The interaction of methanol dehydrogenase and its electron acceptor, cytochrome cL in methylotrophic bacteria.

The interactions of methanol dehydrogenase (MDH, EC1.1.99.8) with its specific electron acceptor cytochrome cL has been investigated in Methylobacterium extorquens and Methylophilus methylotrophus. The MDHs of these two very different methylotrophs have the same alpha 2 beta 2 structure; the interaction of these MDHs with their specific electron acceptor, cytochrome cL, has been studied using a novel assay system. Electrostatic reactions are involved in 'docking' of the two proteins. EDTA inhibits the reaction by a process involving neither metal chelation nor the 'docking' process. Chemical modification studies showed that the two proteins interact by a 'docking' process involving interactions of lysyl residues on MDH and carboxyl residues on cytochrome cL. When 'zero length', two stage cross-linking was done (with proteins from both bacteria), the alpha-subunits of MDH cross-linked with cytochrome cL by way of lysyl groups on MDH and carboxyl groups on the cytochrome. Tuna mitochondrial cytochrome c provided a model for cytochrome cH which is the electron acceptor for cytochrome cL in the 'methanol oxidase' electron transport chain. Tuna cytochrome c was shown to form crosslinked products with carboxyl-modified cytochrome cL. MDH and tuna cytochrome c competed for the same domain on cytochrome cL. It was concluded that MDH reacts with cytochrome cL by an electrostatic reaction which involves carboxyl groups on cytochrome cL and amino groups on the alpha-subunit of MDH. The same domain on cytochrome cL is involved in subsequent 'docking' with its electron acceptor.


On the redox equilibrium between H2 and hydrogenase.

Redox titrations of the nickel ion in active hydrogenase from Methanobacterium thermoautotrophicum and Chromatium vinosum were performed in the absence of artificial redox mediators, by variation of the H2-partial pressure. These experiments revealed a redox behaviour of the nickel ion which differed remarkably from previous redox titrations in the presence of redox mediators. Notably the EPR signal of the species earlier characterized as monovalent nickel with bound hydrogen, behaved as an n = 2 redox component upon reduction under varying H2-partial pressures. The EPR signal was not a transient one and persisted upon removal of hydrogen. Possible redox processes to explain these observations are discussed. A similar behaviour of nickel was also observed in enzyme as present in intact cells of M. thermoautotrophicum. These results suggest that nickel hydrogenases possess a second site for reaction with H2.


Subunit IV of human cytochrome c oxidase, polymorphism and a putative isoform.

As part of our study of isoenzyme forms of human cytochrome c oxidase, we purified subunit IV from human heart and skeletal muscle with reversed-phase HPLC and determined the N-terminal amino acid sequences and the electrophoretic mobility. The N-terminus of human heart subunit IV proved to be ragged with 30% of the protein lacking the first three residues. Also a Tyr/Phe polymorphism was observed at residue 16. No differences in N-terminal sequence and electrophoretic mobility were observed between subunit IV of cytochrome c oxidase from human heart and skeletal muscle. Therefore, our results suggest that identical subunits IV are present in cytochrome c oxidase from human heart and skeletal muscle. A putative isoform of subunit IV with a blocked N-terminus was purified from human heart cytochrome c oxidase, which proved to have a different retention time on a reversed-phase column and also a slightly higher electrophoretic mobility on an SDS-polyacrylamide gel compared to the native subunit IV. We could not demonstrate the existence of isoforms of subunit IV in human skeletal muscle.


A model for the coordinated development of columnar systems in primate striate cortex.

The existence of patchy regions in primate striate cortex in which orientation selectivity is reduced, and which lie in the centers of ocular dominance stripes is well established (Hubel and Livingstone 1981). Analysis of functional maps obtained with voltage sensitive dyes (Blasdel and Salama 1986) has suggested that regions where the spatial rate of change of orientation preference is high, tend to be aligned either along the centers of ocular dominance stripes, or to intersect stripe borders at right angles. In this paper I present results from a developmental model which show that a tendency for orientation selectivity to develop more slowly in the centers of ocular dominance stripes would lead to the observed relationships between the layout of ocular dominance and the map of orientation gradient. This occurs despite the fact that there is no direct connection between the measures of preferred orientation (from which the gradient map is derived) and orientation selectivity (which is independent of preferred orientation). I also show that in both the monkey and the model, orientation singularities have an irregular distribution, but tend to be concentrated in the centers of the ocular dominance stripes. The average density of singularities is about 3/lambda 2 theta, where lambda theta is the period of the orientation columns. The results are based on an elaboration of previous models (Swindale 1980, 1982) which show how, given initially disordered starting conditions, lateral interactions that are short-range excitatory and long-range inhibitory can lead to the development of patterns of orientation or ocular dominance that resemble those found in monkey striate cortex.(ABSTRACT TRUNCATED AT 250 WORDS)


Pulsatile signaling in intercellular communication. Periodic stimuli are more efficient than random or chaotic signals in a model based on receptor desensitization.

The efficiency of various patterns of pulsatile stimulation is determined in a model in which a receptor becomes desensitized in the presence of its stimulatory ligand. The effect of stochastic or chaotic changes in the duration and/or interval between successive pulses in a series of square-wave stimuli is investigated. Before addressing the effect of random variations in the pulsatile signal, we first extend the results of a previous analysis (Li, Y.X., and A. Goldbeter. 1989. Biophys. J. 55:125-145) by demonstrating the existence of an optimal periodic signal that maximizes target cell responsiveness whatever the magnitude of stimulation. As to the effect of stochastic or chaotic variations in the pulsatile stimulus, three kinds of random distributions are used, namely, a Gaussian and a white-noise distribution, and a chaotic time series generated by the logistic map. All these random distributions are symmetrically centered around the reference value of the duration or interval that characterizes the optimal periodic stimulus yielding maximal responsiveness in target cells. Stochastically or chaotically varying pulses are less effective than the periodic signal that corresponds to the optimal pattern of pulsatile stimulation. The response of the receptor system is most sensitive to changes in the time interval that separates successive stimuli. Similar conclusions hold when stochastic or chaotic signals are compared to a reference periodic stimulus differing from the optimal one, although the effect of random variations is then reduced. The decreased efficiency of stochastic pulses accounts for the observed superiority of periodic versus stochastic pulses of cyclic AMP (cAMP) in Dictyostelium amoebae. The results are also discussed with respect to the efficiency of periodic versus stochastic or chaotic patterns of hormone secretion.


Novel voltage clamp to record small, fast currents from ion channels expressed in Xenopus oocytes.

The present report describes a novel technique for voltage-clamping amphibian oocytes in which part of the membrane is isolated by a vaseline gap and the cytoplasmic fluid is exchanged by cutting or permeabilizing the remaining membrane. The main features of this open-oocyte, vaseline-gap voltage clamp are: (a) low current noise (1 nA at 3 kHz), (b) control of the ionic composition of both the internal and external media, (c) fast time resolution (20-100 microseconds time constant of decay of the capacity transient) and (d) stable recordings for several hours. These features allow reliable measurements of tail or gating currents and the new method is especially suitable when either of these currents must be measured to test the effects of mutations introduced into the cDNAs of cloned ion channels.


Voltage-sensitive and solvent-sensitive processes in ion channel gating. Kinetic effects of hyperosmolar media on activation and deactivation of sodium channels.

Kinetic effects of osmotic stress on sodium ionic and gating currents have been studied in crayfish giant axons after removal of fast inactivation with chloramine-T. Internal perfusion with media made hyperosmolar by addition of formamide or sucrose, reduces peak sodium current (before and after removal of fast inactivation with chloramine-T), increases the half-time for activation, but has no effect on tail current deactivation rate(s). Kinetics of ON and OFF gating currents are not affected by osmotic stress. These results confirm (and extend to sodium channels) the separation of channel gating mechanisms into voltage-sensitive and solvent-sensitive processes recently proposed by Zimmerberg J., F. Bezanilla, and V. A. Parsegian. (1990. Biophys. J. 57:1049-1064) for potassium delayed rectifier channels. Additionally, the kinetic effects produced by hyperosmolar media seem qualitatively similar to the kinetic effects of heavy water substitution in crayfish axons (Alicata, D. A., M. D. Rayner, and J. G. Starkus. 1990. Biophys. J. 57:745-758). However, our observations are incompatible with models in which voltage-sensitive and solvent-sensitive gating processes are presumed to be either (a) strictly sequential or, (b) parallel and independent. We introduce a variant of the parallel model which includes explicit coupling between voltage-sensitive and solvent-sensitive processes. Simulations of this model, in which the total coupling energy is as small as 1/10th of kT, demonstrate the characteristic kinetic changes noted in our data.


MyoD and myogenin are coexpressed in regenerating skeletal muscle of the mouse.

The differential expression of genes triggering myogenesis might cause or reflect differences among myoblasts. Little is known about the presence of MyoD1 and myogenin during the process of regeneration. We therefore examined the expression of MyoD1 and myogenin in muscle regeneration after grafting. Immunostaining of regenerating skeletal muscle of the mouse revealed the presence of both MyoD1 and myogenin. In mononucleated cells the proteins were not detected until shortly before fusion into myotubes. They persisted in the nuclei of regenerated muscle fibers for at least 2 weeks. MyoD1 and myogenin were not detected in nonregenerating control muscle.


Patency of side branches after peripheral placement of metallic biliary endoprostheses.

During a 28-month period, the authors placed 91 Wallstent endoprostheses in 55 patients with malignant obstructive jaundice. Five patients developed recurrent jaundice between 2 and 60 weeks after stent insertion due to stent occlusion by tumor overgrowth on seven occasions. To assess long-term segmental side-branch drainage through the walls of such endoprostheses, the cholangiograms obtained following stent occlusion were reviewed. In all five patients, evidence of drainage of intrahepatic ducts through the side of the mesh was observed. Although the number of patients in the series is small, this initial experience suggests that long metallic endoprostheses can be placed peripherally in the intrahepatic bile ducts without the potential risk of infection or occlusion of undrained, noninvolved segments. This policy may delay or prevent endoprosthesis occlusion in many patients.


