Molecular mimicry of trifluoroacetylated human liver protein adducts by constitutive proteins and immunochemical evidence for its impairment in halothane hepatitis. A monospecific antibody (anti-CF3CO antibody) was obtained by affinity chromatography on a N epsilon-trifluoroacetyl-L-lysine (CF3CO-Lys) matrix of a rabbit polyclonal antiserum, directed against trifluoroacetylated protein adducts (CF3CO-proteins). The anti-CF3CO antibody recognized distinct CF3CO-proteins on immunoblots of a liver biopsy obtained from a human individual 10 h after halothane anaesthesia. Cross-reactive proteins of 52 kDa and 64 kDa were recognized on immunoblots of livers obtained from human individuals not exposed to halothane. Recognition of both CF3CO-proteins and the 52-kDa and 64-kDa cross-reactive proteins was abolished in the presence of 1 mM CF3CO-Lys. Anti-CF3CO antibody, affinity-adsorbed to the 52-kDa or the 64-kDa cross-reactive proteins of human liver, recognized the majority of target CF3CO-proteins on immunoblots of the human liver biopsy of an individual exposed to halothane. Liver biopsies of 5 out of 7 (71%) patients with halothane hepatitis exhibited an absence or low amounts of immunorecognizable 52-kDa and/or 64-kDa cross-reactive proteins. In contrast, of 22 control human individuals tested, all liver tissue samples were positive for the 52-kDa and/or the 64-kDa cross-reactive proteins. These data indicate that epitopes on the cross-reactive proteins of 52 kDa and 64 kDa of human liver bear strong immunochemical resemblance to epitopes on human liver CF3CO-proteins. Low-level expression of the cross-reactive proteins of 52 kDa and 64 kDa is discussed as one possible factor in human susceptibility to halothane hepatitis.


Effects of chemical modification of Anabaena flavodoxin and ferredoxin-NADP+ reductase on the kinetics of interprotein electron transfer reactions. The influence of chemical modification of arginine residues (using phenylglyoxal) in ferredoxin-NADP+ reductase (FNR), and of carboxyl groups (using glycine ethyl ester) in flavodoxin (Fld), on the kinetics of electron transfer between FNR and Fld, and between ferredoxin (Fd) and FNR, was examined using laser flash photolysis methods. All proteins were obtained from the cyanobacterium Anabaena PCC7119. Reduction by laser-generated 5-deazariboflavin semiquinone of the FAD moiety of phenylglyoxal-modified FNR occurred with a second-order rate constant 2.5-fold smaller than that obtained for reduction of native FNR, indicating either a small degree of steric hindrance of the cofactor, or a decrease in its redox potential, upon chemical modification. In contrast, no changes were found in the kinetics of reduction of the FMN cofactor of Fld modified by glycine ethyl ester as compared with the native protein. The observed rate constants for reoxidation of Fdred (reduced Fd) by FNRox (oxidized FNR) were dramatically decreased (approximately 100-fold) when phenylglyoxal-modified FNR was used. In contrast to the reaction involving the native proteins, no ionic strength effects on kobs values were found. These results, and those obtained upon varying the protein concentration, indicate that the rate constant for complex formation and the attractive electrostatic interaction between the two proteins were greatly diminished by chemical modification of arginine residues of FNR. When phenylglyoxal-modified FNRsq (FNR semiquinone) was used to reduce Fldox (oxidized Fld), similar inhibitory effects were observed. In this case, the limiting first-order rate constant for Fldsq (Fld semiquinone) formation via intracomplex electron transfer from FNRsq was approximately 12-fold smaller than that obtained for the native FNR (600 s-1 vs 7000 s-1). Again, ionic strength effects were diminished. The glycine-ethyl-ester-modified Fld yielded a limiting first-order rate constant for intracomplex electron transfer from FNRsq to Fldox which was approximately 7-fold smaller (1000 s-1) than that obtained with native Fld, and ionic strength effects were again diminished. These results indicate that complex formation can still occur between modified FNR and native Fld, and between native FNR and modified Fld, but that the geometry of these complexes is altered so as to decrease the effectiveness of interprotein electron transfer. The results are discussed in terms of the specific structural features of the proteins involved.


Binding of Bacillus sphaericus binary toxin to a specific receptor on midgut brush-border membranes from mosquito larvae. The presence of specific receptors for Bacillus sphaericus binary toxin on brush-border membrane fractions (BBMF) from Culex pipiens larvae midgut cells was demonstrated by an in vitro binding assay. Both activated and radiolabelled polypeptides from the 51-kDa and 42-kDa binary toxin of B. sphaericus 1593 specifically bound to BBMF. Direct binding and homologous competition experiments indicated a single class of B. sphaericus toxin receptors, with a dissociation constant (Kd) of approximately 20 nM and a maximum binding capacity (Bmax) of approximately 7 pmol/mg BBMF protein. The sugars GalNAc, GlcNAc and N-acetyl neuraminic acid had no detectable inhibitory effect on toxin binding to C. pipiens BBMF. Binding experiments with the non-susceptible mosquito species Aedes aegypti failed to detect significant binding of B. sphaericus binary toxin to A. aegypti BBMF.


Non-lysosomal degradation of misfolded human lysozymes with and without an asparagine-linked glycosylation site. Human lysozyme is a monomeric secretory protein composed of 130 amino acid residues, with four intramolecular disulfide bonds and no oligosaccharides. In this study, a mutant protein, [Ala128] lysozyme, which cannot fold because it lacks a disulfide bond, Cys6-Cys128, was expressed in mouse fibroblasts and was found to be mostly degraded in the cells, whereas the control wild-type lysozyme was quantitatively secreted into the media. The degradation of [Ala128]lysozyme was independent of the transport from the endoplasmic reticulum to the Golgi apparatus. The degradation was greatly inhibited by incubation of cells at 15 degrees C, but was minimally affected by treatment of cells with the lysosomotropic agent, chloroquine, implying a non-lysosomal process. Additional mutations (Gly48-->Ser or Met29-->Thr) were created to make asparagine-linked (N-linked) glycosylation site in the [Ala128]lysozyme, and the resultant double mutants, [Ser48, Ala128]lysozyme and [Thr29, Ala128]lysozyme, were analyzed with respect to their intracellular degradation. These mutant proteins were susceptible to N-linked glycosylation, and were degraded in a similar manner to that of [Ala128] lysozyme, except that the onset of degradation of [Ser48, Ala128]lysozyme and [Thr29, Ala128] lysozyme, but not of [Ala128]lysozyme, was preceded by a lag period of up to 60 min. Furthermore, the degradative double mutants, [Ser48, Ala128]lysozyme and [Thr29, Ala128]lysozyme, were glycosylated post-translationally as well as co-translationally. These observations suggest that there is some interaction between the mechanisms of glycosylation and degradation.


Oleate stimulation of diacylglycerol formation from phosphatidylcholine through effects on phospholipase D and phosphatidate phosphohydrolase. Hydrolysis of exogenous phosphatidylcholine (PtdCho) to 1,2-diacylglycerol by rat liver plasma membranes was stimulated by oleate concentrations as low as 0.1 mM. In the presence of 75 mM ethanol, the fatty acid also enhanced phosphatidylethanol (PtdEtOH) formation from PtdCho. These effects were also observed with linoleate and arachidonate, but not with saturated fatty acids or detergents, and were minimal in microsomes or mitochondria. Release of [3H]choline from exogenous Ptd[3H]Cho was stimulated by oleate, whereas phosphoryl[3H]choline formation was inhibited. Oleate and other unsaturated, but not saturated, fatty acids also stimulated the conversion of exogenous [14C]phosphatidic acid to [14C]diacylglycerol. These data are consistent with stimulatory effects of these fatty acids on both phospholipase D and phosphatidate phosphohydrolase in liver plasma membranes. The stimulatory effect of guanosine 5'-O-[3-thio]triphosphate) (20 microM) on PtdEtOH and diacylglycerol formation from PtdCho was enhanced by low concentrations of oleate. Phospholipase A2 also stimulated PtdEtOH and diacylglycerol formation from exogenous PtdCho. It is proposed that unsaturated fatty acids may play a physiological role in the regulation of diacylglycerol production through activation of phospholipase D and phosphatidate phosphohydrolase.


Studies on the interaction of alpha subunits of GTP-binding proteins with beta gamma dimers. The interaction of several preparations of purified beta gamma dimers with two types of guanosine-nucleotide-binding-regulatory-(G)-protein alpha subunits, a recombinant bv alpha i3, made in Sf9 Spodoptera frugiperda cells by the baculovirus (bv) expression system, and alpha s, either purified from human erythrocyte Gs-type GTP-binding protein, and activated by NaF/AlCl3, or unpurified as found in a natural membrane, were studied. The beta gamma dimers used were from bovine rod outer segments (ROS), bovine brain, human erythrocytes (hRBC) and human placenta and contained distinct ratios of beta subunits that, upon electrophoresis, migrated as two bands with approximate M(r) of 35,000 and 36,000, as well as distinct complements of at least two gamma subunits each. When tested for their ability to recombine at submaximal concentrations with bv alpha i3, ROS, brain, hRBC and placental beta gamma dimers exhibited apparent affinities that were the same within a factor of two. When bovine brain, placental and ROS beta gamma dimers were tested for their ability to promote deactivation of Gs, brain and placental beta gamma dimers were equipotent and at least 10-fold more potent than that of ROS beta gamma dimers; likewise, brain beta gamma and placental dimers were equipotent in inhibiting GTP-activated and GTP-plus-isoproterenol-activated adenylyl cyclase, while ROS beta gamma dimers were less potent when assayed at the same concentration. The possibility that different alpha subunits may distinguish subsets of beta gamma dimers from a single cell was investigated by analyzing the beta gamma composition of three G proteins, Gs, Gi2 and Gi3, purified to near homogeneity from a single cell type, the human erythrocyte. No evidence for an alpha-subunit-specific difference in beta gamma composition was found. These findings suggests that, in most cells, alpha subunits interact indistinctly with a common pool of beta gamma dimers. However, since at least one beta gamma preparation (ROS) showed unique behavior, it is clear that there may be mechanisms by which some combinations of beta gamma dimers may exhibit selectivity for the alpha subunits they interact with.


Chromatin structure and conserved sequence elements in genes encoding ribosomal proteins in Tetrahymena thermophila. The chromatin structure of the macronuclear genes encoding ribosomal proteins S25 and L1 in the ciliated protozoan Tetrahymena thermophila was analyzed. Using the indirect end-labelling technique, DNase-I-hypersensitive regions were located in the promoter regions as well as in the 3' regions of the genes. The DNase-I-hypersensitive regions were present in chromatin of exponentially growing cells, where the rate of ribosomal-protein gene transcription is high, and in chromatin from starved cells, where transcription of ribosomal-protein genes is severely depressed. Micrococcalnuclease-digestion experiments revealed that the promoter regions of the S25 gene and the L1 gene are devoid of nucleosomes in exponentially growing cells. In starved cells, no nucleosomal organisation of the promoter region of the L1 gene could be detected, whereas nucleosomal structures were discernible in the promoter region of the S25 gene. A conspicuous polypurine sequence motif, AARGGGAAA, is present within or adjacent to the DNase-I-hypersensitive regions in the promoter of the S25 and the L1 gene, and interestingly, the same motif is found also in the promoter regions of the genes encoding ribosomal proteins L21 and L37.


