bay K 8644, as well as by electrophysiological and radioligand binding properties.


Intravascular filarial parasites inhibit platelet aggregation. Role of parasite-derived prostanoids. The nematode parasites that cause human lymphatic filariasis survive for long periods in their vascular habitats despite continual exposure to host cells. Platelets do not adhere to blood-borne microfilariae, and thrombo-occlusive phenomena are not observed in patients with circulating microfilariae. We studied the ability of microfilariae to inhibit human platelet aggregation in vitro. Brugia malayi microfilariae incubated with human platelets caused dose-dependent inhibition of agonist-induced platelet aggregation, thromboxane generation, and serotonin release. As few as one microfilaria per 10(4) platelets completely inhibited aggregation of platelets induced by thrombin, collagen, arachidonic acid, or ionophore A23187. Microfilariae also inhibited aggregation of platelets in platelet-rich plasma stimulated by ADP, compound U46619, or platelet-activating factor. The inhibition required intimate proximity but not direct contact between parasites and platelets, and was mediated by parasite-derived soluble factors of low (less than 1,000 Mr) molecular weight that were labile in aqueous media and caused an elevation of platelet cAMP. Prior treatment of microfilariae with pharmacologic inhibitors of cyclooxygenase decreased both parasite release of prostacyclin and PGE2 and microfilarial inhibition of platelet aggregation. These results indicate that microfilariae inhibit platelet aggregation, via mechanisms that may include the elaboration of anti-aggregatory eicosanoids.


Cyclic nucleotides attenuate thrombin-evoked alterations in parameters of platelet Na/H antiport. The role of cytosolic Ca. In this work, we explored the role of cyclic nucleotides in modulating parameters of the Na/H antiport in human platelets. Sodium nitroprusside and iloprost, as well as cyclic nucleotide analogues, were used to raise cellular levels of cAMP and cGMP. Cyclic nucleotides reversed the thrombin-evoked alkaline shift in cytosolic pH set point and the activity of the Na/H antiport, concurrently with attenuation of thrombin-induced rise in cytosolic free Ca. No effect of cyclic nucleotides was observed in platelets not treated with thrombin, or platelets subjected to phorbol 12-myristate 13-acetate. cAMP did not reverse ionomycin-induced changes in the parameters of the Na/H antiport. Collectively, these observations indicate that cyclic nucleotides modulate the Na/H antiporter in human platelets through their effect on thrombin-evoked changes in cytosolic free Ca. Presumably, this effect holds for other agonists which stimulate phospholipase C, raise cytosolic-free Ca, and activate the Na/H antiport through protein kinase C dependent and protein kinase C-independent mechanisms.


Beta-adrenergic upregulation of the Na(+)-K(+)-2Cl- cotransporter in rat parotid acinar cells. We used the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6')-carboxyfluorescein to monitor the recovery of the intracellular pH (pHi) of rat parotid acini from an NH4(+)-induced alkaline load. This recovery was markedly inhibited by the loop diuretic bumetanide and by Cl- removal, indicating that it is largely due to NH4+ entry via the basolateral Na(+)-K(+)-2Cl- cotransporter. The rate of recovery of pHi was enhanced threefold by pretreatment (37.5 s) with isoproterenol (K1/2 = 21.5 nM) or norepinephrine (in the presence of phentolamine), and blocked by the beta 1-specific antagonist atenolol, indicating an upregulation of cotransport activity by beta 1-adrenergic stimulation. The effect of isoproterenol was prevented by protein kinase inhibitors and mimicked by cAMP analogues, and by maneuvers known to increase cytosolic cAMP levels in these cells, consistent with the involvement of protein kinase A. Physiologically, such an upregulation of the acinar Na(+)-K(+)-2Cl- cotransporter would lead to an increase in acinar chloride uptake across the basolateral membrane, and consequently, an increase in overall chloride and fluid secretion. Prevention of this upregulation by beta-blockers and possibly by other commonly used clinical agents may account for the dry mouth and dry eyes experienced by some patients taking these medications.