Electrophysiological consequences of exposure of hippocampal slices to dihydroxyfumarate, a generator of superoxide radicals.

In an effort to understand the damaging actions of free radicals to neuronal electrophysiology, the superoxide generator, dihydroxyfumarate (DHF), was evaluated in slices of guinea pig hippocampus. Using field potential recording techniques, population spikes and population synaptic potentials were recorded in field CA1. Slices were exposed to 3 mM DHF either alone or in the presence of a protectant. DHF did not alter the ability of the afferent volley to generate a synaptic potential, but it did impair the ability of the synaptic potential to elicit a population spike. In addition, DHF induced lipid peroxidation as measured by the thiobarbituric acid assay. Superoxide dismutase (SOD) provided no protection. Instead, SOD treatment promoted DHF damage to synaptic potentials. Catalase alone mitigated the actions of DHF, but only in SOD plus catalase was the DHF-induced electrophysiological deficit and lipid peroxidation completely antagonized. The iron chelator, Desferal, did not protect but promoted synaptic damage. Desferal may be ineffective because of the nitroxide radical formed upon its reaction with DHF. The hydroxyl radical scavenger, dimethylsulfoxide, prevented lipid peroxidation and reduced the DHF-induced deficit but did not completely prevent the impairment of spike generation. These data suggest that DHF exerts its actions through generation of hydrogen peroxide which would further react with tissue iron to produce hydroxyl radicals.


Ionic responsiveness in third ventricular hypertonic stimulation of antidiuresis in ducks.

Domestic ducks were chronically equipped with a device probing the third cerebral ventricle (VIII) for localized intracerebroventricular (i.c.v.) perfusion. In conscious animals made diuretic by intravenous water loading with 1.0 ml/min hypoosmotic glucose solution (200 mOsm/kg), hyperosmotic i.c.v. stimulations were tested for antidiuretic actions. Artificial cerebrospinal fluid made hypertonic (400 mOsm/kg) by adding sucrose, mannitol, NaCl, LiCl, choline chloride, NaI, NaNO3, LiNO3, CaCl2 or MgCl2 was perfused i.c.v. for 10-15 min at rates of 10-15 microliters/min. Arterial pressure and heart rate were monitored continuously. Hyperosmotic stimulations with non-electrolytes did not induce antidiuresis. Approximately equivalent degrees of antidiuresis were elicited by Na(+)-, Li(+)- and choline salts with a tendency for moderate rises in arterial pressure. Compared to Cl(-)- and I(-)-salts, the effects of NO3(-)-salts were attenuated. Divalent cations caused prolonged antidiuresis, sometimes preceded by initial diuresis, with circulatory side effects unrelated to the changes in renal fluid excretion. It is concluded that the observed antidiuretic effects were mediated by cation-sensitive, rather than osmosensitive neurons on the brain side of the blood-brain-barrier. Their transduction mechanism might consist of poorly selective membrane channels permeable to cations but not to anions.


Increased hypothalamic [3H]flunitrazepam binding in hypothalamic-pituitary-adrenal axis hyporesponsive Lewis rats.

We have previously demonstrated that susceptibility of Lewis (LEW/N) rats to inflammatory disease, compared to relatively resistant Fischer (F344/N) rats, is related to deficient glucocorticoid counter-regulation of the immune response resulting from deficient corticotropin-releasing hormone (CRH) responsiveness to inflammatory and other stress mediators. The GABA/benzodiazepine receptor complex is an important negative modulator of CRH secretion and responsiveness to excitatory stimuli. In this study, we have examined in vitro binding of [3H]flunitrazepam to hypothalamic membrane preparations from LEW/N and F344/N rats. LEW/N rats had significantly more hypothalamic benzodiazepine binding sites (Bmax) than F344/N rats, but there were no differences in benzodiazepine binding affinities (Kd) between these two strains. The differences in benzodiazepine receptor number were consistent with the respective plasma corticosterone levels in the two strains, and with previous work indicating a negative correlation between corticosterone levels and benzodiazepine binding site number. Adrenalectomy of F344/N rats increased benzodiazepine binding to levels comparable to LEW/N animals and treatment of adrenalectomized F344/N rats with DEX resulted in lowering of benzodiazepine Bmax to levels that did not differ significantly from those of intact F344/N rats. There was no significant change in receptor number in either adrenalectomized or DEX-treated LEW/N rats. These findings suggest that basal benzodiazepine receptor differences between these strains may be partially related to strain differences in corticosterone levels, however that additional factors may contribute to maintenance of these differences in LEW/N rats. Since benzodiazepines attenuate hypothalamic CRH secretion through GABAergic inhibition, we suggest that strain differences in receptor number could also augment strain differences in hypothalamic-pituitary-adrenal axis function through differential sensitivity to GABA-mediated feedback.


Type Ia glycogenosis associated with hepatocellular carcinoma.

The predisposition of preexisting cirrhotic metabolic liver disease to the development of hepatocellular carcinoma is well established. However, the association between glycogenoses (a form of noncirrhotic metabolic liver disease) and the formation of benign and malignant liver tumors is less well known. The sixth case of simultaneous occurrence of glycogenosis type Ia and hepatocellular carcinoma, which occurred in a 34-year-old man referred for advice on therapy for his malignant neoplasm, is reported. Cases previously described in the literature are reviewed and management of this entity in light of the current knowledge in the diagnosis and treatment of hepatocellular carcinoma is discussed.


Combined hepatoblastoma and yolk sac tumor of the liver.

The authors report a liver tumor that occurred in a 6-month-old boy in which areas of yolk sac and hepatoblastoma were identified. To the best knowledge of the authors, this morphologic pattern has not been reported previously. Theories of histogenesis are discussed.


The cutaneous form of adult T-cell leukemia/lymphoma in a woman from the Ivory Coast. Clinical, immunovirologic studies and a review of the African adult T-cell leukemia/lymphoma cases.

A 36-year-old woman from Ivory Coast, who has lived in France since 1976, had multiple cutaneous nodules and tumors in 1988. Histopathologic studies showed a massive infiltration of the dermis and hypodermis by a diffuse proliferation of mature activated T-cells (CD4-positive, CD25-positive, HLA-DR-positive) with irregular nuclei. The patient did not present with a leukemic picture and only few lymphoid cells with abnormally shaped nuclei were present in the blood. Human T leukemia/lymphoma virus type I (HTLV-I) antibodies were present in the serum and specific HTLV-I pol sequences were detected in the DNA extracted from the tumor nodules and peripheral blood mononuclear cells (PBMC) using the polymerase chain reaction technique. Whereas only a polyclonal integration of HTLV-I provirus was detectable in the PBMC, a clonal integration of three HTLV-I proviruses was demonstrated in the tumor nodules DNA, establishing with certainty the diagnosis of HTLV-I-induced adult T-cell leukemia/lymphoma (ATL). This case illustrates the need for molecular studies to differentiate without ambiguity an ATL from any other type of cutaneous lymphoproliferation, even when it occurs in a HTLV-I-seropositive individual. The situation of HTLV-I-associated ATL in Africa is reviewed.


Peritumoral lymphatic invasion in patients with node-negative mammary duct carcinoma.

Five hundred six consecutive cases of ductal infiltrating carcinoma of the breast (T1-T2,N0,M0) were evaluated to define the frequency of peritumoral lymphatic invasion (PLI) and verify its possible prognostic significance. Histologically, PLI was characterized by the presence of neoplastic emboli within vascular lumina lined by recognizable endothelial cells, adjacent to but outside the margins of the carcinoma. In routine histopathologic assessment the frequency of PLI was 68% whereas in a randomly selected group of 234 reviewed cases the frequency rose to 20%. Patients with routinely evaluated PLI had a worse prognosis than those without PLI with reference both to disease-free survival (P = 0.0001) and total survival rates (P = 0.0001). The difference for local recurrences was prognostically highly significant (P = 0.0001) and also significant for the development of metastases (P = 0.0576). In the reviewed material the difference in prognosis between PLI-positive and PLI-negative cases was not confirmed for total survival whereas the significance for the disease-free interval persisted. The assessment of PLI, carried out following strict histopathologic criteria, appears to select a group of node-negative breast cancer patients who have an increased risk of recurrences and might benefit from a treatment different from that reserved for node-negative and PLI-negative patients.


Organic delusional syndrome associated with tamoxifen treatment.

The authors describe two cases of tamoxifen-induced organic delusional syndrome that resulted from administration of 40 mg daily for the adjuvant treatment of breast carcinoma. After discontinuation of the tamoxifen therapy, the psychiatric episode resolved within 2 to 4 weeks. Because tamoxifen might cause delusional syndrome, the authors recommend full psychiatric evaluation in appropriate cases.


The value of flow cytometric analysis in multicentric glomus tumors of the head and neck.

Glomus tumors of the head and neck include those arising from the carotid body, jugular vein, and vagus nerve. Because these cannot be differentiated histologically, when encountering a large tumor mass involving more than one structure in the carotid sheath, one often cannot be sure whether the tumors are from one or more of these structures. The authors performed DNA flow cytometric analysis on a patient with a multicentric glomus tumor on the right side of the neck involving the carotid body, jugular vein, and vagus nerve, in an effort to determine the separate or similar origin of her tumor mass. Different DNA indices, including a double peak for the carotid body tumor, were obtained. There were three aneuploid tumors and one diploid tumor (DNA indices: carotid body 1.78, 2.04; jugular vein 2.20; vagus nerve 1.82). Different synthetic phase fractions were calculated for each aneuploid tumor except the second carotid body peak (carotid body 7.2; jugular vein 3.6; vagus nerve 4.8). The authors conclude that DNA flow cytometry may be useful in confirming the multicentric origin of tumors that encompass more than one histologically similar structure.


Carcinoembryonic antigen and carbohydrate 19-9 antigen as markers for colorectal carcinoma in children and adolescents.