Evidence for two histidine ligands at the diiron site of methane monooxygenase. Circular dichroism spectroscopy has shown the hydroxylase component of methane monooxygenase to have a high helical content. The apoprotein has the same secondary structure as the holoenzyme. Chemical modification shows 12 histidines to be reactive with diethylpyrocarbonate in the holoenzyme, whereas 14 are reactive in the apoenzyme. Two histidine residues are implicated as iron ligands. Further chemical modification results suggest a cysteine residue is in close proximity to the diiron centre.


The kinetic mechanism of sheep liver sorbitol dehydrogenase. The relations between the kinetic parameters for both sorbitol oxidation and fructose reduction by sheep liver sorbitol dehydrogenase show that a Theorell-Chance compulsory order mechanism operates from pH 7.4 to 9.9. This is supported by many parallels with the kinetics of horse liver alcohol dehydrogenase, which operates by this classical mechanism. An isotope-exchange study using D-(2H8)sorbitol confirmed the existence of ternary complexes and that, under maximum velocity conditions, their interconversion is not rate-determining. Substrate inhibition at high concentrations of D-sorbitol or D-fructose confirmed rate-determining enzyme--coenzyme product dissociation, slowed by the existence of more stable abortive ternary enzyme-coenzyme product complexes with substrate. The effect of the inhibitor/activator 2,2,2-tribromoethanol showed the existence of enzyme-NAD-CBr3CH2OH complexes inhibiting the first phase of reaction and enzyme-NADH-CBr3CH2OH complexes dissociating more rapidly than the usual rate-determining enzyme-NADH coenzyme product dissociation in the final phase. Inhibition studies with dithiothreitol also confirmed an ordered binding of coenzymes and second substrates to sorbitol dehydrogenase. Neither D-sorbitol nor D-fructose had any effect on enzyme inactivation by the affinity labelling reagent DL-2-bromo-3-(5-imidazolyl)propionic acid, thus giving no evidence for their existence as binary enzyme-substrate complexes. Several alternative polyol substrates for sorbitol dehydrogenase gave the same maximum velocity as sorbitol. This indicated a common rate-limiting binary enzyme-NADH product dissociation and a similarity of mechanism. An enzyme assay for pH 7.0 and 9.9 is given which enables the concentration of sorbitol dehydrogenase to be determined from initial rate measurements of enzyme activity.


Radical prostatectomy in the management of stage A carcinoma of the prostate. A total of 115 patients (29 with stage A1 and 86 with stage A2 prostate cancer) underwent radical retropubic prostatectomy. Residual cancer was found in the radical prostatectomy specimens in 11 of the 29 stage A1 patients (38%) and in 66 of the 86 stage A2 patients (77%). Fourteen percent of the stage A1 patients and 29.5% of the stage A2 patients had pathological evidence of disease extension beyond the confined prostate. No perioperative death occurred and no patient suffered rectal injury or was totally incontinent. Early postoperative complications occurred in 6 patients (5%). There were no late complications. Complete urinary control was achieved in 111 patients (96.5%) and stress urinary incontinence was present in 4 patients (3.5%). Sexual function was preserved in 21 of the 26 patients (81%) who underwent a nerve-sparing radical prostatectomy. Follow-up ranged from 12 to 84 months with a mean of 30 months. All patients were alive at the time of this study and only 3 patients suffered disease progression. These 3 patients were among the stage A2 group and had pathological evidence of tumor extension beyond the prostatic capsule. Twenty-six patients who also had evidence of disease extension were alive without evidence of disease. These data demonstrate that patients with stage A disease are at risk for disease progression if left untreated and with a morbidity as low as that achieved in our series, radical prostatectomy should remain an optimal option for tumor control in these patients.


Buserelin treatment of advanced prostatic carcinoma: prognostic factor analysis. From August 1986 to August 1990, 116 patients with prostatic carcinoma, advanced disease (stage C-D1 only in patients older than 75 years, or D2) were treated with Buserelin (0.5 mg 3 times/day subcutaneously for 7 days, followed by 0.4 mg 3 times/day intranasally) until progression. No concomitant antiandrogens were administered. Of the 108 evaluable patients, 10 had complete remission (CR), 49 partial remission (PR), 46 remained stable while 3 progressed (response rate = 54.6%). Median duration of response was 31 months, median survival was 34 months. The toxicity of treatment was mild and mainly related to the hormonal effect of the drug. Castrate testosterone levels were obtained in all patients except 7. Slight, transient pain increase was noted at day 8 in 12 patients. Absence of symptoms at the start of treatment, well- or moderately differentiated tumor and serum testosterone negativization following Buserelin were associated with a significantly higher response rate as compared to presence of symptoms, poorly differentiated tumor and failure to obtain castrate testosterone levels, respectively. The following prognostic factors were found, at univariate analysis, to be associated with a prolonged survival: stage (C-D1 versus D2), PS (greater than 80 versus equal or less than 80), symptoms (absent versus present) and histological grade (G1 + G2 versus G3). Age and basal T levels did not influence survival. Those patients who obtained a CR or PR survived significantly longer than those with stable disease or progression.(ABSTRACT TRUNCATED AT 250 WORDS)


Value of electrical dispersion as a cause of urethral stenosis after endoscopic surgery. We have evaluated the incidence of iatrogenic urethral strictures in a prospective study of 125 patients with a diagnosis of initial, superficial bladder tumors (without prior urethral instrumentation) and who were treated with endoscopic surgery. A global incidence of 12% was found after 24 months of follow-up with the strictures detected a mean of 9.5 months after the endoscopic procedure. These patients were divided into two groups. Group 1 was treated using standard transurethral resection (TUR) and group 2 underwent Nd:YAG laser photocoagulation. Both populations of patients underwent cystoscopic controls every 4 months as part of our standard follow-up protocol in bladder tumors. In the group of patients who underwent diathermy using a 24-F sheath (85 patients), the percentage of stenosis was 16.4%. In the patients treated with laser via a 21-F panendoscope (40 patients), the incidence of postoperative urethral stenosis was only 2.5%. We believe that the electrical dispersion generated by the unipolar electric current as well as the caliber of the operating instruments are factors which may influence the development of iatrogenic endoscopic urethral strictures.


Clinical study of the longitudinal deformation of the flaccid penis and of its variations with aging. The length changes of the flaccid penis provoked by a maximum manual stretching of the glans have been measured in 905 men in order to study its biomechanical qualities. Our study shows that the flaccid penis is deformable, extensible and elastic in its longitudinal axis. The analysis of the variations of these biomechanical properties with aging shows a significant decrease. Thus, the biomechanical behavior of the flaccid penis during stretching is highly different in young men and old men. This distortion difference according to age proves the physiological importance of the penile distortion since impotence significantly increases with age. As the vascular mechanisms may be disregarded during flaccidity, these penile physical features of the flaccid penis are only due to tissue mechanisms. Consequently, any distortion loss would reflect a tissue impairment, very likely a progressive senile fibrosis of cavernous tissues.


Incidentally diagnosed renal cell carcinoma. We analyzed the incidence, sex and age distribution, diagnostic methods and survival rate of incidentally detected renal cell carcinomas (RCCs) and compared these factors with those of symptomatic RCCs. Of 141 patients with RCC treated between 1980 and 1989, 44 cases (31.4%) were incidentally detected. Thirty-one of these 44 cases were diagnosed by abdominal ultrasonography. The age of the incidental cases was significantly higher than that of the symptomatic ones (p = 0.045), particularly in male patients (p = 0.049). The tumor size in incidental cases was smaller and tumor stage earlier (p less than 0.0001). Moreover, the grade of malignancy was significantly lower, and clear cell type tumors were more frequently detected in the incidental cases. No difference was observed between the survival rates of incidental and symptomatic cases with stage 1 or 2 tumors. Of the incidental cases with stage 1 or 2, however, no patient with a tumor 3 cm or less in diameter has died. In conclusion, abdominal ultrasonography is a useful tool to detect RCC at an early stage, and patients with a relatively small tumor tend to have a good prognosis.


Studies of temperature rise in bladder cancer and surrounding tissues during radiofrequency hyperthermia. Temperatures in the tumor on the bladder wall, tumor margin, intravesical urine, rectal cavity and pubic subcutaneous fat tissue (SCFT) were measured in a total of 48 bladder tumor patients during radiofrequency (RF)-capacitive heating of the tumors. There were close correlations (p less than 0.01) between the temperature of the tumor margin and intratumor temperature, between the intravesical temperature and the temperature of the tumor margin, between the RF power applied and the intravesical temperature and also between the thickness and the temperature of SCFT. As long as the thickness of SCFT was 20 mm or less, it was possible to raise the temperature of bladder cancer to 42.5 degrees C or above. The intravesical temperature could be regarded as an indicator for hyperthermia treatment since the intravesical urine temperature of over 42 degrees C implied a therapeutic temperature of 42.5 degrees C or more in the margin of the tumor. Since the intravesical temperature was closely related to the output power, the therapeutic magnitude of the output power could be defined at least as 400 W, by which intravesical urine temperature could exceed 42 degrees C.


Immunophenotypic characterization of the bladder mucosa infiltrating lymphocytes after intravesical BCG treatment for superficial bladder carcinoma. The lymphocytes infiltrating the bladder mucosa of 28 patients treated with bacillus Calmette-Guérin (BCG) for superficial bladder carcinoma were characterized using an immunohistochemical technique on frozen sections of biopsy specimens obtained during cystoscopy. The inflammatory response induced by BCG consisted mainly of T lymphocytes (CD3+), most of which had the helper/inducer phenotype (CD4+), with a CD4/CD8 ratio greater than 1. A minor subset of lymphocytes were of B phenotype (CD22+). These findings persisted for the whole follow-up period (6-12 months) in spite of a progressive decrease of the inflammatory infiltrate. No difference in the lymphocyte phenotype was observed between nonresponding patients and those who responded to BCG in the short term. It is concluded that, although intravesical BCG therapy does affect the immunocompetent cells of the bladder wall, the BCG-induced antitumor activity is unlikely to depend exclusively on a local immune mechanism.