cAMP stimulates bicarbonate secretion across normal, but not cystic fibrosis airway epithelia. Adenosine 3',5'-cyclic monophosphate stimulates chloride (Cl-) secretion across airway epithelia. To determine whether cAMP also stimulates HCO3- secretion, we studied cultured canine and human airway epithelial cells bathed in a HCO3-/CO2-buffered, Cl(-)-free solution. Addition of forskolin stimulated an increase in short-circuit current that was likely a result of bicarbonate secretion because it was inhibited by a HCO3(-)-free solution, by addition of the carbonic anhydrase inhibitor, acetazolamide, or by mucosal addition of the anion channel blocker, diphenylamine 2-carboxylate. The current was dependent on Na+ because it was inhibited by removal of Na+ from the submucosal bathing solution, by addition of the Na+ pump inhibitor, ouabain, or by addition of amiloride (1 mM) to the submucosal solution. An increase in cytosolic Ca2+ produced by addition of a Ca2+ ionophore also stimulated short-circuit current. These data suggest that cAMP and Ca2+ stimulate HCO3- secretion across airway epithelium, and suggest that HCO3- leaves the cell across the apical membrane via conductive pathways. These results may explain previous observations that the short-circuit current across airway epithelia was not entirely accounted for by the sum of Na+ absorption and Cl- secretion. The cAMP-induced secretory response was absent in cystic fibrosis (CF) airway epithelial cells, although Ca(2+)-stimulated secretion was intact. This result suggests that HCO3- exist at the apical membrane is through the Cl- channel that is defectively regulated in CF epithelia. These results suggest the possibility that a defect in HCO3- secretion may contribute to the pathophysiology of CF pulmonary disease.


Preferential mRNA expression of prostromelysin relative to procollagenase and in situ localization in human articular cartilage. An imbalance between extracellular proteinases and their inhibitors is thought to underlie cartilage degradation. In cultures of adult cartilage, prostromelysin mRNA levels were much higher than those for procollagenase and this differential was increased in cultures stimulated with IL-1 beta. Analysis of mRNA prepared from freshly isolated chondrocytes showed abundant amounts of prostromelysin mRNA in normal adult cartilage but low levels in the neonate. Not all adult cartilage may possess such high levels of prostromelysin mRNA, as the message levels in the cartilage remaining on late-stage osteoarthritic joints were lower than those in normal adult cartilage. Relative to prostromelysin mRNA, little procollagenase and TIMP mRNA were found in the adult cartilage. In situ hybridization revealed that metalloproteinase mRNAs were localized in chondrocytes of the superficial zone in adult cartilage. However, upon IL-1 beta treatment, chondrocytes in all cartilage zones were observed to express prostromelysin mRNA. Relative to the neonate, the normal adult cartilage appears to have a high degradative potential, if one accepts that steady-state mRNA levels reflect prostromelysin production. As the adult cartilage is not apparently undergoing rapid turnover, it would appear that control of prostromelysin activation may be the major regulatory step in stromelysin-induced cartilage degradation.


Autoantibodies to the ribosomal P proteins react with a plasma membrane-related target on human cells. Autoantibodies to ribosomal P-proteins are present in 12-16% of patients with systemic lupus erythematosus and are associated with neuropsychiatric disease. As the ribosomal P proteins are located in the cytoplasm, the pathogenic effects of their cognate autoantibodies are unclear. In this study affinity-purified anti-P autoantibodies were used to explore the cell surface of several types of human and animal cells. Immunofluorescence as well as EM immunogold analysis demonstrated, on the surface of human hepatoma cells, the presence of an epitope that is antigenically related to the immunodominant carboxy terminus of P-proteins. The presence of this epitope was also demonstrated on the surface of human neuroblastoma cells and, to a lesser extent, on human fibroblasts. Furthermore, the Western blot technique revealed in purified human and animal plasma membranes a 38-kD protein that is closely related or identical with ribosomal P0 protein. The availability of reactive P peptide on the surface of cells makes possible the direct effect of autoantibodies on the function and viability of cells that express this antigenic target. This delineates one of the possible impacts of anti-P antibodies in disease expression.


Effect of acute changes in glomerular filtration rate on Na+/H+ exchange in rat renal cortex. Studies were undertaken in Munich-Wistar rats to assess the influence of changes in filtered bicarbonate (FLHCO3), induced by changes in GFR, on Na+/H+ exchange activity in renal brush border membrane vesicles (BBMV). Whole-kidney and micropuncture measurements of GFR, FLHCO3, and whole-kidney and proximal tubule HCO3 reabsorption (APRHCO3) were coupled with BBMV measurements of H+ gradient-driven 22Na+ uptake in each animal studied. 22Na+ uptake was measured at three Na+ concentration gradients to allow calculation of Vmax and Km for Na+/H+ exchange. GFR was varied by studying animals under conditions of hydropenia, plasma repletion, and acute plasma expansion. The increase in GFR, FLHCO3, and APRHCO3 induced by plasma administration correlated directly with an increase in the Vmax for Na+/H+ exchange in BBMV. The Km for sodium was unaffected. In the plasma-expanded rats, the Vmax for Na+/H+ exchange was 22% greater than in the hydropenic rats (P less than 0.025) whereas APRHCO3 was 86% greater (P less than 0.001). These results indicate that increases in FLHCO3, induced by acute increases in GFR, stimulate Na+/H+ exchange activity in proximal tubular epithelium. This stimulation is a mechanism which can, in part, account for the delivery dependence of proximal bicarbonate reabsorption.