Colorectal cancer is rare in patients younger than 20 years of age (incidence, 1 in 10 million). Although carcinoembryonic antigen (CEA) and carbohydrate 19-9 antigen (CA 19-9) have been used widely as markers for the efficacy of therapy or detection of recurrent colorectal carcinomas in adults, no studies evaluating their efficacy in children and adolescents have been performed. Between 1986 and 1989, serial measurements of serum CEA and CA 19-9 levels were obtained from 11 patients (aged 8 to 18 years) treated at the institution of the authors. In contrast to the experience in adults, most (9 of 11) of the tumors were localized in the right or transverse colon. Histologically, 10 of 11 carcinomas were mucinous. Most patients had either Dukes' C (5 of 11) or D (5 of 11) lesions. After surgery, all patients were treated with a combination of 5-fluorouracil with Leucovorin (Lederle Laboratories, Pearl River, NY). With the use of the adult normal standards (CEA less than 3.0 ng/ml and CA 19-9 less than 37 ng/ml)6 for the patients, the specificity and sensitivity of CEA were 77% and 64%, respectively, whereas the specificity of CA 19-9 was 79% and the sensitivity was significantly low (10%). The combined specificity of the two markers was 71%, and the combined sensitivity was 60%. On the basis of these results, the authors believe that CEA and CA 19-9 are not effective markers for detection of recurrent or progressive colorectal carcinomas in children and adolescents.


Molecular markers for the diagnosis and prognosis of lung cancer.

Molecular probes for lung cancer have greatly increased the understanding of the biology of this disease and the preneoplastic changes that precede it. They have confirmed and extended the clinical, pathologic, and biologic reasons for the primary division of lung cancers into small cell and non-small cell lung cancer types. Many molecular changes are present in lung cancers and involve dominant oncogenes and recessive growth regulatory genes. Clinical application of these markers will aid diagnosis, classification, and clinical management.


Insulin like growth factor I is an autocrine regulator of human colon cancer cell differentiation and growth.

The polyanionic compound suramin triggers enterocyte-like differentiation of the human colic adenocarcinoma cell clone HT29-D4. We now demonstrate that suramin interferes with the binding of IGF-I to its receptor at the surface of HT29-D4 cells. Half-maximum inhibition of 125I-IGF-I binding was obtained in the presence of 25 micrograms/ml suramin. Moreover, the drug was able to dissociate 125I-IGF previously bound to its cell surface receptor. Affinity labeling HT29-D4 cells were cultured in the presence of 10 micrograms/ml of alpha-IR3, a monoclonal antibody directed against the binding site of IGF-I, an inhibition of cell proliferation and a stimulation of cell differentiation was observed. After 10 days of treatment with alpha-IR3, HT29-D4 cells formed a regular monolayer of enterocyte-like cells exhibiting an apical brush border and tight junctions delimiting two domains of the plasma membrane (apical and basolateral). Furthermore, we show that IGF-I significantly increased the initial rate of glucose uptake by HT29-D4 cells, while we have previously shown that suramin decreased glucose consumption. From these data we conclude that IGF-I secreted by the cells themselves, stimulates proliferation of HT29-D4 cells via an autocrine mechanism. Blockade of this stimulation by suramin or by a specific monoclonal antibody inhibits cell growth, glucose uptake and triggers the process of enterocytic differentiation.


Steroid and growth factor modulation of aromatase activity in MCF7 and T47D breast carcinoma cell lines.

The effect of a number of steroids, growth factors, and peptides on aromatase activity in two estrogen receptor positive breast cancer cell lines (MCF7 and T47D) was investigated. The cells were incubated in Dulbecco's minimum essential medium containing phenol red and 10% fetal calf serum. Pronounced differences in basal aromatase activity and different responses to the addition of experimental agents were found in the two cell lines. Aromatase activity in MCF7 cells was significantly stimulated by phorbol 12,13-diacetate [PDA], dibutyryl cyclic AMP [(Bu)2cAMP], transforming growth factor alpha, and epidermal growth factor individually and PDA and (Bu)2cAMP in combination, while it was inhibited by dexamethasone and unaffected by transforming growth factor beta, fibroblast growth factor, platelet-derived growth factor, prolactin, and tamoxifen. Addition of cortisol to MCF7 cells had no effect on aromatase activity at 1 nM, caused suppression of activity at 10 nM and stimulated activity at 100 nM. Aromatase activity in T47D cells was stimulated by transforming growth factor alpha, epidermal growth factor, platelet-derived growth factor, prolactin, dexamethasone, and cortisol individually and PDA and (Bu)2cAMP in combination. It was unaffected by transforming growth factor beta, PDA, (Bu)2cAMP, and fibroblast growth factor. These findings suggest that aromatase activity is induced by agents which stimulate cyclic AMP-dependent protein kinases [e.g., (Bu)2cAMP] and that this effect is potentiated by factors which stimulate protein kinase C [e.g., PDA]. The effect on aromatase activity of growth factors, the actions of which are believed to be mediated by receptors linked to tyrosine kinase activity, is not as clearly defined, with a factor causing stimulation, inhibition, and no change in activity depending on the tissue concerned. Further insight into these differences will require resolution of the molecular mechanisms that mediate the actions of stimulatory and repressive growth factors on aromatase activity of oestrogen-producing cells.


Tumor invasion-inhibiting factor 2: primary structure and inhibitory effect on invasion in vitro and pulmonary metastasis of tumor cells.

The primary structure of tumor invasion-inhibiting factor 2 (IIF-2) purified from bovine liver (A. Isoai et al., Jpn. J. Cancer Res., 81:909-914, 1990) was determined. A computer homology search of the National Biomedical Research Foundation data bank revealed that IIF-2 is identical to the carboxyl-terminal region, residue number [69-89], of high mobility group 17 which is a DNA-binding non-histone protein. IIF-2 synthesized by an automated peptide synthesizer showed similar invasion-inhibitory activity as compared with the purified factor, when tested with the monolayer invasion assay system using highly invasive rat ascites tumor cells. When examined with the other in vitro assay systems using a modified Boyden chamber, the synthetic IIF-2 suppressed the chemotactic migration of highly metastatic B16 melanoma (B16FE7) cells to fibronectin or laminin and invasion through Matrigel. The IIF-2 inhibited neither the cell proliferation nor the binding of cells to fibronectin or Matrigel and also showed no significant inhibition of Mr 90,000 type IV collagenase (gelatinase) obtained from human schwannoma (YST-3) cells. The formation of lung colonies in mice given injections of B16FE7 and Lewis lung carcinoma cells was significantly reduced by the coinjection of the IIF-2. These results suggest that IIF-2 suppresses tumor invasion by impairing cell motility and inhibits the migration of metastasizing cells through extracellular matrix (extravasation steps) following their arrest in the capillary bed of the lung in vivo.


A gene from human chromosome region 3p21 with reduced expression in small cell lung cancer.

A combination of cytogenetic and molecular studies has implicated the p21 region of human chromosome 3 as the probable site of a gene the loss of which contributes to the development of small cell lung cancer. We report here the isolation of a gene from this region which is expressed in normal lung tissue and in cell lines derived from a number of different types of tumor, but the expression of which in small cell lung cancer cell lines is undetectable by RNA blot analysis. Although the more sensitive polymerase chain reaction did detect transcripts, a novel quantitative polymerase chain reaction assay showed that their concentration in small cell lung cancer cell lines is less than 3% of that in normal lung.


Expression of high-affinity laminin receptor mRNA correlates with cell proliferation rather than invasion in human papillomavirus-associated cervical neoplasms.

Induction of the expression of the Mr 67,000 high-affinity laminin receptor gene has been postulated as playing a role in the progression of human tumors to invasive cancers. We tested this hypothesis by examining histopathological sections of a large number of epithelial lesions of the genital tract associated with human papillomaviruses. In situ hybridization was performed with a riboprobe generated from a laminin receptor complementary DNA. Laminin receptor mRNA was expressed primarily in the less differentiated cells in normal squamous tissues and in a spectrum of squamous neoplasms. There was no net induction of mRNA per cell in intraepithelial or invasive squamous neoplasms relative to normal tissue. In contrast, laminin receptor mRNA was not expressed at a detectable level in normal glands of the uterine cervix but was dramatically induced in morphologically abnormal, human papillomavirus-positive glands, irrespective of the genotype of human papillomaviruses present. The induction occurred before any evidence of invasion, and there was no further increase during the transition from adenocarcinoma in situ to invasive carcinoma. We conclude that induction of high-affinity laminin receptor gene expression is associated with the development of malignancies of cervical glandular epithelia, but the increased expression appears to correlate with the proliferative rather than the invasive properties of these cells.


Molecular characterization of areas with low grade tumor or satellitosis in human malignant astrocytomas.

Malignant astrocytomas often display histopathological heterogeneity. In the present study, we have molecularly characterized different areas within 4 such tumors to determine whether the tissue heterogeneity can be explained by differences in DNA constitution. Two tumors contained low grade areas, and the other 2 had areas with satellitosis. The tumors were examined for loss of heterozygosity with markers from chromosomes 9p, 10, and 17p and for amplification of the epidermal growth factor receptor gene. In each case, the high grade portion of the tumor displayed at least one of these structural alterations. However, identical alterations were found in the associated low grade or satellitosis areas of each tumor. Our data suggest that: (a) genetic alterations associated with tumor progression already occur in histopathologically low grade areas of high grade astrocytoma; (b) satellitosis associated with a high grade astrocytoma has to be considered as part of that tumor; and (c) tissue heterogeneity within a high grade astrocytoma is not a consequence of differences in DNA constitution at the loci that were examined.


Tumorigenic transformation and neoplastic progression of human uroepithelial cells after exposure in vitro to 4-aminobiphenyl or its metabolites.