Postoperative long-term course of peripheral blood immune parameters and immunomodulating effects of keyhole limpet hemocyanin in patients with nonmetastatic renal cell carcinoma. Therapeutic approaches to metastatic renal cell carcinoma (RCC) often focus on the application of immune modulators; the success rates, however, are not satisfactory. Up to the time of this study, no diagnostic tool has been available to select those patients who might profit from immunotherapy. Starting from this point, we have been assessing the immune status of patients suffering from RCC, intending to find markers that would characterize the more favorable prognosis. Our interest is focused not only on the metastatic but also on the nonmetastatic disease, i.e., the disease with the better prognosis. In the present study, we have assessed both the postoperative long-term course of several immune parameters of the peripheral blood and the reactivity of the immune system to immunostimulation with keyhole limpet hemocyanin (KLH) in patients with nonmetastatic RCC. In a prospectively randomized study, the verum group (n = 8) got 1 mg KLH per month up to 1 year while the control group (n = 9) got no immunostimulator after tumor nephrectomy. Both patient groups had stable or even increasing cell counts of lymphocyte subpopulations (T, B, natural killer, T4, T8 cells), and the humoral immunoactivation markers neopterin, beta 2-microglobulin and tumor necrosis factor increased considerably after tumor nephrectomy. An effect of KLH is evident 4-8 months postoperatively: here, the neopterin values in the KLH group are more than twice as high as in the control group. Thus, while patients with metastatic disease had turned out to be immunosuppressed (previous study), in patients with nonmetastatic RCC, both the long-term course indicating postoperative immunostimulation and the reactivity to KLH give evidence of immunocompetence.


Renal insufficiency induced by parathyroid hormone: influence of the calcium antagonist Gö 6070. To study the pathogenesis of parathyroid-hormone-induced renal insufficiency and the influence of the new calcium channel antagonist Gö 6070 (Gödecke AG, Berlin, FRG) rats were assigned to three groups (n = 8 each): (1) control, (2) PTH, (3) PTH plus Gö 6070. PTH (30 micrograms/24 h, a nonhypercalcemic dose) was administered i.p. and Gö 6070 (1 mg/kg/24 h) p.o. for 6 days. PTH did not change plasma or urinary calcium and did not induce nephrocalcinosis. The glomerular filtration rate (GFR), however, was decreased (4.9 vs. 10.3 ml/min/kg). Concomitant administration of Gö 6070 attenuated the PTH-induced fall in GFR (6.2 ml/min/kg).


Role of the autonomic nervous system in catheter-induced urethral inflammation. We have studied role of the autonomic nervous system on experimentally induced urethral inflammation in the rat. Urethral inflammation was produced by inserting latex strips into the urethra. The effects of different experimental procedures were assessed by using a 4-grade inflammation scale based on histological findings. Rats pretreated with the nonspecific catecholamine depletors reserpine or quanethidine had a significantly less severe urethral inflammation than vehicle-treated controls. The severity of urethral inflammation was increased in spontaneous hypertensive rats, which have an increased sympathetic tone as compared to the normotensive rats. Propranolol, a nonselective beta-adrenergic receptor antagonist reduced and butoxamine, a beta 2-antagonist, significantly reduced the urethral inflammation. Neither phenoxybenzamine (nonselective) nor prazosin (alpha 1) or yohimbine (alpha 2), alpha-adrenergic receptor antagonists, affected the degree of urethral inflammation. These data taken together indicate that the autonomic innervation of the urethral mucosa is critically involved in the inflammatory reaction and that the use of beta 2-antagonist may be a treatment alternative in the future for the treatment of catheter-induced inflammation.


Blind-ending bifid ureters--report of five pediatric cases. We report 5 cases of blind-ending ureters according to Culp's criteria. Occurrence of vesicoureteral reflux allowed us to make the diagnosis of retrograde cystography in all our patients. We wish to emphasize that this procedure must be done as the first step of urinary tract examination in children.


Polyorchidism: an exceptional case of three homolateral testes. Polyorchidism is an uncommon congenital anomaly defined as the presence of more than two histologically proved testes. The present case of polyorchidism was discovered casually in an 11-year-old boy while performing an orchiopexy. Three testes were found on the left side; we have been unable to find this exceptional anomaly previously published in the medical literature.


Intrascrotal extratesticular malignant schwannoma. We report a rare case of intrascrotal extratesticular malignant schwannoma with rhabdomyoblastic features in a 48-year-old man. Malignant schwannomas, which arise from the peripheral nerves, usually occur in the extremities, the trunk or paravertebral areas. The patient had no manifestation of von Recklinghausen's disease. To our knowledge, there has been no intrascrotal extratesticular malignant schwannoma reported.


A practical and theoretical guide to measurement invariance in aging research. We describe mathematical and statistical models for factor invariance. We demonstrate that factor invariance is a condition of measurement invariance. In any study of change (as over age) measurement invariance is necessary for valid inference and interpretation. Two important forms of factorial invariance are distinguished: "configural" and "metric". Tests for factorial invariance and the range of tests from strong to weak are illustrated with multiple group factor and structural equation modeling analyses (with programs such as LISREL, COSAN, and RAM). The tests are for models of the organization and age changes of intellectual abilities. The models are derived from current theory of fluid (Gf) and crystallized (Gc) abilities. The models are made manifest with measurements of the WAIS-R in the standardization sample. Although this is a methodological paper, the key issues and major principles and conclusions are presented in basic English, devoid of technical details and obscure notation. Conceptual principles of multivariate methods of data analysis are presented in terms of substantive issues of importance for the science of the psychology of aging.


Modeling incomplete longitudinal and cross-sectional data using latent growth structural models. In this paper we describe some mathematical and statistical models for identifying and dealing with changes over age. We concentrate specifically on the use of a latent growth structural equation model approach to deal with issues of: (1) latent growth models of change, (2) differences in longitudinal and cross-sectional results, and (3) differences due to longitudinal attrition. This is a methodological paper using simulated data, but we base our models on practical and conceptual principles of modeling change in developmental psychology. Our results illustrate both benefits and limitations using structural models to analyze incomplete longitudinal data.


Types of change: application of configural frequency analysis in repeated measurement designs. Many researchers are concerned both with intraindividual change patterns and interindividual differences and similarities in those change patterns. Configural Frequency Analysis (CFA) provides a way to identify overrepresentations (types) and underrepresentations (antitypes) in the frequencies of multiple variable classifications organized to reflect patterns of change. Three methods of CFA for analyzing repeated measures data are considered. To establish trends, two of them require at least ordinal data and the third requires interval data. Data analysis and the interpretation of results are illustrated. CFA is compared with residual analysis from log-linear modeling.


Increased tissue concentrations of the gastrin precursor in patients treated with omeprazole. The main form of gastrin in antral mucosa, the amidated heptadecapeptide G17, is generated from an inactive precursor, progastrin, by steps involving endopeptidase cleavage and amidation. Gastrin cells are normally inhibited by gastric acid and in this study we have examined how suppression of acid by treatment with omeprazole for 6-8 weeks influences gastrin production in patients with oesophagitis. Plasma concentrations of total amidated gastrins in the fasting state increased from 18 to 43 pmol l-1; assays specific for G17-immunoreactivity indicated that the plasma concentrations of this form increased from 6 to 12 pmol l-1. In endoscopic biopsies of antral mucosa there was no change with omeprazole treatment in the concentrations of total amidated gastrins, or their immediate precursors, the Gly-extended gastrins. However, assays using an antibody that reacts with progastrin, together with size exclusion chromatography, indicated that tissue progastrin concentration increased 6-fold. The data suggest a modest net increase in gastrin production with omeprazole-treatment; because the ratio of tissue concentrations of total amidated gastrins to Gly-extended gastrins did not change, it would seem that the amidating capacity of the gastrin cell was maintained. However, the increase in progastrin concentrations suggests a relative failure of the initial steps of post-translational processing, and consequently that in certain circumstances endopeptidase cleavage of progastrin may be rate limiting.


Multitrait-multimethod models in aging research. The multitrait-multimethod matrix is a versatile tool for structuring the design and analysis of studies in many areas of psychology, including the aging of psychological processes. The basic goal for which the multitrait-multimethod matrix was developed is the establishment of the construct validity of measures. With newer forms of analyzing multitrait-multimethod data, particularly those using structural equation modeling, it is possible to specify models and employ model comparisons for an even broader goal: the more adequate generalization of conclusions across the facets included in the study. Topics addressed in this paper include the design of multitrait-multimethod studies, the specification of an array of structural models for such data, model comparisons that allow the estimation of the degree of convergent validity, discriminant validity, and method variance exhibited by a set of measures, and the extension of structural models to more than a single group, which allows the testing of questions regarding means, as well as the traditional tests of differences in variance and covariance. The procedures discussed are then explicated by describing the representation of data from a hypothetical four-group study of memory employing a form of design that is common in research on aging. Strengths and weaknesses of the structural modeling approach to the analysis of multitrait-multimethod data are discussed.


Age, cohort, and time development muddles: easy in practice, hard in theory. The debate among developmental psychologists over how best to combine longitudinal and cross-sectional data sequences can be traced back at least four decades. During the 1970s, a variation of this theme received much attention: could the developmental influences of age, cohort, and time be unraveled by sufficiently ingenious application of combined data sequences? We believe discussion of this question has been needlessly parochial and confused. Substantive and methodological contributions from other disciplines have, until recently, been largely ignored by developmental psychologists. Moreover, the solutions debated by psychologists have generally been formulated in language that obscured, rather than explicated, the formal indeterminacy implicit in models of age, cohort, and time parameters. Methodologists have outlined formal solutions to problems of indeterminacy in the contexts of model identification and estimability theory. Sociologists have proposed solutions, based on explicitly theoretical assumptions, that permit model identification and unambiguous interpretation. We review these contributions, and suggest a hierarchy of solutions to the problem of age, cohort, and time indeterminacy.


In vitro immune modulation of polymorphonuclear leukocyte adhesiveness by sodium fluoride. We investigated the influence of sodium fluoride on polymorphonuclear leukocyte (PMN) adhesiveness in a healthy subject with low serum levels of fluoride. The PMN were separated from venous blood, and the percentages of adhered and unadhered cells were determined in vitro in plastic culture plates. The cells were cultured with five different fluoride concentrations ranging from 6.25 10(-2) microM to 4.0 microM in the presence and absence of autologous serum. PMN adhesiveness in both the presence and in the absence of autologous serum was 98.5%; the addition of fluoride had no effect on the results in the absence of serum. However, in the presence of autologous serum, PMN adhesiveness decreased significantly with the addition of fluoride (P < 0.05) from 0.5 microM. The decrease was smaller (1.1%) at a concentration of 0.125 microM, and larger at 12 times this concentration of fluoride (52.7%). We conclude that sodium fluoride reduces PMN adhesiveness in a dose-dependent manner. The effect is not direct, but should be modulated by a seric factor.