Cloned platelet thrombin receptor is necessary for thrombin-induced platelet activation. Platelet activation by thrombin is critical for hemostasis and thrombosis. Structure-function studies with a recently cloned platelet thrombin receptor suggest that a hirudin-like domain in the receptor's extracellular amino terminal extension is a thrombin-binding determinant important for receptor activation. We now report that a peptide antiserum to this domain is a potent and specific antagonist of thrombin-induced platelet activation. This study demonstrates that the cloned platelet thrombin receptor is necessary for platelet activation by thrombin, and provides a strategy for developing blocking monoclonal antibodies of potential therapeutic value.


Reinfection of liver graft by hepatitis C virus after liver transplantation. We have investigated hepatitis C virus (HCV) viremia before and after orthotopic liver transplantation (OLT). 38 patients were examined; 16 were anti-HCV positive and 22 anti-HCV negative pre-OLT in a RIBA-2 test (Ortho Diagnostic Systems Inc., Westwood, MA). HCV-RNA was detected using a modified nested polymerase chain reaction in 14/38 and 10/38 patients before and after OLT, respectively. 7 of these 14 subjects who were HCV-RNA positive before OLT were also positive for serum hepatitis B surface antigen. After OLT, six patients became HCV-RNA positive, likely as a result of transfusions, while four developed a probable recurrence of HCV infection. Infection of the liver graft by the same strain of HCV was indeed demonstrated by sequence analysis of a hypervariable domain (in the envelope region) in two cases. This establishes the possibility of HCV recurrence and shows the usefulness of polymerase chain reaction as the only assay currently capable of identifying HCV infection after OLT.


Synchronous squamous and glandular neoplasia of the anal canal. A 48 year old man presented with invasive adenocarcinoma in the wall of a non-healing anal fistula. The subsequent abdomino-perineal resection specimen showed residual invasive carcinoma coexisting with in situ carcinoma of anal glands as well as in situ squamous carcinoma of the anal canal. The epithelium of the anal canal had koilocytotic features. DNA hybridisation studies by the dot blot technique showed weak positivity for human papillomavirus (HPV) subtypes 16, 18. This case illustrates a number of important points--namely, anal fistulas, particularly non-healing fistulas should be biopsied to exclude malignancy; some adenocarcinomas of the anal arise in anal glands; the coexistence of glandular and squamous carcinoma with evidence of HPV infection is highly reminiscent of similar synchronous lesions of the uterine cervix and suggests that HPV may have an aetiological role in both squamous and glandular carcinomas of the anal canal.


Primary biphasic synovial sarcoma of the orbit. Synovial sarcoma is one of the most common soft tissue malignancies of adolescents and young adults. Despite its name, it is no longer thought to be histogenetically derived from the synovium. What seems to be the first case of synovial sarcoma to arise in the orbit presented in a 21 year old woman as a slowly enlarging subconjunctival mass. Although this tumour was typically biphasic, the monophasic spindle cell variant arising at this site could easily be confused with less aggressive orbital connective tissue neoplasms.


Chicken pox infection (varicella zoster virus) and acute monoarthritis: evidence against a direct viral mechanism. A 9 year old boy developed acute monoarthritis of the left knee concurrent with the appearance of a varicella zoster virus (VZV) rash. Repeated VZV DNA hybridisation of the cells within the synovial fluid and synovial membrane failed to show any evidence of intracellular virus. Virus was isolated from synovial fluid 24 hours after the start of clinical infection but not later. These findings suggest that the mechanism of the arthritis is not due to viral replication inside the swollen joint.


Clinical efficacy of a low abrasive dentifrice for the relief of cervical dentinal hypersensitivity. 2 strontium chloride hexahydrate-containing dentifrices (SCH), similar except for their respective abrasive systems, were compared in a 2-month randomised double-blind parallel clinical study to evaluate their comparative effectiveness in terms of cervical dentinal hypersensitivity. 2 groups of 20 subjects, each with cervical dentinal hypersensitivity, were evaluated for tactile sensitivity by Yeaple probe, air sensitivity using a dental air syringe and subjective perception of pain by means of a visual analogue scale. There was no difference between the dentifrices as regards reduction of cervical dentinal hypersensitivity at each time point. The response to both dentifrices was evident within 4 weeks of use and the degree of improvement increased throughout the 8-week study period. The results support the conclusion that changing the abrasive component of SCH dentifrices did not significantly increase or decrease the (desensitizing) activity of the original product.