A multistep in vitro/in vivo transformation system was used to test the transforming effect(s) of the human bladder procarcinogen 4-amino-biphenyl (ABP) and two putative proximate carcinogenic metabolites, N-hydroxy-4-aminobiphenyl (N-OH-ABP) and N-hydroxy-4-acetylamino-biphenyl (N-OH-AABP), on a clonally derived nontumorigenic SV40-immortalized human uroepithelial cell line, SV-HUC. SV-HUC were exposed in vitro to concentrations of ABP, N-OH-ABP, or N-OH-AABP that caused a range of cytotoxicity from 5 to 76%. Tumorigenic transformation of SV-HUC, as assessed by the ability of the exposed cells to form carcinomas when inoculated s.c. into athymic nude mice, was achieved after a single exposure to ABP, N-OH-ABP, or N-OH-AABP. In the tumorigenic transformation experiments, 28 of 45 mice representing all 15 carcinogen-exposed observation groups formed carcinomas, whereas none of 9 mice from control groups formed carcinomas (P = 0.001). Neoplastic progression of a low grade regressive squamous cell carcinoma, MC-T11, was also achieved in this system after in vitro exposure to ABP, N-OH-ABP, or N-OH-AABP. In these progression experiments, 11 of 33 mice representing 7 of 12 carcinogen-exposed observation groups formed persistent, high grade nongressing tumors, while only 1 of 19 untreated MC-T11 controls spontaneously progressed on reinoculation (P = 0.022). Forty independent carcinomas generated in athymic nude mice recapitulated diverse cancer phenotypes (including different growth kinetics and histopathological subtypes and grades) represented in clinical bladder cancers. These results demonstrate for the first time the transforming effects of the potent human carcinogen ABP and two of its proximate N-hydroxy metabolites on a prime human target cell type, HUC.


Induction of thioguanine-resistant mutations in human uroepithelial cells by 4-aminobiphenyl and its N-hydroxy derivatives.

The mutagenic potentials of the human bladder carcinogen 4-amino-biphenyl (ABP) and three of its proximate carcinogenic metabolites, N-hydroxy-4-aminobiphenyl (N-OH-ABP), N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) and N-acetoxy-4-acetylaminobiphenyl (N-OAc-AABP) were tested on a prime human target cell type for carcinogenesis, human uroepithelial cells (HUC). SV-HUC (PC), a near diploid, clonally derived, nontumorigenic SV40-immortalized human uroepithelial cell line that is transformable to tumorigenicity after exposure to ABP and its metabolites, was used for quantitative mutation assays. The end point used was the induction of mutations in the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus, selected using 6-thioguanine resistance (TGr). A single, 24-h exposure of SV-HUC to ABP, N-OH-ABP, N-OH-AABP, or N-OAc-AABP caused a statistically significant, dose-dependent increase in mutation frequency resulting in a 2-30-fold increase in the number of TGr mutants in carcinogen-exposed groups compared to untreated controls. These chemicals were similarly mutagenic towards MC-T11, an SV-HUC-derived low grade tumor cell line that was also shown to be responsive to transformation (in a separate study) by ABP, N-OH-ABP, or N-OH-AABP as judged by the generation of higher grade tumors. In contrast, the mutagenic potencies of ABP and N-OH-ABP were lower when tested on a subclone of SV-HUC (BC) that is refractory to transformation by these chemicals. Thus, these data support a model of transformation in which ABP as well as its metabolites contribute to tumorigenic transformation and neoplastic progression of HUC by inducing mutations in susceptible target cell genes.


Loss of 3p13----p21.2 in tumorigenic reversion of a hybrid between isogeneic nontumorigenic and tumorigenic human uroepithelial cells.

Previous studies in our laboratory showed nonrandom losses of chromosome 3p in association with tumorigenic transformation of SV40-immortalized human uroepithelial cells (HUC) to high grade cancers. To test the hypothesis that genes on 3p suppress HUC tumorigenesis, somatic cell hybrids were formed between nontumorigenic SV40-immortalized HUC and an isogeneic derivative transitional cell carcinoma line, MC-T16, that lost 3p on initial transformation. All hybrids were initially tumorigenically suppressed and reversion was always associated with genetic losses, including losses of 3p (Klingelhutz et al., Somatic Cell Mol. Genet., 17: 551-565, 1991). In this paper, we report that the smallest 3p region lost in a tumorigenic hybrid revertant (THR-X) in this system was an unusual interstitial deletion of 3p13----p21.2. Restriction fragment length polymorphism analysis confirmed this loss by showing that THR-X was reduced to homozygosity for D3S30, a 3p13 probe, but remained heterozygous for the distal 3p21.3 probe, D3F15S2. These data, along with our previous report identifying loss of 3p13----p14.2 as the smallest 3p region deleted in association with SV40-immortalized HUC tumorigenic transformation (Klingelhutz et al., Genes Chromosomes Cancer, 3: 346-357, 1991), provide compelling new evidence for a bladder cancer suppressor gene in the 3p13----p21.2 region.


p53 mutations cluster at codon 249 in hepatitis B virus-positive hepatocellular carcinomas from China.

DNA samples from 36 hepatocellular carcinoma (HCC) patients from China were screened for a specific mutation affecting codon 249 of the p53 gene, recently identified as a hotspot mutation in some HCCs. We detected the tumor-specific p53 codon 249 mutation in 21 (58%) of 36 HCCs examined. Thirteen patients with the specific codon 249 mutation had lost the remaining allele of p53, whereas the remaining eight patients appeared to have retained both copies of the gene. These results suggest that alterations of p53 may be important events in the genesis of HCCs and that point mutation may precede allele loss.


Na+/Ca2+ exchange in plasma membrane vesicles from a glucose-responsive insulinoma.

Plasma membrane vesicles from a glucose-responsive insulinoma exhibited properties consistent with the presence of a membrane Na+/Ca2+ exchange. The exchange was rapid, reversible, and was dependent on the external Ca2+ concentration (Km = 4.1 +/- 1.1 microM). External Na+ inhibited the uptake in a dose-dependent manner (IC50 = 15 mM). Dissipation of the Na+ gradient by 10 microM monensin decreased Na+/Ca2+ exchange from 0.74 +/- 0.17 nmoles/mg protein/s to 0.11 +/- 0.05 nmoles/mg protein/s. Exchange was not influenced by veratridine, tetrodotoxin and ouabain, or by modifiers of cAMP. No effect was seen using the calcium channel blockers, nitrendipine or nifedipine. Glucose had no direct effect on Na+/Ca2+ exchange, while glyceraldehyde, glyceraldehyde-3-phosphate and dihydroxyacetone inhibited the exchange. Na+ induced efflux of calcium was seen in Ca2+ loaded vesicles and was half maximal at [Na+] of 11.1 +/- 0.75 mM. Ca2+ efflux was dependent on [Na+], with a Hill coefficient of 2.7 +/- 0.07 indicating that activation of Ca2+ release involves a minimum of three sites. The electrogenicity of this exchange was demonstrated using the lipophilic cation tetraphenylphosphonium [( 3H]-TPP), a membrane potential sensitive probe. [3H]-TPP uptake increased transiently during Na+/Ca2+ exchange indicating that the exchange generated a membrane potential. These results show that Na+/Ca2+ exchange operates in the beta cell and may be an important regulator of intracellular free Ca2+ concentrations.


Calcium pump isoforms: diversity, selectivity and plasticity. Review article.

Ca2+ pumps are essential for removing cytosolic Ca2+ either across the plasma membrane (PM) or into internal organelles such as the sarcoplasmic reticulum (SR). Four genes (PMCA1, PMCA2, PMCA3 and PMCA4) have been reported to encode the PM Ca2+ pumps and three (SERCA1, SERCA2 and SERCA3) to encode the SR Ca2+ pumps. The PM Ca2+ pumps are stimulated by calmodulin, the SR Ca2+ pumps encoded by SERCA1 and SERCA2 are stimulated by phospholamban while the product of SERCA3 may be regulated directly by cAMP-dependent protein kinase. Alternative splicing of the primary transcripts of several of these genes has been reported to occur in a tissue selective manner and for others to alter during ontogeny. For the PM Ca2+ pump, alternative RNA splicing may result in isoforms with altered cyclic nucleotide dependent protein kinase sensitivity. The diversity in distribution of Ca2+ pump isoforms and their regulatory factors when coupled with different Ca2+ entry mechanisms allows for tissue selectivity and plasticity in stimulus-response coupling. The roles of various Ca2+ pump isoforms, the rationale behind their tissue selective expression and the plasticity in this expression are among the new challenges to researchers in this field.


Evidence for a gelsolin-rich, labile F-actin pool in human polymorphonuclear leukocytes.

Filamentous (F) actin is a major cytoskeletal element in polymorphonuclear leukocytes (PMNs) and other non-muscle cells. Exposure of PMNs to agonists causes polymerization of monomeric (G) actin to F-actin and activates motile responses. In vitro, all purified F-actin is identical. However, in vivo, the presence of multiple, diverse actin regulatory and binding proteins suggests that all F-actin within cells may not be identical. Typically, F-actin in cells is measured by either NBDphallacidin binding or as cytoskeletal associated actin in Triton-extracted cells. To determine whether the two measures of F-actin in PMNs, NBDphallacidin binding and cytoskeletal associated actin, are equivalent, a qualitative and quantitative comparison of the F-actin in basal, non-adherent endotoxin-free PMNs measured by both techniques was performed. F-actin as NBDphallacidin binding and cytoskeletal associated actin was measured in cells fixed with formaldehyde prior to cell lysis and fluorescent staining (PreFix), or in cells lysed with Triton prior to fixation (PostFix). By both techniques, F-actin in PreFix cells is higher than in PostFix cells (54.25 +/- 3.77 vs. 23.5 +/- 3.7 measured as mean fluorescent channel by NBDphallacidin binding and 70.3 +/- 3.5% vs. 47.2 +/- 3.6% of total cellular actin measured as cytoskeletal associated actin). These results show that in PMNs, Triton exposure releases a labile F-actin pool from basal cells while a stable F-actin pool is resistant to Triton exposure. Further characterizations of the distinct labile and stable F-actin pools utilizing NBDphallacidin binding, ultracentrifugation, and electron microscopy demonstrate the actin released with the labile pool is lost as filament. The subcellular localization of F-actin in the two pools is documented by fluorescent microscopy, while the distribution of the actin regulatory protein gelsolin is characterized by immunoblots with anti-gelsolin. Our studies show that at least two distinct F-actin pools coexist in endotoxin-free, basal PMNs in suspension: 1) a stable F-actin pool which is a minority of total cellular F-actin, Triton insoluble, resistant to depolymerization at 4 degrees C, gelsolin-poor, and localized to submembranous areas of the cell; and 2) a labile F-actin pool which is the majority of total cellular F-actin, Triton soluble, depolymerizes at 4 degrees C, is gelsolin-rich, and distributed diffusely throughout the cell. The results suggest that the two pools may subserve unique cytoskeletal functions within PMNs, and should be carefully considered in efforts to elucidate the mechanisms which regulate actin polymerization and depolymerization in non-muscle cells.