Component analysis in multivariate aging research. A method of components analysis, related to principal components analysis, is described for applications in multivariate cross-sectional or longitudinal data. The method may be useful to researchers seeking to reduce the number of observed variables through scale construction. The method creates component variables as weighted sums of the observed variables using weights that are identical across groups and occasions. The statistical and conceptual properties of these components are discussed. The method is contrasted with traditional principal components analysis and factor analysis. An application of the method is presented using longitudinal WAIS and WAIS-R data.


Supplementation with n-3 fatty acids reduces triglycerides but increases PAI-1 in non-insulin-dependent diabetes mellitus. The effects of dietary supplementation with n-3 fatty acids on lipid and glucose metabolism and on fibrinolysis were evaluated in 14 non-insulin-dependent diabetic patients who were given 10 g of MaxEPA (3 g n-3 fatty acids) or placebo (olive oil) per day in a randomized double-blind cross-over study during two consecutive 8-week periods. The serum triglyceride (TG) concentrations decreased by 27% (P < 0.01) after addition of MaxEPA with a reduction of VLDL TG by 36% (P < 0.05) while LDL cholesterol increased by 6% (P = 0.05). The fasting blood sugar and HbA1c concentrations increased significantly after addition of MaxEPA but the changes were not significantly different from those during the placebo period. The highest glucose concentrations at fasting and after an i.v. glucose injection were seen after MaxEPA while the serum insulin concentrations were unchanged. The peripheral insulin sensitivity, as measured by a euglycaemic, hyperinsulinaemic clamp technique, did not change during the study. The mean plasminogen inhibitor-1 (PAI-1) activity of the patients was elevated compared with healthy controls. In spite of the reduction of the triglyceride concentrations and unchanged insulin levels, there was a significant increase of the activity of PAI-1 (+21%, P < 0.01) after MaxEPA suggesting a possible impairment of the fibrinolytic capacity. In many situations there seems to be a reduction of PAI-1 when the triglycerides are lowered. In the diabetic patients given n-3 fatty acids this was not the case.


Antibody mediated enhancement of HIV-1 infection of an EBV transformed B cell line is CD4 dependent. Low levels of anti-viral antibodies may facilitate virus infection of Fc-receptor bearing cells. For human immunodeficiency virus (HIV) it has been reported that antibodies can enhance infection of phagocytic cells. We show that HIV-1 can infect an Epstein-Barr virus transformed B cell line and that low levels of anti-HIV antibodies enhance infection. The enhanced infection was characterized by an increase in viral DNA and increased HIV p24 protein production. Detection of cell surface antigen expression of CD4, the receptor for HIV, Fc-receptor type II for IgG, but not of type I and III could be demonstrated by immunofluorescence cytometry. The enhancement was abrogated when infection was performed in presence of a monoclonal antibody directed against CD4. Based on these results we conclude that antibody mediated enhancement of HIV-1 infection can also occur in non-phagocytic cells in a CD4 dependent manner and that IgG Fc-receptors other than types I or III are involved in this process.


Age-based construct validation using structural equation modeling. In this paper we describe some mathematical and statistical models based on structural equation modeling (SEM) using computer programs like LISREL. We focus on SEM methodology for the simultaneous examination of the internal validity of psychological constructs and the external validity represented by age relations. To illustrate these ideas we use a latent variable path model to examine the organization of intellectual abilities measured by the WAIS-R in the standardization sample. We also examine different ways in which age can be used to structure this organization. This is primarily a methodological paper, but we try to integrate conceptual principles of modeling with some substantive issues of research on the psychology of aging.


Circulating inhibitor of interleukin 1 activity in patients with chronic renal diseases. The effects of sera derived from patients suffering from chronic renal diseases (endemic Balkan nephropathy, glomerulonephritis and pyelonephritis) on T cell proliferative response was studied. It was found that these sera contained factors which affect interleukin 1 (IL-1) dependent events in T cell proliferative response. The factors prevent costimulatory effects of IL-1 on T cells but do not bind to IL-1, nor do they affect interleukin 2 (IL-2) dependent T cell proliferation. These findings indicate that immuno-suppression observed in some kidney disorders may be partially due to serum immunoinhibitory factors affecting IL-1 activity.


The effects of dietary supplementation with n-3 polyunsaturated fatty acids in patients with rheumatoid arthritis: a randomized, double blind trial. To determine the effect of dietary supplementation with n-3 polyunsaturated fatty acids (n-3 PUFA) on disease variables in patients with rheumatoid arthritis. Multicenter, randomized, placebo controlled, double blind. Three Danish hospital Departments of Rheumatology. Fifty-one patients with active rheumatoid arthritis. Random allocation to 12 weeks of treatment with either six n-3 PUFA capsules (3.6 g) or six capsules with fat composition as the average Danish diet. Significant improvement of morning stiffness and joint tenderness. No significant effect on the four other assessed clinical parameters. No serious side effects. Dietary supplementation with n-3 PUFA in patients with rheumatoid arthritis improved two out of six patient reported disease parameters. Further studies are needed to clarify the more precise role of n-3 PUFA in the treatment of rheumatoid arthritis.


Insulin sensitivity and secretion in healthy elderly human subjects with 'abnormal' glucose tolerance. Glucose tolerance deteriorates dramatically with advancing age. It is not known whether the underlying pathophysiology is different in older subjects. We employed a two step hyperinsulinaemic euglycaemic glucose clamp with [6(14)C] glucose infusion to compare peripheral and hepatic insulin sensitivity in eight elderly (EAGT) with eight young (YAGT) subjects with abnormal (matched) glucose tolerance and nine elderly subjects with normal glucose tolerance (ENGT). There was no difference in basal HGO (EAGT 14.5 +/- 0.9, YAGT 15.3 +/- 1.1 mumol kg-1 min-1). Glucose turnover was similar in both groups at step 1 (EAGT 13.2 +/- 0.8, YAGT 13.4 +/- 0.8 mumol kg-1 min-1) and step 2 (EAGT 25.1 +/- 3.1, YAGT 27.2 +/- 2.7 mumol kg-1 min-1). HGO was lower in the EAGT subjects at step 1 (2.3 +/- 0.4 vs. 4.3 +/- 0.6 mumol kg-1 min-1 P = 0.01). Incremental serum insulin response to oral glucose was comparable (EAGT 66.8 +/- 11.6 YAGT 57.8 +/- 12.2 mU l-1.h). Compared to the ENGT group the EAGT group was insulin resistant with a lower MCR of glucose at step 1 (2.03 +/- 0.28 vs. 3.23 +/- 0.44 ml kg-1 min-1 P = 0.04) and at step 2 (6.18 +/- 0.83 vs. 9.64 +/- 0.38 ml kg-1 min-1 P = 0.004) and had a lower early insulin response (AUC 0-30 min 5.9 +/- 1.1 vs. 9.8 +/- 1.4 mU l-1.h P = 0.04).(ABSTRACT TRUNCATED AT 250 WORDS)


Increased GPIIB/IIIA expression and altered DNA-ploidy pattern in megakaryocytes of diabetic BB-rats. Hyperactive platelets contribute to angiopathic complications in diabetes mellitus. It is unclear whether the increased platelet function is a primary pathogenetic factor in diabetes or follows vascular injury. Increased platelet size and numbers of glycoprotein receptors on diabetic platelets suggest that thrombopoiesis is altered in diabetes mellitus. For further support of this hypothesis we studied whether megakaryocytes are changed with regard to the DNA-ploidy pattern and the GPIIB/IIIA expression in 10 acute diabetic (AD) and 24 insulin treated diabetic (ITD) BB rats in comparison with 22 diabetes resistant (ND) BB rats. In the AD group megakaryocyte size (P = 0.035) and the modal DNA-ploidy distribution dropped (P = 0.0001) concomitant with increased TNF-alpha activity (P = 0.001). GPIIB/IIIA expression and the peripheral platelet status were unchanged. After 4 weeks of insulin substitution metabolic parameters (glucose, cholesterol, triglycerides) were lowered, but remained still elevated. As compared to the AD group the modal DNA-ploidy pattern reversed, but the relative percentage of 64n megakaryocytes increased 2.3-fold and GPIIB/IIIA expression increased 1.6-fold. Simultaneously, the peripheral platelet count and size increased. From these results we conclude that alterations of the megakaryocyte compartment occur at early onset of diabetes. These changes could reflect a response to increased systemic cytokine production during inflammatory islet cell destruction. The peripheral platelet thrombotic potency increased with insulin treatment. This was associated with an increase of 64n-megakaryocytes with upregulated GPIIB/IIIA expression and could reflect a mitogenic effect of insulin upon the endomitotic cycle of the megakaryocytes.


Correspondence between plasma mevalonic acid levels and deuterium uptake in measuring human cholesterol synthesis. To assess the validity of two techniques capable of identifying immediate changes in human cholesterol production, plasma mevalonic acid levels and the rate of uptake of deuterium into plasma free cholesterol were compared in 5 healthy individuals over 48 h. The free-living subjects self-selected three meals per day prior to and during study. At t = 0, deuterium oxide was administered orally. Blood samples were collected before and every 4 h after dosing. Total cholesterol and mevalonic acid levels were determined in plasma at each timepoint. Deuterium enrichment changes in plasma free cholesterol, relative to plasma water content, were used to calculate free cholesterol fractional synthetic rates (FSR) at each timepoint. Total plasma cholesterol levels remained constant, whereas significant circadian rhythmicity was observed in both plasma mevalonic acid and deuterium uptake methods, with nadir and peak formation rates indicated at 14.00 to 16.00 h and about midnight, respectively. It is suggested that plasma mevalonic acid levels and free cholesterol deuterium uptake rate techniques are both suitable techniques for short-term measurement of human cholesterol synthesis.


Effect of lipid A on the deformability, membrane rigidity and geometry of human adult red blood cells. Lipid A is responsible for the activities of endotoxin and may cause circulatory failure and haemolysis. This study evaluated the effects of different lipid A concentrations on red blood cell (RBC) deformation (rheoscope), the aspiration pressure required to aspirate RBC into 3.3 microns pipettes, the membrane shear elastic modulus (i.e. membrane rigidity) and cellular geometry (micropipette system) after 15 min of incubation. Lipid A concentrations of 10 and 100 micrograms ml-1 of RBCs decreased RBC deformability by 26% and 39%, respectively. The aspiration pressure for RBCs into a 3.3 microns micropipette increased by 235% at a lipid A concentration of 10 micrograms ml-1 and by 586% at a concentration of 100 micrograms ml-1. The elastic shear modulus almost doubled at a lipid A concentration of 10 micrograms ml-1 and tripled at 100 micrograms ml-1. At a lipid A concentration of 100 micrograms ml-1, 37% of RBCs showed spicules. These echinocytes were less deformable than discocytes. Mean corpuscular volume, RBC volume and surface area were not affected by lipid A. We conclude that lipid A causes marked reduction of RBC deformability due to increasing membrane rigidity.


Colchicine analogues: effect on amyloidogenesis in a murine model and, in vitro, on polymorphonuclear leukocytes. Colchicine has been used in diverse clinical settings such as gout, familial Mediterranean fever, liver cirrhosis, Behcet's disease and pericarditis. It also has an antimitotic potential hitherto unexplored due to its narrow therapeutic toxic ratio. The aim of the present study was to compare the effectiveness and the toxicity of colchicine and three analogues: thiocolchicine, 2,3 dimethyl-colchicine and 3-dimethylthiocolchicine in the blockage of amyloid synthesis in a murine model. 3-demethylthiocolchicine was equipotent to colchicine in the blockage of casein induced amyloidogenesis. However, it was markedly less toxic (LD50 11.3 mg kg-1 vs. 1.6 mg kg-1). Thiocolchicine was toxic (LD50 1.0 mg kg-1) and 2,3 didemethyl-colchicine was far less effective. The effect of 3-dimethylthiocolchicine on polymorphonuclear leukocytes was then compared to colchicine. The effect of this analogue on inhibition of chemotaxis was equivalent to that of colchicine whereas the latter was superior to the analogue in the suppression of phagocytosis (by a ratio of 2:1) and in the inhibition of bactericidal activity (by a ratio of 10:1). Since in therapeutic concentrations the only detectable effect of colchicine on PMNs is inhibition of chemotaxis, our data may point to 3-demethylthiocolchicine as an optional, perhaps superior alternative to colchicine for some of its therapeutic indications.


Neuropsychological impairment in auto-immune disease. Estimations of the prevalence of central nervous system involvement in patients with systemic lupus erythematosus (SLE) vary from 25 to 75%. In order to assess possible deficits in memory, attention, and/or reaction time, a battery of neuropsychological tests was administered to 15 patients with SLE and to two matched groups of control subjects; eight patients on corticosteroids and 15 healthy controls. The test battery included the Multiple Choice Word Fluency Test (MWT-B), Benton Visual Memory Scale, Syndrome Short Test (SKT; attention and memory), Attention and Concentration Test (d2), Wechsler Adult Intelligence Scale (WAIS) subtests for Comprehension, Digit Span, Block Design, a Computer Controlled Reaction Time task (CCRT), and a Learning and Memory Test (LGT-3). Results revealed a statistically significant short-term memory deficit (for all six relevant subtests used) and a marked delayed simple reaction time in SLE patients as compared with scores in healthy subjects. An attention deficit as well as the expected difference between the estimated premorbid and actual level of intelligence could not be demonstrated. With three exceptions (SKT-9 figural recognition, Benton Visual Memory, and LGT-3 figural recognition), patients treated with corticosteroids did not differ from healthy controls. They also did not differ from SLE patients in any of the parameters tested. It therefore appears that the demonstrated deficit in short-term memory and reaction time in SLE patients may not only be due to the neuropsychiatric manifestation of SLE but possibly also result from medication side-effects.


Modulation of the immune response to stress in patients with systemic lupus erythematosus: review of recent studies. Physical and psychological stress in healthy subjects leads to characteristic alterations of plasma hormone concentrations and the lymphocyte subset distribution. After short-term bicycle ergometry or acoustic stress, and after a 2-h-long neuropsychological examination, a rise in B- and T-suppressor/cytotoxic lymphocytes and a decrease in T-helper lymphocytes has been shown to occur. This cell mobilization was accompanied by an elevation in plasma catecholamines. Although the rise in catecholamine levels was similar in patients with systemic lupus erythematosus (SLE) and in healthy subjects, the alterations in the lymphocyte subsets were less pronounced in SLE patients after bicycle ergometry as well as after psychological testing than in healthy subjects, patients with sarcoidosis, and patients without collagenosis under treatment with corticosteroids. Differences between short-term physical exercise and the acoustic stress test on the one hand, and the 2-h-long neuropsychological test on the other, were observed only for the absolute lymphocyte count. After short-term stress, a rise in the leukocyte and lymphocyte count was noted, whereas after long-term testing, only an increase in leukocytes and a decrease in lymphocytes could be found. In conclusion, sympathomimetic stimulation by bicycle ergometry or by psychological stress led to a characteristic cell mobilization in all the groups investigated. The extent of cell mobilization was attenuated in SLE patients despite the fact that comparable increases of plasma catecholamines were observed.


Modulation of lymphocyte subsets due to psychological stress in patients with rheumatoid arthritis. The influence of a 1-hour neuropsychological stress test on the distribution of lymphocyte subpopulations and on plasma catecholamine levels was investigated in 18 patients with rheumatoid arthritis (RA) and 14 sex- and age-matched controls. Despite significant increases in lymphocyte counts in both groups, lymphocyte subsets did not change accordingly. A wide scattering of catecholamine levels in plasma before and after stress was observed. Plasma levels of lymphokines such as interleukin (IL)-1 beta and IL-6 could not be detected in RA patients. Enzyme immunoassay of markers of lymphocyte activation such as HLA-DR and cell-bound IL-2 receptor showed only a significant elevation of HLA-DR marked cells in RA patients at baseline. Significantly higher amounts of the soluble IL-2 receptor were detected in patients with RA before the stress test, but stress testing did not alter this parameter. In conclusion, lymphocyte activation in RA and a defect in the expression of IL-2 receptor on the cell surface of lymphocytes were confirmed in the present study.


Bidirectional communication between the brain and the immune system. There is now a substantial body of data available indicating that the brain, the nervous system, and the immune system are functionally connected. Consequently, environmental conditions of a psychosocial or physical nature may influence the body's defence. The magnitude of the change in immune reactivity is partly determined by the individual's evaluation of the psychological or physical stimulus. This is illustrated by pharmacological modification of perception resulting in more pronounced stress-induced immunomodulation. In man, it has been demonstrated that recently experienced life stress, particularly daily hassles, can codetermine the effect of an experimental stressor applied for a short period of time on the immune system. An intriguing finding relates to the synthesis and secretion of hormones and neuropeptides by immune cells. These chemical messengers act as autocrine or paracrine immunoregulatory molecules on the one hand, and as messengers for communication with the brain and peripheral nervous system on the other. Animal research indicates that this dialogue is of biological significance.


Interferon inducer, polyriboguanylic.polyribocytidylic acid, inhibits experimental hepatic metastases in mice. The antitumour activity of an interferon inducer, the double-stranded complex of polyriboguanylic.polyribocytidylic acid was studied on murine lymphosarcoma LS/BL. The antitumour effect was determined with the aid of an experimental liver-colony model and compared to that exhibited by another synthetic RNA, polyriboinosinic.polyribocytidylic acid. We found that both polynucleotide complexes decreased the number of liver colonies and prolonged the survival of the tumour-bearing mice. This effect was only observed if the complexes were applied in an appropriate dose schedule which included the administration of the drug prior to tumour cell inoculation and subsequent continuous treatment. We have also verified that the polynucleotide complexes did not exert their antitumour effect by a direct action on tumour cells.


Effect of muscarinic cholinergic drugs on ischemia-induced decreases in glucose uptake and CA1 field potentials in rat hippocampus slices. To clarify the role of muscarinic acetylcholine receptors in the hypoxia/hypoglycemia (ischemia)-induced functional deficit in hippocampal neurons, we examined the effect of cholinergic drugs on ischemia-induced impairments of glucose uptake and CA1 field potentials in hippocampus slices. Muscarinic receptors were subdivided into M1 (high affinity for pirenzepine) and M2 (low affinity for pirenzepine) subtypes. The M1 receptor subtype is coupled to an increase in phosphoinositide hydrolysis and the M2 receptor subtype is associated with inhibition of adenylate cyclase. The greater potency of carbachol in stimulating phosphoinositide hydrolysis resulted in exacerbated ischemia-induced deficits. Treatment with the muscarinic receptor antagonists scopolamine and pirenzepine (M1 receptor-selective antagonist) had a strong dose-dependent protective effect against ischemia-induced deficits. Oxotremorine and McN-A-343, weak stimulators of phosphoinositide hydrolysis and strong inhibitors of adenylate cyclase, had a weak neuroprotective action against ischemia-induced deficits. These results suggest that stimulation of M1 muscarinic receptors coupled with an increase in phosphoinositide hydrolysis may play a facilitatory role in ischemia-induced deficits. Stimulation of M2 muscarinic receptors may play an inhibitory role in ischemia-induced neuronal deficits.


A rodent model of focally evoked self-sustaining status epilepticus. We describe a novel model of status epilepticus produced by the focal application of bicuculline methiodide into the deep prepiriform cortex of rats pretreated with lithium chloride. Three out of eight rats pretreated with one dose of lithium (3 mmol/kg) 24 h prior to induction of seizures by focal bicuculline, and eight out of 12 rats pretreated with two doses of lithium (at 24 and 48 h) prior to seizure induction, exhibited continual uninterrupted convulsive seizure activity (status epilepticus) lasting between 10 min and > 2 h. This status epilepticus which was manifest both behaviorally and electroencephalographically, was sensitive to reversal by diazepam (5 mg/kg i.p.) given as long as 2 h after the onset of sustained status epilepticus. Pilocarpine (25 mg/kg) pretreatment also predisposed to status epilepticus in response to the focal application of bicuculline, but diazepam (5 or 10 mg/kg i.p.) was ineffective in suppressing the status epilepticus in the presence of pilocarpine.


Pharmacology of the 5-hydroxytryptamine-induced depolarization of the ferret vagus nerve in vitro. Grease-gap recordings revealed that 5-hydroxytryptamine (5-HT) depolarized the ferret vagus nerve (pEC50 = 4.9). This response was mimicked by 2-methyl-5-HT and 1-phenylbiguanide, but not by 5-carboxamidotryptamine. Paroxetine (1 microM) or ketamine (10 microM) did not potentiate the response. Ketanserin (1 microM) did not reduce the depolarization, but four 5-HT3 receptor antagonists did. It is concluded that 5-HT depolarizes the ferret vagus nerve via 5-HT3 receptors, but these receptors may differ pharmacologically from those in other species.