Increased calmodulin affects cell morphology and mRNA levels of cytoskeletal protein genes.

We have previously described stable mouse C127 cell lines in which a CaM mini-gene has been expressed in a bovine papilloma virus-based expression vector (Rasmussen and Means: EMBO J. 6:3961-3968, 1987). Elevation of CaM to levels five-fold higher than in control cells caused an acceleration in cell cycle progression by reducing the length of the G1 period. When these cell lines were originally isolated it was observed that cells in which CaM levels were increased had a flattened morphology. In this study we have examined the localization of actin, vimentin, and tubulin in these cells as compared to the BPV-transformed control cell line in order to determine if changes in shape were accompanied by differences in the cytoskeletal organization. Cell-cycle-dependent changes in the levels of mRNAs for histone H4, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, vimentin, and beta-tubulin have also been examined. Our results indicate that increased CaM causes differences in the organization of microfilaments, intermediate filaments, and microtubules and that these changes are accompanied by selective differences in the cell-cycle-dependent expression of some mRNAs. Elevated CaM was also correlated with a reduced stability of beta-tubulin mRNA. These studies indicate that CaM has pleiotropic effects on cell function and suggest that stable cell lines with altered CaM levels may provide a useful model system for understanding the molecular basis of CaM-dependent regulation of cellular processes.


Enhancement of spermidine/spermine N1-acetyltransferase activity by treatment with lithium chloride in Ehrlich ascites tumor cells.

The activity of spermidine/spermine N1-acetyltransferase (SAT) was enhanced in Ehrlich ascites tumor cells by the addition of lithium chloride. Na+ did not affect the enzyme activity. Total RNA was isolated from cells treated with LiCl and the relative abundance of the SAT mRNA was measured by Northern blot analysis. The levels in cells treated with LiCl were comparable to those in control cells. In the treated cells, the biological half-life of SAT was approximately 20 min, which was the same as for control cells. When LiCl and H-7, a protein kinase inhibitor, were added simultaneously to culture, the elevation caused by LiCl of SAT activity was reduced. LiCl did not cause maximum enhancement of the enzyme in cells treated beforehand with a higher concentration of TPA. These results suggest that treatment of Ehrlich ascites tumor cells with LiCl enhanced SAT activity during translation, not during transcription or after translation and that the enhancement of SAT by LiCl is probably mediated by protein kinase C.


Hepatocyte cytotoxicity induced by various hepatotoxins mediated by cytochrome P-450IIE1: protection with diethyldithiocarbamate administration.

The objective of this study was to determine whether the thiol drug, diethyldithiocarbamate (DEDC) and its two metabolites, disulfiram (DS) and carbon disulfide (CS2) could be used as inhibitors of cytochrome P-450IIE1 to protect hepatocytes from cytotoxic xenobiotics. (1) Hepatocytes isolated from rats following pyrazole administration to induce cytochrome P-450IIE1 were much more susceptible to carbon tetrachloride (CCl4) and dimethylnitrosamine (DMN) than hepatocytes from untreated rats. Microsomes isolated from P-450IIE1-induced liver were also much more effective at catalysing a NADPH-dependent metabolism of CCl4 and DMN. The activities of aniline hydroxylase and p-nitroanisole-O-demethylase increased whereas ethoxyresorufin-O-dealkylase activity was much less induced and pentoxyresorufin-O-dealkylase activity was decreased. The P-450IIE1 antibody markedly inhibited the NADPH-dependent metabolism of these compounds indicating that IIE1 is a major catalyst of the microsomal metabolism of CCl4 and DMN. (2) Hepatocytes isolated from rats treated with DEDC or its metabolites, DS and CS2, on the other hand, were resistant to CCl4 and DMN. Microsomes isolated from the liver of animals treated with DEDC or DS or CS2 were also much less effective at catalysing the NADPH-dependent metabolism of the above compounds. DEDC markedly decreased the activities of aniline hydroxylase, p-nitroanisole-O-demethylase and pentoxyresorufin-O-dealkylase but had no effect on ethoxyresorufin-O-dealkylase activity. (3) Hepatocytes isolated from pyrazole-treated rats were also more susceptible to bromobenzene (BB) and naphthalene-induced cytotoxicity than hepatocytes from untreated rats. Furthermore, DEDC or CS2 administration beforehand significantly protected hepatocytes against both xenobiotics. (4) By contrast, hepatocytes isolated from P-450IIE1 induced rats were not more susceptible to lactonitrile or cyclophosphamide. Instead, cyclophosphamide was activated by phenobarbital-induced P-450 isozymes whereas lactonitrile was activated by alcohol dehydrogenase. Hepatocytes isolated from DEDC-treated rats were also resistant to cyclophosphamide but not lactonitrile. (5) The above results suggest that P-450IIE1 catalyses the cytotoxic activation of CCl4, DMN, BB and naphthalene but not of lactonitrile or cyclophosphamide. Furthermore, the administration of DEDC and its metabolites, disulfiram or CS2, inactivates P-450IIE1 so that the hepatocytes become resistant to these hepatotoxins.


Improving accuracy of glucose oxidase procedure for glucose determinations on discrete analyzers.

We describe a rapid kinetic glucose oxidase (EC 1.1.3.4) procedure for quantifying glucose. Glucose oxidase concentration was reduced from the more usual 20 kU/L to 4 kU/L, and pH was reduced from 7.0 to 6.6. Potassium ferrocyanide (20 mumol/L) and ascorbate oxidase (1 kU/L) were incorporated in the procedure. The assay results vary linearly with glucose concentration from 0 to 50 mmol/L and are unaffected by bilirubin concentrations less than or equal to 600 mumol/L, hemoglobin less than or equal to 12 g/L, Intralipid less than or equal to 4 g/L, urate less than or equal to 1 mmol/L, and ascorbate less than or equal to 2.0 mmol/L. The assay is readily adaptable to most open-system analyzers.


Strategies and difficulties in dietary intervention in myocardial infarction patients.

Randomized controlled trials along with 'within group' studies of patients with symptomatic coronary artery disease suggest that dietary and other lifestyle changes can halt progression of coronary atherosclerosis, induce regression of pre-existing severe lesions, and reduce the severity or frequency of angina. Varying combinations have been tested, including restriction of dietary total, saturated and polyunsaturated fat using lean meat or vegetarian diets, fish oil supplements, smoking cessation, stress management, and exercise training. The relative importance of each of these remains unclear. In patients with recent myocardial infarction high fish diets appeared effective in reducing both ischaemic heart deaths and total death rates over two years, whereas modest changes in dietary saturated fats or fibre had no influence on outcome. These results suggest that a far more active dietary approach is needed for patients with symptomatic coronary disease. Further research is required into the possible complementary role of dietary measures and drug treatment for reversing the disease process and improving outcome, using new techniques for achieving behavioural change.


Modulation of human IgE synthesis by transforming growth factor-beta.

Exogenous transforming growth factor-beta 2 (TGF-beta 2) markedly inhibits the interleukin 4 (IL4)-stimulated synthesis of human IgE in three models where the B cell co-stimulation signals are contact dependent. This concerns T cell-dependent IgE production by (i) unfractionated peripheral blood mononuclear cells (PMBC) cultured with IL4 and (ii) highly purified B cells cocultured with irradiated EL4 thymoma cells in the presence of IL4 and phorbol myristate acetate, as well as monocyte-dependent IgE production by rigorously T cell-depleted PBMC cultured with IL4 and hydrocortisone. The suppression is not isotype specific. TGF-beta exerts its effect by inhibiting the proliferation of B cells and perhaps also the differentiation of proliferating B cells. However, at a later stage of differentiation, IgE B cells are refractory to the inhibitory effect of TGF-beta, as shown by the slight but significant increase of the spontaneous secretion of IgE by PBMC of atopic patients. This enhancement is due to the suppression of endogenous interferon-gamma production. Most interestingly the synthesis of IgE by highly purified B cells costimulated with IL4 and Epstein-Barr virus is unaffected by TGF-beta. It is concluded that TGF-beta mainly acts by inhibiting IL4-supported B cell proliferation; however, its effects depend upon the B cell costimulation signals that are required together with IL4 for the induction of IgE synthesis.


Renal haemodynamic, hormonal and excretory effects of low-dose atrial natriuretic factor infusion in renal transplant recipients.