Evidence that 5-HT2 receptors predominantly mediate the contraction of the rat basilar artery to 5-hydroxytryptamine. We characterised the 5-hydroxytryptamine (5-HT) receptor subtype which mediates the contraction of the rat isolated basilar artery, mounted in a myograph, by using agonists and antagonists for 5-HT1-like, 5-HT2, 5-HT3 and 5-HT4 receptors. The rank order of potency for a range of selective agonists was: 5-HT > alpha-methyl-5-HT > 5-carboxamidotryptamine > 2-methyl-5-HT > sumatriptan. The maximum contractions for these agonists (Emax) was less than for 5-HT while 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was devoid of contractile activity. Ketanserin antagonised the contractile effect of 5-HT in the rat basilar artery with a provisional pA2 estimate of 9.3 +/- 0.2. A similar antagonism was observed against alpha-methyl-5-HT. Ergometrine (0.01-1 microM) was devoid of any agonist activity but antagonised the contractile effect of 5-HT on the rat basilar artery with a provisional pKB of 8.7 (7.8-9.8, 95% confidence limits). The 5-HT3 and 5-HT4 receptor antagonist ICS 205930 (10 microM) did not alter the response to 5-HT. The high potency and efficacy of alpha-methyl-5-HT, poor effect of sumatriptan and antagonism of 5-HT by ergometrine and ketanserin, support the conclusion that the 5-HT2 receptors are primarily responsible for the 5-HT-induced contraction of rat basilar artery.


Capsazepine inhibits low pH- and lactic acid-evoked release of calcitonin gene-related peptide from sensory nerves in guinea-pig heart. In the isolated perfused guinea-pig heart low-pH buffer (pH 5), lactic acid (5 mM), capsaicin (10(-7) M) and nicotine (10(-4) M) all evoked a clear-cut release of calcitonin gene-related peptide (CGRP) from sensory nerves. Incubation with the proposed capsaicin receptor antagonist, capsazepine (10(-5) M), significantly reduced the CGRP release evoked not only by capsaicin but also by low pH and lactic acid, indicating a common mechanism of C-fibre activation, while the nicotine response remained unchanged.


Unmasking the vasoconstrictor response to neuropeptide Y and its interaction with vasodilating agents in vitro. Neuropeptide Y (NPY) is a powerful vasoconstrictor in vivo but is usually much less active on isolated blood vessels. The contractile effect of NPY was examined in the isolated rat femoral artery exposed to various degrees of vasoconstriction. The effects of NPY on the relaxation induced by vasodilator agents was also studied. NPY (< or = 1 microM) had no contractile effect. In vessels pretreated with a low concentration of phenylephrine (0.3-1.0 microM), NPY evoked a concentration-dependent contraction, which was similar in intact and in endothelium-deprived vessels. Other vessels were contracted with phenylephrine (3-10 microM) and relaxed with histamine (0.1 mM). Subsequent addition of NPY elicited a contraction which was much greater than that observed in vessels pretreated with phenylephrine only. The Y1 receptor agonist, [Pro34]NPY, but not the Y2 receptor agonist, NPY 13-36, evoked a concentration-dependent contraction in phenylephrine-pretreated vessels. Acetylcholine (ACh) induced endothelium-dependent relaxation in vessels contracted with phenylephrine. NPY (0.1 microM) induced a rightward shift of the concentration-response curve and a lower maximum relaxation in response to ACh. NPY was without effect on the dilatation evoked by nitroprusside, histamine or forskolin. In conclusion, under appropriate vasoconstrictor and vasodilator influence, NPY can act at Y1 receptors to evoke vasoconstriction in the femoral artery via endothelium-independent mechanisms. In addition, NPY seems to attenuate the endothelium-dependent relaxation induced by ACh. These actions of NPY may contribute to explain the strong vascular effects of the peptide in vivo.


Protective effect of a selective endothelin receptor antagonist, BQ-123, in ischemic acute renal failure in rats. To elucidate the pathophysiological role of endogenous endothelin (ET), we examined the effects of the newly synthesized ETA receptor-selective antagonist, BQ-123, on ischemic acute renal failure induced by bilateral clamping of renal artery and vein followed by reperfusion in rats. BQ-123, when given by i.v. infusion of 0.5 mg/kg per min for 2.5 h during the pre- and post-ischemic period, was found to prevent the decrease in creatinine clearance and increases in blood urea nitrogen, plasma creatinine and the fractional excretion of sodium. Morphological observation also showed an effect of BQ-123, i.e. prevention of proximal tubular (S3 segment) necrosis. At 2 h after the start of reperfusion, the ET-1 content in the kidney increased to its maximal level. At this time, the Ca2+ content in the mitochondrial fraction of the renal cortex increased, with a concomitant increase in blood urea nitrogen. However, these increases were limited by treatment with BQ-123. Thus, BQ-123 was effective to both prevent mitochondrial Ca2+ accumulation in the early phase of ischemic acute renal failure and protect proximal tubular cells from post-ischemic degeneration. We conclude that ET may be at least partially involved in the pathogenesis of tubular cell injury in this acute renal failure model.


Histamine H1 receptors mediate vasodilation in guinea-pig ileum resistance vessels: characterization with computer-assisted videomicroscopy and new selective agonists. Histamine receptors on guinea-pig ileum submucosal arterioles (outside diameter 40-80 microns) were studied in vitro using a computer-assisted videomicroscopy system (Diamtrak). Histamine receptor agonists investigated in this study were histamine, the H1 receptor-selective compound, 2-[2-(3-fluorophenyl)-4-imidazolyl]ethanamine (VZ 20), the H2 receptor-selective compounds, dimaprit, impromidine, (+/-)-N1-[3-(4-fluorophenyl)-3-(pyridin-2-yl)propyl]- N2-[3-(1H-imidazol-4-yl)propyl]guanidine (arpromidine) and (+/-)-N1-[3-(3,4-difluorophenyl)-3-(pyridin-2-yl)propyl]- N2-[3-(1H-imidazol-4-yl)propyl]guanidine (BU-E-75), as well as the H3 receptor-selective drug, (R)-alpha-methylhistamine ((R)-alpha-MeHA). Applied to vessels at resting tone, the agonists (1 nM-300 microM) did not change arteriolar diameter. Vessels preconstricted by 10 microM noradrenaline showed similar concentration-dependent vasodilations with histamine and VZ 20 (pD2 = 5.38 and 5.36, respectively). This histamine-induced vasodilation was not affected by tetrodotoxin (0.5 microM) or indomethacin (1 microM), but was completely abolished in the presence of 1 microM of the H1 receptor antagonist, mepyramine. Calculation of the antagonist affinity of mepyramine for the histamine receptors in submucosal arterioles yielded a pA2 of 9.46. In contrast to histamine and VZ 20, the H2 receptor agonist, dimaprit, and the H3 receptor agonist, (R)-alpha-MeHA, were ineffective at preconstricted arterioles. The guanidine-type H2 receptor agonists, impromidine, apromidine and BU-E-75, produced vasodilation at noradrenaline-preconstricted arterioles (-log EC50 = 4.47, 5.30 and 5.39, respectively) but, in contrast to histamine, were ineffective at arterioles preconstricted by U-46619 (300 nM).(ABSTRACT TRUNCATED AT 250 WORDS)


Tetronothiodin, a novel CCKB receptor ligand, antagonizes cholecystokinin-induced Ca2+ mobilization in a pituitary cell line. We found a novel nonpeptide CCKB receptor antagonist, tetronothiodin (Ro 09-1468), in the culture broth of Streptomyces sp. NR0489. The structure of the compound (C31O8H38S), which has a 19-membered ring with an alpha-acyltetronic acid and tetrahydrothiophene moiety, is completely different from that of any known CCK receptor antagonist. Tetronothiodin inhibited [125I]CCK-8 binding to rat brain CCKB receptors with an IC50 of 3.6 nM, whereas it showed only weak affinity for rat CCKA receptors (IC50 = 70 microM). As demonstrated autoradiographically, tetronothiodin concentration dependently inhibited [125I]CCK-8 binding to CCKB receptors in rat forebrain slices. The effects of tetronothiodin on cytosolic Ca2+ concentrations in GH3 cells, a rat anterior pituitary tumor cell line, were investigated with the fura-2 method. Tetronothiodin inhibited CCK-8-induced Ca2+ mobilization without affecting basal cytosolic Ca2+ concentrations. In conclusion, tetronothiodin is a new, potent and highly selective CCKB receptor antagonist. It is a useful tool for investigating the pharmacological and physiological roles of CCKB receptors.


Effects of exogenous wild-type p53 on a human lung carcinoma cell line with endogenous wild-type p53. Several studies have shown that expression of exogenous wild-type p53 is detrimental to the growth of cell lines with absent or mutant p53. In this study, wild-type p53 cDNA expression plasmids were transfected into A549 lung carcinoma cells which had previously been shown by sequencing to contain wild-type p53. When a constitutively expressed wild-type p53 plasmid containing the neomycin resistance gene was transfected into these cells, no G418-resistant colonies contained the exogenous p53 cDNA even though the neomycin resistance gene was integrated. When cells were transfected with a dexamethasone-inducible wild-type p53 cDNA expression plasmid, induction of p53 expression resulted in a decreased growth rate and a decreased proportion of S-phase cells. Continuous treatment with dexamethasone resulted in continued p53 expression for 16 days, but beyond that time expression ceased and could not be reinduced. These data indicated that although the A549 cell line could proliferate in the presence of endogenous wild-type p53 there was a strong selection pressure against continued expression of additional exogenous wild-type p53.


Analysis of competence in cultured sea urchin micromeres. Sea urchin embryo micromeres form the primary mesenchyme, the skeleton-producing cells of the embryo. Almost nothing is known about nature and timing of the embryonic cues which induce or initiate spicule formation by these cells. A related question concerns the competence of the micromeres to respond to the cues. To examine competence in this system we have exposed cultured sea urchin micromeres to an inducing medium containing horse serum for various periods of time and have identified a period when micromeres are competent to respond to serum and form spicules. This window, between 30 and 50 h after fertilization, corresponds to the time when mesenchyme cells in vivo are aggregating and beginning to form the syncytium in which the spicule will be deposited. The loss of competence after 50 h is not due to impaired cell health since protein synthesis at this time is not significantly different from controls. Likewise the accumulation of a spicule matrix mRNA (SM 50) and a cell surface glycoprotein (msp 130), both indices of micromere/mesenchyme differentiation, still occurs in cells that have lost competence to respond to serum by forming spicules. These experiments demonstrate that the acquisition and loss of competence in these cells are regulated developmental events and establish an in vitro system for the identification of the molecular basis for inductive signal recognition and signal transduction.