1. In experimental studies, activation of renal sympathetic nerves attenuates the natriuretic response to atrial natriuretic factor. We therefore investigated the response to low-dose infusion of atrial natriuretic factor in renal transplant recipients. 2. Eight male cyclosporin-treated renal transplant recipients received human-alpha atrial natriuretic factor (1-28) at a dose of 1.2 pmol min-1 kg-1 or placebo for 2 h in a placebo-controlled, randomized, cross-over study. The plasma atrial natriuretic factor concentration rose from 18.5 to 49.2 pmol/l in association with an immediate natriuresis (a rise of 49.1 mumol/min in the first 30 min, P less than 0.05; peaking at a 61% increase from baseline, P less than 0.01), diuresis (from 3.37 to 7.46 ml/min) and a threefold rise in urinary cyclic GMP excretion. 3. In response to infusion of atrial natriuretic factor, the packed cell volume rose by 4.2% (P less than 0.001) and the filtration fraction by 5% (from 22 to 27%, P less than 0.05), but there was no significant change in renal plasma flow, glomerular filtration rate or mean arterial blood pressure. Likewise, the plasma catecholamine concentrations, plasma renin activity and serum erythropoietin concentration remained unchanged. 4. The sensitivity of the renal allograft to plasma atrial natriuretic factor concentrations in the high physiological range suggests a role for endogenous atrial natriuretic factor in the modulation of graft function. Furthermore, the immediate natriuretic response to atrial natriuretic factor in the effectively denervated transplant kidney, in contrast to the delayed response seen in normal subjects, may imply that sympathetic nerves have an inhibitory effect on the renal response to atrial natriuretic factor in normal man.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of haemodialysis on plasma levels of brain natriuretic peptide in patients with chronic renal failure.

1. Plasma human brain natriuretic peptide-like immunoreactivity (hBNP-li) was measured in ten patients with chronic renal failure before and after 4 h of haemodialysis. 2. Plasma hBNP-li was elevated in all patients before dialysis (mean +/- SEM 21.0 +/- 3.8 pmol/l) compared with healthy control subjects (1.3 +/- 0.2 pmol/l, n = 11), but showed considerable inter-patient variability. Before dialysis plasma hBNP-li bore no relationship to the serum creatinine level or to the mean blood pressure. 3. Plasma hBNP-li fell significantly (P = 0.04) during 4 h of haemodialysis. The fall in plasma hBNP-li correlated significantly with the degree of postural blood pressure drop (r2 = 0.44, P = 0.05) and with the fall in body weight (r2 = 0.64, P less than 0.01) after haemodialysis. In all patients, plasma hBNP-li at the end of treatment remained above that in healthy subjects. 4. There was no significant correlation between the fall in plasma hBNP-li and the fall in serum creatinine level, and between the fall in plasma hBNP-li and the fall in supine systolic or diastolic blood pressure, during haemodialysis. 5. We have shown that plasma hBNP-li is elevated in patients with chronic renal failure and is decreased during haemodialysis. The fact that the plasma hBNP-li was not reduced to normal by haemodialysis despite restoration to normovolaemia gives tentative support to the view that, in addition to hypervolaemia, another factor may also be responsible for the elevated plasma hBNP-li seen in these patients.


Coronary haemodynamic effects of angiotensin II in mild essential hypertension in man.

1. The present investigation was carried out to elucidate the possible role of the renin-angiotensin system in modulating coronary vasomotor responses in eight patients with uncomplicated mild essential hypertension with no electrocardiographic-echocardiographic evidence of left ventricular hypertrophy. 2. Systemic and coronary haemodynamics were monitored at baseline and during intravenous infusion of angiotensin II at a subpressor dose (3 ng min-1 kg-1 for 15 min) and at a pressor dose (13 ng min-1 kg-1 for 15 min) both at rest and during handgrip exercise. Infusion of the subpressor dose of angiotensin II decreased coronary sinus blood flow at rest (207 +/- 10 versus 182 +/- 9 ml/min, P less than 0.05) without a significant change in mean arterial pressure, heart rate or mean right atrial pressure. The performance of handgrip at baseline and during infusion of the subpressor dose of angiotensin II resulted in 55% (321 +/- 13 versus 207 +/- 10 ml/min) and 44% (263 +/- 16 versus 182 +/- 9 ml/min) increases in coronary sinus blood flow, respectively, in response to comparable increments in the rate-pressure product. At rest, infusion of the pressor dose of angiotensin II increased both coronary sinus blood flow (235 +/- 11 versus 207 +/- 10 ml/min, P less than 0.01) and the rate-pressure product (134 +/- 5 versus 111 +/- 8 mmHg beats/min, P less than 0.01). The increase in coronary sinus blood flow during isometric exercise was less than control (309 +/- 18 versus 321 +/- 13 ml/min, P less than 0.01). 3. It is thus concluded that (1) the opposite effects of angiotensin II on coronary blood flow are dose-dependent, and that (2) angiotensin II competes with the ability of the coronary arteries to dilate during handgrip exercise.


Mutual interactions of respiratory sinus arrhythmia and the carotid baroreceptor-heart rate reflex.

1. In six healthy subjects the amplitude and phase of respiratory sinus arrhythmia were determined at five different respiratory cycle lengths ranging from 3 to 9.5 s. 2. At each respiratory cycle length the carotid baroreceptor-heart rate reflex response was determined by cyclical neck suction at -40 mmHg at five different cycle lengths covering the same range of 3-9.5 s. 3. The application of cyclical neck suction increased the amplitude of respiratory sinus arrhythmia in all but the longest respiratory cycle lengths. 4. With increasing respiratory cycle length the amplitude of sinus arrhythmia increased, and R-R intervals were at their longest at an earlier phase of the respiratory cycle. Similarly, with increasing suction cycle length the amplitude of the cardiac interval response increased and the phase angle decreased. 5. The cardiac interval responses to respiration and to neck suction at different frequencies were independent of each other, the heart rate at any moment resulting from the algebraic summation of the two responses.


Pressor response to a postural change in cirrhosis: an experimental study in the CCl4-treated rat.

1. Systemic hypotension, blunted cardiovascular responsiveness to noradrenaline and an abnormal hypertensive pressor response to a postural change have been described in cirrhotic patients. 2. We have examined the role of blunted responsiveness in these abnormalities by studying basal arterial blood pressure and its response to a postural change (vertical head-up 90 degrees tilting) in conscious and pithed CCl4-treated (cirrhotic) rats, as well as assessing the pressor response to noradrenaline in vivo and the vascular contractile response to noradrenaline in vitro. 3. A diminished hypotensive response to a change in posture was found in pre-cirrhotic portal hypertensive rats, whereas an inverted hypertensive pressor response in the face of systemic hypotension occurred in the cirrhotic rats with portal hypertension. 4. The inverted pressor response was abolished in the pithed portal hypertensive cirrhotic rats. 5. The pressor response to noradrenaline in vivo in conscious cirrhotic rats and the vascular contractile responsiveness to noradrenaline in vitro were intact. 6. We conclude that blunted responsiveness to noradrenaline is not a contributory factor to the development of systemic hypotension or the inverted pressor response to a change in posture in cirrhosis.


Comparative study of pressor and heart rate responses to angiotensin II and noradrenaline in pregnant and non-pregnant women.

1. Twenty-eight healthy non-pregnant women and 28 women in the first or second trimester of pregnancy were studied. They were given an incremental intravenous infusion of either noradrenaline or angiotensin II. Pressor and heart rate responses were documented. 2. Dose-pressor response curves were constructed for the two agents in pregnant and non-pregnant women (n = 14 in each group). The regression parameters of slope and intercept were calculated, and were used to derive the variables of dose required to elicit a 10 mmHg rise in systolic or diastolic blood pressure. 3. The pressor response to angiotensin II was diminished in pregnancy, with approximately twice the dose being required to raise the systolic or diastolic arterial blood pressure as in non-pregnant subjects. 4. The systolic pressor response to noradrenaline was slightly diminished in pregnancy, but the diastolic pressor response was unchanged. There were no significant differences between the doses of noradrenaline required to elicit a 10 mmHg rise in systolic or diastolic arterial blood pressure in pregnant or non-pregnant subjects. 5. There was a diminution in the bradycardia evoked in response to both hormones in pregnancy. 6. We conclude that the well-documented pressor insensitivity to angiotensin II during pregnancy is a specific phenomenon, not a manifestation of a generalized reduction in vascular reactivity.


Location of enkephalinase and functional effects of [Leu5]enkephalin and inhibition of enkephalinase in the feline main pancreatic and bile duct sphincters.

1. Morphological studies have demonstrated enkephalinergic nerve fibres in proximity to the sphincter of Oddi, and opiates are known to contract this sphincter. In this study, the flow resistances in the common bile duct and main pancreatic duct sphincters were studied simultaneously in anaesthetized cats using a perfusion technique. 2. Naloxone did not affect the activity of these sphincters under basal conditions, indicating that there is no basal enkephalinergic tone. 3. The response to [Leu5]enkephalin (0.015-15 micrograms/kg), morphine (1 mg/kg) and ketamine (10 mg/kg) was a naloxone-sensitive increased activity in the sphincters with a raised frequency of phasic contractions. The threshold dose for an effect of [Leu5]enkephalin on the sphincter of Oddi was 0.015 microgram/kg and a maximal response was observed at 0.75 microgram/kg. There were no differences in the response of the main pancreatic duct sphincter and the bile duct sphincter to the different drugs. 4. Immunoautoradiographic studies demonstrated enkephalinase in the sphincter++ of Oddi. 5. Acetorphan (3 mg/kg intravenously), which inhibits endogenous enkephalinase both in the peripheral and the central nervous system when administered parenterally, caused a naloxone-sensitive contraction, whereas thiorphan (3-20 mg/kg), an enkephalinase inhibitor that does not easily penetrate the blood-brain barrier, had no effect on the sphincter of Oddi. 6. These results show that endogenous and exogenous opiates influence the function of the feline sphincter of Oddi and that enkephalins may be involved in the physiological control of this sphincter, although not under basal conditions.


Isolation and purification of human biliary vesicles with potent cholesterol-nucleation-promoting activity.

1. Cholesterol nucleation is a critical step in the formation of cholesterol gallstones. This nucleation takes place after aggregation and fusion of cholesterol-rich biliary vesicles, a process probably modulated by biliary proteins. The present study was conducted to identify specific proteins associated with native cholesterol-rich biliary vesicles and to explore their effect on the cholesterol-nucleation time of supersaturated artificial bile. 2. Hepatic bile was obtained from six patients with cholesterol gallstone disease. Biliary vesicles were isolated by ultracentrifugation and were purified by gel filtration chromatography. A small amount of protein (less than 1% by weight) remained associated with the purified cholesterol-rich biliary vesicles. The electrophoretic profile of these proteins was remarkably similar in all six patients, showing the presence of at least six polypeptides (of molecular mass from 52 to 200 kDa), five of them having carbohydrate residues (except the 52 kDa one). The effect of reconstituted biliary vesicle solutions, containing their specific vesicular proteins, on cholesterol-nucleation time was studied by mixing the vesicle solution with artificial supersaturated bile. A potent cholesterol-pronucleating activity, reflected in a 20-70% reduction in nucleation time, was present in the biliary vesicle solutions compared with control solutions having a similar lipid composition. The pronucleating activity disappeared on heating and was not detected in the micellar fraction containing the major proportion of biliary proteins. 3. These results indicate that cholesterol-rich biliary vesicles containing a unique and defined glycoprotein profile can be isolated and purified from human hepatic bile. The potent cholesterol-pronucleating activity of the biliary vesicles from patients with gallstones was unrelated to their lipid composition or cholesterol content.(ABSTRACT TRUNCATED AT 250 WORDS)


Differences in ouabain-induced natriuresis between isolated kidneys of Milan hypertensive and normotensive rats.

1. Several observations support the hypothesis that in rats of the Milan hypertensive strain elevated levels of a circulating ouabain-like factor might normalize the elevated Na+ reabsorption, but, on the other hand, might contribute to the development of hypertension. 2. As the receptor occupancy of this endogenous factor seems to be reversible, the aim of our study was to test, in vitro, the hypothesis of its presence in isolated kidneys from Milan hypertensive rats by studying the response to exogenous ouabain before and after prolonged washing. 3. The kidneys were isolated from adult Milan hypertensive rats and from age-matched normotensive controls and ouabain was given at two different experimental time intervals: shortly (15 min) after washout or after a further 60 min of washout (75 min in total). Comparative experiments with the diuretic hydrochlorothiazide were performed using the same protocol. 4. Ouabain given after 15 min of perfusion caused an increase in renal vascular resistance, diuresis and natriuresis; these haemodynamic and tubular responses were similar in kidneys from both Milan hypertensive and Milan normotensive rats. If given after the washout period, ouabain caused a comparable increase in renal vascular resistance, but a significantly greater natriuresis in kidneys from Milan hypertensive rats as compared with kidneys from Milan normotensive rats. On the other hand, hydrochlorothiazide caused similar natriuresis in kidneys from both strains after washout. 5. These results support the hypothesis that a factor, capable of interacting with the ouabain receptor on the Na+/K(+)-ATPase of tubular cells, is present in the kidney of adult Milan hypertensive rats and that it can be removed by prolonged washout.


Adaptation to a diet low in protein: effect of complex carbohydrate upon urea kinetics in normal man.

1. Urea kinetics were measured by using prime/intermittent oral doses of [15N15N]urea in five healthy men taking formula diets adequate in energy and containing either 70 or 35 g of protein/day. In some studies the low-protein diet was supplemented with non-starch polysaccharides in the form of ispaghula husk or ripe bananas. 2. On the 70 g of protein/day diet urea production was 132% of intake. Only 54% of the urea produced was excreted in the urine with 46% being salvaged in the colon; 90% of the salvaged nitrogen was retained in the metabolic nitrogen pool. 3. On the 35 g of protein/day diet the small decrease in urea production rate compared with that on the 70 g of protein/day diet was not significant, but only 36% of the urea produced was excreted in urine, with the majority, 64%, being salvaged. 4. The extent of urea-nitrogen salvaging on the 35 g of protein/day diet was similar in magnitude to the decrease in nitrogen intake, with the effect that the sum of intake and salvaged nitrogen did not differ between the 35 and the 70 g of protein/day diets. This implies that quantitative control is exerted over the rate at which urea nitrogen is salvaged. 5. The addition of non-starch polysaccharides to the 35 g of protein/day diet had a demonstrable effect upon faecal weight and composition, but did not exert any significant influence upon urea kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of early amino acid administration during total parenteral nutrition on protein metabolism in pre-term infants.

1. We investigated the effects of starting amino acid administration on post-natal day 2 on protein turnover and nitrogen balance in appropriate-for-gestational-age, very-low-birth-weight infants. Eighteen infants were divided into two groups. Group A received from day 2 onwards an amino acid solution, whereas group B started on this solution after day 4. Both groups were exclusively parenterally fed, 200 kJ day-1 kg-1 on post-natal days 3 and 4. Group A (birth weight 1.5 +/- 0.3 kg) received 4.6 g of glucose, 1.9 g of fat and 2.3 g of amino acids day-1 kg-1 body weight. Group B (birth weight 1.4 +/- 0.2 kg) received 7.0 g of glucose and 1.9 g of fat day-1 kg-1 body weight. 2. At post-natal day 3, a primed constant infusion of 3 mg of [15N]glycine day-1 kg-1 was given. Protein flux, protein synthesis and protein breakdown were calculated from the 15N enrichment in urinary ammonia. In five out of nine infants in group B no plateau of 15N enrichment in urinary urea could be detected, whereas in group A two out of nine infants did not reach a plateau. For this reason we did not use the end product urea for our calculations. 3. The administration of the amino acids resulted in a higher protein flux (6.9 +/- 1.5 g day-1 kg-1 versus 5.2 +/- 0.9 g day-1 kg-1) and a higher protein synthesis rate (6.0 +/- 1.4 g day-1 kg-1 versus 4.6 +/- 0.8 g day-1 kg-1) in group A.(ABSTRACT TRUNCATED AT 250 WORDS)


Acute administration of tumour necrosis factor-alpha or interleukin-1-alpha does not mimic the hypoketonaemia associated with sepsis and inflammatory stress in the rat.

1. Administration of tumour necrosis factor (cachectin) and of interleukin-1-alpha increased the plasma level of nonesterified fatty acids in fed rats, and in the case of interleukin-1-alpha the blood glycerol level was also increased, suggesting stimulation of adipose tissue lipolysis. There were parallel increases in the plasma level of triacylglycerols. Neither cytokine had significant effects on blood or liver total ketone body (acetoacetate plus 3-hydroxybutyrate) concentrations. 2. In starved rats, the higher plasma non-esterified fatty acid concentration was not increased further by the cytokines. The plasma triacylglycerol level was increased, although the absolute change was less than in fed rats. The ketonaemia associated with starvation tended to be increased by the cytokines, but this was only significant in the case of interleukin-1-alpha. Parallel changes occurred in hepatic ketone bodies. 3. It is concluded that tumour necrosis factor-alpha and interleukin-1-alpha are not responsible for the hypoketonaemia associated with sepsis or other inflammatory states.


Danazol induces resistance to both insulin and glucagon in young women.

1. Danazol elevates plasma insulin, plasma glucagon and serum low-density lipoprotein concentrations and reduces the serum high-density lipoprotein concentration. 2. Associations between these disturbances were studied in 17 women receiving danazol therapy for endometriosis. Eleven women underwent intravenous glucose tolerance tests with measurement of plasma glucose, insulin, C-peptide and glucagon concentrations and modelling analysis of intravenous glucose tolerance test concentration profiles. Six women underwent glucagon sensitivity tests. Serum concentrations of lipids and lipoproteins were measured in all cases. 3. Danazol reduced the fasting plasma glucose and insulin concentrations, but markedly raised the fasting plasma glucagon concentration. The insulin and C-peptide responses to the intravenous glucose tolerance test were increased twofold and the net decrement in glucagon concentration was increased tenfold. The glucose response to the intravenous glucose tolerance test was unaffected. Insulin sensitivity was reduced by 55%. Both first-phase plasma insulin responsiveness and net first-phase pancreatic insulin secretion were increased; insulin half-life was prolonged. The glucose response to the glucagon sensitivity test was reduced on treatment. The calculated low-density lipoprotein cholesterol level rose by 20%, whereas high-density lipoprotein cholesterol level fell by 47%. None of these changes in serum lipoprotein levels correlated with changes in insulin metabolism. In general, metabolic changes normalized after 3 months. 4. Danazol increases the sensitivity of pancreatic insulin and glucagon secretion to glucose. Danazol-induced insulin and glucagon resistance could be due to receptor down-regulation resulting from hypersecretion of insulin and glucagon.


Effect of increased free fatty acid supply on glucose metabolism and skeletal muscle glycogen synthase activity in normal man.

1. Experimental elevation of plasma non-esterified fatty acid concentrations has been postulated to decrease insulin-stimulated glucose oxidation and storage rates. Possible mechanisms were examined by measuring skeletal muscle glycogen synthase activity and muscle glycogen content before and during hyperinsulinaemia while fasting plasma non-esterified fatty acid levels were maintained. 2. Fasting plasma non-esterified fatty acid levels were maintained in seven healthy male subjects by infusion of 20% (w/v) Intralipid (1 ml/min) for 120 min before and during a 240 min hyperinsulinaemic euglycaemic clamp (100 m-units h-1 kg-1) combined with indirect calorimetry. On the control day, 0.154 mol/l NaCl was infused. Vastus lateralis muscle biopsy was performed before and at the end of the insulin infusion. 3. On the Intralipid study day serum triacylglycerol (2.24 +/- 0.20 versus 0.67 +/- 0.10 mmol/l), plasma nonesterified fatty acid (395 +/- 13 versus 51 +/- 1 mumol/l), blood glycerol (152 +/- 2 versus 11 +/- 1 mumol/l) and blood 3-hydroxybutyrate clamp levels [mean (95% confidence interval)] [81 (64-104) versus 4 (3-5) mumol/l] were all significantly higher (all P less than 0.001) than on the control study day. Lipid oxidation rates were also elevated (1.07 +/- 0.07 versus 0.27 +/- 0.08 mg min-1 kg-1, P less than 0.001). During the clamp with Intralipid infusion, insulin-stimulated whole-body glucose disposal decreased by 28% (from 8.53 +/- 0.77 to 6.17 +/- 0.71 mg min-1 kg-1, P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)


Contractile properties and susceptibility to exercise-induced damage of normal and mdx mouse tibialis anterior muscle.