Isolation and characterization of a brefeldin A-resistant mutant of monkey kidney Vero cells. Brefeldin A (BFA) is a fungal antibiotic which disrupts protein transport between the endoplasmic reticulum and the Golgi. A BFA-resistant mutant of monkey kidney Vero cells, BER-40, which exhibited about a 90-fold increase in the LD50 of BFA (5.2 ng/ml for Vero cells versus 460 ng/ml for BER-40 cells), has been isolated. The increased resistance of BER-40 cells toward BFA was also manifested in a greatly reduced inhibition of protein secretion by BFA in the mutant and a lack of protection by BFA of the mutant cells from ricin cytotoxicity. Somatic cell hybridization between the Vero and BER-40 cells showed that the BFA-resistance in BER-40 behaved as a codominant trait. The structure of the Golgi region, as examined by immunofluorescence microscopy with antibodies against Golgi markers (the 110-kDa protein and mannosidase II) or with fluorescent lipid NBD-ceramide, was unchanged in the mutant cells as compared to that in the wild-type cells. Treatment of Vero cells with BFA (1 micrograms/ml) or with 2-deoxyglucose plus sodium azide resulted in a rapid release of the 110-kDa protein, mannosidase II, and NBD-ceramide from the Golgi membrane to a more diffuse distribution in the cytosol. In contrast, these three Golgi markers remained to be Golgi-associated following treatment of BER-40 cells with BFA or with 2-deoxyglucose plus sodium azide. Immunoblotting of cell extracts from Vero and BER-40 cells with monoclonal antibody against the 110-kDa protein did not reveal any significant difference in the level of this Golgi marker in the mutant cells. These data suggest that the BFA-resistance mutation in BER-40 has rendered the cyclic pathway of the 110-kDa protein assembly to the Golgi membrane resistant to both BFA and 2-deoxyglucose plus sodium azide.


Evidence that oocyte maturation induced by an oncogenic ras-p21 protein and insulin is mediated by overlapping yet distinct mechanisms. We have recently shown that a peptide (residues 35-47) from a functional region of the ras p21 protein, thought to be involved in the binding of p21 to GTPase activating protein, the antibiotic azatyrosine, known to induce the ras-recision gene, and the selective protein kinase C inhibitor, CGP 41,251, all inhibit oncogenic p21 protein-induced maturation of oocytes in a dose-dependent manner. We now show that these three agents only partially inhibit insulin-induced oocyte maturation, known to be dependent on activation of cellular p21 protein. On the other hand, the anti-p21 protein antibody Y13-259 completely inhibits both insulin- and oncogenic p21 protein-induced maturation as does a tetrapeptide, CVIM, known to block the enzyme farnesyl transferase which covalently attaches the farnesyl moiety to the p21 protein allowing it to attach to the cell membrane. Our results suggest that while the oncogenic and insulin-activated normal p21 proteins share certain elements of their signal transduction pathways in common, these pathways diverge and allow for selective inhibition of the oncogenic pathway.


Metabolic consequences of adenine-phosphoribosyl transferase deficiency in V79 hamster fibroblasts. Two APRT- clones (V79-E3 and V79-E1A) were isolated from V79 hamster fibroblasts treated with ethyl methanesulfonate. Selection involved sequential exposure of the mutagenized cells to the adenine analogues 8-azaadenine and 2,6-diaminopurine. To examine the influence of APRT deficiency on cell metabolism we determined the size and turnover of adenine ribonucleotide pools, the deoxyribonucleoside triphosphate pools, the rate of DNA synthesis, and the length of the cell cycle. Clone V79-E3 was hemizygous for aprt and carried a new chromosome, 3p-. Clone V79-E1A was quasi-tetraploid with a cell volume more than twice that of the WT cells. When the difference in size was taken into account, both clones behaved similarly. While WT V79 cells released no adenine into the medium, they excreted adenine at a rate of 6 pmol/min. This did not affect the size of the ATP pool. The main change in the deoxynucleotide pools was a marked decrease of the concentration of dCTP. The rate of DNA synthesis was the same in WT cells and in the diploid V79-E3 clone. APRT is known to recycle adenine produced during polyamine synthesis, but the enzyme apparently contributes little to the maintenance of adenine ribonucleotide pools of V79 fibroblasts.


Localization by high resolution in situ hybridization of the ribosomal minichromosomes during the nucleolar cycle of Physarum polycephalum. We have used biotinylated rDNA probes to localize by in situ hybridization the extrachromosomal genes for ribosomal RNA in the slime mold Physarum polycephalum. We established conditions that allow for highly specific hybridization at the ultrastructural level and determined that the 60-kb palindromic rDNA molecules are confined to the nucleolus in interphase. Our study definitively locates these extrachromosomal genes in mitosis in the form of thin DNA fibers contained within nucleolar remnants. We further show that these rDNA minichromosomes do not condense and that they segregate as entities independent of the condensed chromosomal DNA. In telophase, these minichromosomes migrate from the poles toward the equatorial region of the nucleus in a direction opposite that of the chromosomes. Our results illustrate the discontinuous nature of the nucleolar organizing region in Physarum.


Cellular interactions with the extracellular matrix are coupled to diverse transmembrane signaling pathways. Extracellular matrix (ECM) glycoproteins such as laminin, fibronectin, or collagen IV play a major role in cell behavior regulation. The molecular mechanisms taking place at the interface between the ECM and the cell surface are now rather well defined; however, very little is known about intracellular signals induced by these interactions. In order to get insights into the transduction pathways involved in cell-ECM interactions we have investigated the effects of several intracellular kinase inhibitors. Calmodulin-dependent kinase inhibitors, W-7 and sphingosine, have negative effects on cell-matrix interactions. They inhibit adhesion of several cell lines to laminin (IC50 = 4-10 microM), fibronectin and collagen IV (IC50 = 7-25 microM). The effects are immediate, reversible, and also cell specific, certain combinations of cell line-substrate being irresponsive to these inhibitors. In contrast, two inhibitors, H-7 and staurosporine, for which protein kinase C is a common target, increase two- to fourfold the attachment of HT1080, OVCAR-4, and B16F10 cells to laminin but not to fibronectin. Another inhibitor, HA-1004, known to inhibit protein kinase A at low concentrations, has an activating effect only at high concentration (> 200 microM) when it becomes an inhibitor of protein kinase C. These inhibitors are without effect on RuGli and Saos-2 cell adhesion on the three substrates. Altogether these results suggest that calmodulin-dependent kinases and protein kinase C could be separately involved in ECM-induced cellular responses. However, the effects of kinase inhibitors are substrate-specific and cell type-specific, suggesting that the intracellular signals induced by the extracellular matrix vary with the nature of integrin involved in signal transmission.


Nuclear colocalization of c-myc protein and hsp70 in cells transfected with human wild-type and mutant c-myc genes. Using immunofluorescence and electron microscopy we have studied the localization of wild-type and mutant c-myc proteins transiently expressed in CV-1 cells. In agreement with our previous observations, wild-type c-myc protein accumulated in large amorphous globules in the nucleus. All mutant proteins tested accumulated in the nucleus as well, but gave rise to morphologically different inclusion bodies. Many small globules appeared in cells transfected with D145-262 (deletion of amino acids 145-262), while cells transfected with D371-412 or D414-433 generated structures looking like a fine network or like beads on a string. In addition, a particulate cytoplasmic staining appeared in some cells transfected with the wild-type gene and in cells transfected with mutants D145-262 or D414-433. Since the c-myc protein has been reported to stimulate expression of exogenous hsp70 protein, we also examined the intracellular distribution of hsp70 in the transfected cells. Double immunofluorescence microscopy revealed that hsp70 codistributed with the c-myc protein in distinct globules in the nucleus of many but not all myc-positive cells. However, the levels of hsp70 transcripts were not significantly raised compared to nontransfected and vector-transfected cells. Likewise, the levels of hsp70 protein did not vary significantly. These findings indicate that overexpression of c-myc stimulates translocation of preexisting hsp70 from the cytoplasm into the nucleus, rather than influencing hsp70 expression. Conceivably, this may represent one of several mechanisms whereby the cell deals with excessive amounts of c-myc protein.


The effect of staurosporine, a protein kinase inhibitor, on asialoglycoprotein receptor endocytosis. Receptor-mediated endocytosis via coated pits is modulated by the activity of protein kinases and protein phosphorylation. We examined the effects of the potent protein kinase inhibitor staurosporine (SSP) on endocytosis of the asialoglycoprotein (ASGP) receptor in HepG2 cells. Staurosporine caused a rapid (< 2 min) inhibition of ligand internalization from the cell surface. In contrast the rate of receptor exocytosis from intracellular compartments to the cell surface was not altered (t1/2 = 8 min). This resulted in increased ASGP receptors at the plasma membrane (140% of control) while the total number of receptors per cell was unchanged. Receptor up-regulation was half-maximal at 30 nM SSP. At this concentration staurosporine also inhibited the internalization of iodinated transferrin by HepG2 cells and SK Hep-1 cells, another human hepatoma-derived cell line. Staurosporine was without effect on the non-receptor-mediated uptake of Lucifer yellow by pinocytosis. We investigated the possible involvement of protein kinase C in the inhibitory effects of staurosporine on receptor endocytosis. The active protein kinase C inhibitor H7 did not inhibit ASGP receptor internalization. Furthermore depletion of cellular protein kinase C by overnight incubation with 1 microM phorbol myristate acetate did not abrogate the SSP effect. Together these data suggest that the mechanism of SSP action is independent of the inhibition of protein kinase C. In conclusion staurosporine is a potent and rapid inhibitor of receptor trafficking which is specific for receptor internalization from the plasma membrane.


ORS12, a mammalian autonomously replicating DNA sequence, is present at the centromere of CV-1 cell chromosomes. ors12, an 812-bp-long sequence, previously isolated by extrusion of nascent DNA from replication bubbles active at the onset of S phase (G. Kaufmann, M. Zannis-Hadzopoulus, and R. G. Martin Mol. Cell. Biol. 5, 721-727, 1985), has been shown to function as an origin of DNA replication in autonomously replicating plasmids (L. Frappier and M. Zannis-Hadjopoulos Proc. Natl. Acad. Sci. USA 84, 6668-6672, 1987) and in a cell-free system (C. E. Pearson, L. Frappier, and M. Zannis-Hadzopoulos Biochim. Biophys. Acta 1090, 156-166, 1991). A portion of ors12 (nucleotides 1-168) consists of the highly reiterated alpha-satellite sequence (B. S. Rao et al. Gene 87, 233-242, 1990). We have estimated the copy number of the non-alpha-satellite portion of ors12 in CV-1 cells to be < 9 copies per haploid genome and have used it as a probe to generate a genomic map of ors12 on CV-1 DNA. In situ hybridization of CV-1 metaphase chromosomes, using a biotinylated probe of the entire ors12 sequence, positively identified the centromeres of all chromosomes. However, when the non-alpha-satellite portion of ors12 was used as a probe, it positively identified the centromeric region of only six chromosomes, namely, B4, C11, D14, D24, E25, and E27, as well as that of a marker chromosome. The results suggest that ors12 represents a centromeric putative replication origin that is present on a subset of CV-1 chromosomes and is activated at the onset of S phase.


Polyoma middle T antigen or v-src desensitizes human epidermal growth factor receptor function and interference by a monensin-resistant mutation in mouse Balb/3T3 cells. Epidermal growth factor (EGF)-induced down-regulation of its receptor is an obligatory pathway for cellular regulation of EGF-specific receptor (EGF-R) in normal and malignant cells. BNER4 cells are mouse Balb/3T3 cells transfected with the human EGF-R complementary DNA (cDNA). Polyoma middle T antigen-transfectants of BNER4, B4/MT-2, B4/MT-13, B4/MT-23, and B4/MT-24, showed diminished down-regulation of cell surface human EGF-R in response to EGF relative to the parental BNER4 cells. Also, the v-src-transfectants B4/SRC-13 and B4/SRC-24 showed much less down-regulation than BNER4 cells, whereas H-ras-transfectants of BNER4, B4/RAS-24 and B4/RAS-25, showed EGF-induced down-regulation of the cell surface EGF-R similar to that of BNER4. EGF induced DNA synthesis more than 20-fold in BNER4, but induced only about a 1.5- to 6-fold increase in the middle T antigen- and v-src-transfectants. EGF-Rs of the middle T antigen-transfectants were metabolically stable in the presence of EGF in comparison with their parental BNER4 cells. EGF-Rs of BNER4 cells degraded with half-lives of about 2 h in the presence of EGF, but those of the middle T antigen transformants were found to be highly stabilized in the presence of EGF. On the other hand, transfection with polyoma middle T antigen (MTAg) cDNA causes malignant transformation of Balb/3T3 cells, but not its monensin (an ionophoric antibiotic)-resistant mutant MO-5 cells, which have no significant EGF binding activity. Transfection of human EGF-R cDNA into MO-5 leads to the expression of high levels of human EGF-R in MNER31. Unlike the polyoma MTAg transfectants of BNER4, EGF-R in polyoma MTAg cDNA-transfectants into MNER31, M31/MT-13 and M31/MT-14, were down-regulated to levels similar to those of their parental MNER31. Exposure to EGF induced a more than 10-fold increase in DNA synthesis of quiescent BNER4, MNER31, M31/MT-13, and M31/MT-14 cells. Polyoma middle T antigen or v-src appears to modulate EGF-induced down-regulation of EGF-R, possibly through interaction of the receptor with the viral oncogenes, and this interaction may be altered in the mutant.


Deficiency in integrin-mediated transmembrane signaling and microfilament stress fiber formation by aging dermal fibroblasts from normal and Down's syndrome patients. Previous evidence has shown a deficiency in microfilament stress fiber formation upon short-term cycloheximide treatment of cultured human dermal fibroblasts while cytoplasmic spreading appeared completely normal and other cytoskeletal networks organized normally. This deficiency applied to collagen substrata (not fibronectin substrata) and was specific for in vitro-aged normal fibroblasts and for fibroblasts from three different Down's syndrome patients at any passage level. To identify the mechanism(s) for matrix receptor deficiency in aging cells, cells were evaluated for amounts and distributions of several integrin subunits using specific monoclonal antibodies and two complementary experimental approaches. Flow cytometric analyses have shown that all these cells at all passage levels have large amounts of alpha 3 and beta 1 integrin subunits and smaller amounts of the alpha 5 subunit, directed to fibronectin, which are minimally affected in their cell surface availability by cycloheximide treatment. In contrast, cycloheximide treatment leads to the loss from surface availability of most of the alpha 2 subunit, directed to collagen, in late-passage papillary and reticular normal fibroblasts and in all three Down's patient cells at all passages. Prior growth of cells in ascorbate-supplemented medium, which overcomes the deficiency in stress fiber formation, conserves the large amounts of cell surface-available alpha 2 subunit detectable by flow cytometry. When amounts of integrin subunits were evaluated by immunoprecipitation of [35S]methionine-radiolabeled cells, there was no diminution of the alpha 2 subunit or any other subunit for any cells upon cycloheximide treatment; however, there was much less alpha 2 subunit complexed with beta 1 in aging normal and Down's cells. Therefore, cycloheximide treatment does not lead to loss in the amounts of the alpha 2 subunit but rather to its masking at the cell surface and inability to transmit signals across the plasma membrane to effect stress fiber formation. This aging-related deficiency in integrin-mediated signaling can now be studied mechanistically with a variety of approaches to determine the nature of cell-surface molecules interacting with integrins (cis- and/or trans-acting molecules) that discriminate functional from nonfunctional receptors.


Inhibition of gliding movement by calcium in doublet microtubules on Tetrahymena ciliary dyneins in vitro. We examined the effects of Ca ions on the gliding movement of Tetrahymena ciliary doublet microtubules induced by 14S or 22S dyneins in an in vitro motility assay system. The doublet microtubule appeared as circular-arc in solution, about 5 to 6 microns in length [1]. The doublet microtubules glided distal-end first on a 14S or 22S dynein-coated glass surface either clockwise or counterclockwise following the addition of ATP. The diameter of the circular path changed according to Ca concentration in the solution. Gliding velocity was from 1 to 5 microns/s. The addition of 0.1% Nonidet P-40 was necessary to induce the gliding movement on 22S dynein. This movement on 22S dynein was strongly inhibited above 0.5 mM ATP in the presence of 10(-9) M Ca, and at 0.05 to 1 mM ATP in the presence of 10(-3) M Ca. Many studies have indicated that Ca ions regulate ciliary movement [2-8] in which dyneins and doublet microtubule in the axoneme may play an essential role. The inhibition of the gliding movement of doublet microtubule on dyneins at appropriate concentrations of Ca and ATP as observed in this study may be the key for understanding Ca regulation of ciliary motility.


The short-term increase in extracellular potassium concentration causes DNA replication onset in density-inhibited cells. The high potassium concentration effect on the human diploid fibroblasts (HDF) and 3T3 cells was investigated. The incubation of confluent cultures of HDF or 3T3 Swiss cells in the medium with 50 mM K+ for 35 min induced, 12 h later, the onset of DNA replication in a significant proportion of culture cell population. The same treatment had no effect upon the sparce cell cultures. No stimulation of DNA replication was observed in the absence of serum in culture medium.


Radiolabeling of DNA can induce its fragmentation in HL-60 human promyelocytic leukemic cells. Incorporation of radiolabeled thymidine is commonly used to investigate DNA damage. Using a filter-binding assay, we observed that the addition of various doses of [methyl-3H]thymidine (0.2 and 2 microCi/ml) or [2-14C]thymidine (0.02 and 0.2 microCi/ml) in the culture medium for 2 days, a standard method for cell-labeling, induces DNA fragmentation in HL-60 human promyelocytic cells. This effect was dose- and time-dependent and the DNA fragments were not protein-linked since the levels of DNA fragmentation were identical in the presence and in the absence of proteinase K (0.5 mg/ml). Radiolabeled thymidine-induced DNA fragmentation was associated with an inhibition of cell growth, but cells remained able to exclude trypan blue, suggesting that plasma membrane integrity was conserved, except at very high doses of [methyl-3H]thymidine (2 microCi/ml). By agarose-gel electrophoresis, the DNA-fragmentation was demonstrated to be internucleosomal with a typical ladder pattern. Addition of unlabeled thymidine to the culture medium prevented DNA fragmentation in a dose-dependent manner, indicating that radiolabeled thymidine incorporation in DNA was directly responsible for DNA fragmentation. We conclude that radiolabeling of DNA using thymidine incorporation can induce DNA fragmentation in some cell lines such as HL-60. This observation must be taken into account in methods using radiolabeling to study DNA damage in these cells.


Basic FGF and TGF-beta differentially modulate integrin expression of human microvascular endothelial cells. Basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta) are known to alter the migratory and proliferative capacity of endothelial cells in vitro and to stimulate angiogenesis in vivo. One mechanism by which these cytokines induce their effects may be through the regulation of integrin adhesion receptor expression and activity. We examined the ability of these growth factors to modulate the expression of specific integrins in human microvascular endothelial cells (MEC). Immunoprecipitation of metabolically labeled MEC showed that bFGF upregulated the biosynthesis of alpha 2, alpha 5, beta 1, and beta 3. bFGF induced an increase in the levels of mRNA for alpha 2 and beta 1. TGF-beta increased synthesis of alpha 2, alpha 5, and beta 1. These results suggest that bFGF and TGF-beta selectively alter integrin profiles and influence interactions of MEC with the extracellular matrix during neovascularization. In particular, the upregulation of the collagen/laminin receptor, alpha 2 beta 1, by bFGF may provide activated endothelial cells with an enhanced capacity to migrate through both their underlying basement membrane and the interstitial matrix.


Functional coupling of the stabilizing eye and head reflexes during horizontal and vertical linear motion in the cat. The otolith contribution and otolith-visual interaction in eye and head stabilization were investigated in alert cats submitted to sinusoidal linear accelerations in three defined directions of space: up-down (Z motion), left-right (Y motion), and forward-back (X motion). Otolith stimulation alone was performed in total darkness with stimulus frequency varying from 0.05 to 1.39 Hz at a constant half peak-to-peak amplitude of 0.145 m (corresponding acceleration range 0.0014-1.13 g). Optokinetic stimuli were provided by sinusoidally moving a pseudorandom visual pattern in the Z and Y directions, using a similar half peak-to-peak amplitude (0.145 m, i.e., 16.1 degrees) in the 0.025-1.39 Hz frequency domain (corresponding velocity range 2.5 degrees-141 degrees/s). Congruent otolith-visual interaction (costimulation, CS) was produced by moving the cat in front of the earth-stationary visual pattern, while conflicting interaction was obtained by suppressing all visual motion cues during linear motion (visual stabilization method, VS, with cat and visual pattern moving together, in phase). Electromyographic (EMG) activity of antagonist neck extensor (splenius capitis) and flexor (longus capitis) muscles as well as horizontal and vertical eye movements (electro-oculography, EOG) were recorded in these different experimental conditions. Results showed that otolith-neck (ONR) and otolith-ocular (OOR) responses were produced during pure otolith stimulation with relatively weak stimuli (0.036 g) in all directions tested. Both EMG and EOG response gain slightly increased, while response phase lead decreased (with respect to stimulus velocity) as stimulus frequency increased in the range 0.25-1.39 Hz. Otolith contribution to compensatory eye and neck responses increased with stimulus frequency, leading to EMG and EOG responses, which oppose the imposed displacement more and more. But the otolith system alone remained unable to produce perfect compensatory responses, even at the highest frequency tested. In contrast, optokinetic stimuli in the Z and Y directions evoked consistent and compensatory eye movement responses (OKR) in a lower frequency range (0.025-0.25 Hz). Increasing stimulus frequency induced strong gain reduction and phase lag. Oculo-neck coupling or eye-head synergy was found during optokinetic stimulation in the Z and Y directions. It was characterized by bilateral activation of neck extensors and flexors during upward and downward eye movements, respectively, and by ipsilateral activation of neck muscles during horizontal eye movements. These visually-induced neck responses seemed related to eye velocity signals. Dynamic properties of neck and eye responses were significantly improved when both inputs were combined (CS).(ABSTRACT TRUNCATED AT 400 WORDS)