1. The functional properties of tibialis anterior muscles of normal adult (C57BL/10) and age-matched dystrophin-deficient (C57BL/10 mdx) mice have been investigated in situ. Comparisons were made between tibialis anterior muscle strength, rates of force development and relaxation, force-frequency responses and fatiguability. Subjecting mdx and C57 muscles to a regimen of eccentric exercise allowed the hypothesis to be tested that dystrophin-deficient muscles are more susceptible to exercise-induced muscle damage. 2. mdx muscles were, on average, 30% stronger than C57 muscles and almost 80% heavier, but both had similar muscle lengths. Thus, although mdx muscles were stronger in absolute terms, their estimated force per unit cross-sectional area was significantly less than that of C57 muscles. 3. The force-frequency relationships of C57 and mdx muscles differed in that whilst, at 40 Hz, the former developed 70% of the force developed at 100 Hz, the latter developed only 55% of the maximal force. Twitch force was normal in mdx muscles, but contraction time was shortened, and the consequent reduction in fusion frequency probably explains the force-frequency differences observed between the two groups. 4. mdx muscles were less fatiguable than normal muscles when stimulated repeatedly at a frequency of 40 Hz. It is possible that the lower relative force at 40 Hz in mdx muscles entailed a lower energy demand and thus a slower rate of fatigue than seen in normal muscles. 5. Eccentrically exercised C57 muscles showed a large loss of maximal force for up to 12 days after exercise. Maximal force loss occurred 3 days after exercise (55% of non-exercised tibialis anterior muscle), which also corresponded with the period of greatest fibre necrosis. C57 muscles showed a significantly reduced 40 Hz/100 Hz force-frequency ratio at 1 and 3 days after exercise. This was primarily due to a reduced twitch amplitude rather than to a change in the time course of the twitch. It is unlikely, therefore, that the altered contractile characteristics of mdx muscle were a result of the presence of damaged but otherwise normal fibres. 6. C57 and mdx tibialis anterior muscles displayed similar degrees of force loss after exercise. Furthermore, the rate of recovery after the nadir of force loss was very similar for the two groups. By 12 days after exercise, force recovered to 76% and 80% of control in C57 and mdx muscles, respectively. Our findings do not support the hypothesis that dystrophin-deficient muscle is more susceptible to exercise-induced muscle damage.


Granulocyte margination in the human lung and its response to strenuous exercise.

1. It is widely believed that the lung is an important site of granulocyte margination and releases most of the granulocytes of the peripheral neutrophilia of exercise. 2. We measured granulocyte margination in the lung in terms of the lung total blood granulocyte pool and the lung circulating granulocyte pool in eight patients without inflammatory disease or evidence of lung pathology by comparing the regional gamma-camera lung count rate of 111In-labelled granulocytes with that of 111In-labelled erythrocytes. According to the respective 111In activities in peripheral blood samples taken between 5 and 40 min after granulocyte injection, the lung marginating granulocyte pool was 0.78 (SEM 0.045) of the lung total blood granulocyte pool or 4.6 (0.92) of the lung circulating granulocyte pool 5 min after injection, decreasing to plateau values of 0.57 (0.053) and 1.53 (0.28) from 20 min after injection. This compared with corresponding whole-body ratios of about 0.6 and 1.5, respectively. 3. After 4 min of maximal exercise in four normal subjects given 111In-labelled granulocytes 60 min before exercise, the 111In-labelled granulocyte count rate over the lung increased to 1.23 (0.05) of the pre-exercise value with a time course that was essentially identical with the time course of the peripheral native neutrophilia. The spleen 111In signal decreased with the same time course, reaching a minimum of 0.63 (0.05) of the pre-exercise level at 5-10 min after the end of exercise.(ABSTRACT TRUNCATED AT 250 WORDS)


Lactic acidosis treated with continuous hemodiafiltration and regional citrate anticoagulation.

To evaluate the effectiveness of continuous arteriovenous hemodiafiltration (CAVHD) using citrate as the anticoagulant for the treatment of lactic acidosis in patients with renal failure. Case series with careful monitoring of the clinical course of patients being treated in a medical or surgical ICU. University hospital ICU. Two patients with lactic acidosis are described, along with our experience using CAVHD and citrate in other clinical settings. CAVHD was used to manage renal failure, while a continuous infusion of citrate was administered to maintain patency of the extracorporeal circuit. Total and ionized serum calcium concentrations and citrate concentrations were monitored. CAVHD with citrate as the anticoagulant proved to be a convenient means of managing vascular volume, serum electrolyte concentrations, acid-base balance, and replacement renal function requirements in the setting of severe lactic acidosis, oliguric renal failure, and hemorrhagic diathesis. CAVHD with citrate as the anticoagulant can be recommended as effective therapy for selected patients, but careful monitoring is needed to avoid serious complications.


Cystic mucinous adenocarcinoma of the lung. Two cases of cystic variant of mucus-producing lung adenocarcinoma.

Two previously unreported cases of mucus-producing lung adenocarcinoma are presented as uncommon tumors, which are clinicopathologically different from other histologic types of lung adenocarcinoma. The tumors, showing apparently rapid development on chest roentgenograms, were tightly packed with copious mucus and resembled cystic lesions. Because they contained very few cancer cells, and these were only at the periphery, it was impossible to diagnose malignant neoplasms preoperatively through cytologic examination. The present tumors, which we described as cystic mucinous adenocarcinoma, are considered to be a cystic variant of mucus-producing lung adenocarcinoma that expands grossly by storing mucus.


Effects of the addition of nedocromil sodium to maintenance bronchodilator therapy in the management of chronic asthma.

To assess the efficacy and safety of nedocromil sodium metered dose aerosol as an adjunct to sustained-released theophylline therapy in adult theophylline-dependent asthma patients and to examine the ability of nedocromil sodium to substitute for theophylline. Randomized double-blind placebo-controlled parallel group study. Two-week baseline, eight-week treatment period. Out-Patient Clinic. Sequential sample of 35 adult chronic asthmatic patients maintained on a regimen of sustained-release theophylline (dose range, 400 to 800 mg daily) and on-demand inhaled beta 2-bronchodilators. All patients completed the study. 2 x 2-mg nedocromil sodium metered dose aerosol twice daily or matching placebo randomly allocated after two-week baseline. Theophylline dose reduced by half or one third after four weeks of test treatments, then stopped for final two weeks. Use of inhaled beta 2-bronchodilators permitted throughout trial period. The following results were in favor (statistically significant findings, p less than 0.05) of nedocromil sodium compared with placebo: all diary card efficacy variables (nighttime asthma, morning tightness, daytime asthma, cough, twice daily peak expiratory flow [PEF], inhaled beta 2 use) during all periods of assessment (weeks 1 to 2, 3 to 4, 5 to 6, and 7 to 8) with the exception of cough and nighttime beta 2 use during weeks 1 to 2; patient and clinician opinion of treatment efficacy (end of weeks 4 and 8); ability to reduce the theophylline dose; clinician assessment of asthma severity at the end of the study, and clinic FEV1 at weeks 4, 5, 6, and 8. One placebo-treated patient reported transient moderately severe nausea and taste loss. No clinically significant changes were seen in the laboratory data. Nedocromil sodium, 4 mg twice daily, conferred significant benefit when added to sustained-release theophylline therapy. The results suggest that nedocromil sodium may permit a reduction in theophylline dosage and possibly substitute for theophylline in previously dependent patients.


Resection of pulmonary metastasis to the azygous lobe from a malignant fibrous histiocytoma.

A 55-year-old man developed a pulmonary metastasis to the azygous lobe from a malignant fibrous histiocytoma of the thigh. The azygous lobe was not identified at the initial resection. A simple technique for the identification and mobilization of the azygous lobe is presented. Preoperative identification of this anatomic variant may assist in resection of parenchymal neoplasms.


The effects of amylin on insulin secretion from Rin m5F cells and glycogen synthesis and lipogenesis in rat primary cultured hepatocytes.

The purpose of the study was to determine the physiological actions of amylin, a novel 37-amino acid peptide isolated from pancreatic islet amyloid deposits. Our results showed that an infusion of amylin reduced fasting plasma insulin levels and impaired glucose tolerance in mice. Amylin significantly reduced insulin secretion in rat insulinoma cell lines (Rin m5F cells) that were stimulated by either isoproterenol and forskolin, but it did not affect insulin secretion stimulated by isobutyl-methylxanthine (IBMX) or dibutyryl cyclic-adenosine monophosphate (db-cAMP). Amylin also reduced cAMP levels in Rin m5F cells in response to isoproterenol, but did not affect cAMP levels in cells pretreated with pertussis toxin. These results suggest that the reduction of cAMP by amylin may be mediated through pertussis toxin-sensitive Gi proteins. Amylin significantly reduced basal and insulin-stimulated glycogen synthesis in rat primary cultured hepatocytes. Amylin stimulated basal and insulin-stimulated lipogenesis in hepatocytes. Amylin did not affect DNA synthesis in hepatocytes. These results suggest that amylin conducts dispersion actions on in vivo glucose metabolism in rat, and in vitro insulin secretion from Rin m5F cells and metabolism in rat hepatocytes.


Nitrobenzo[a]pyrene-induced DNA amplification in SV40-transformed Chinese hamster embryo cells.

Nitrobenzo[a]pyrenes (NBaPs) are ubiquitous environmental pollutants that produce mutations in Salmonella typhimurium and Chinese hamster ovary cells. In this study, 1-, 3-, and 6-NBaP induced amplification of SV40 DNA sequences in an SV40-transformed Chinese hamster embryo cell line which is sensitive to DNA amplification by various known carcinogens. Of the three isomers, 3-NBaP produced the highest level of gene amplification, which was 4.8 relative to untreated controls at a dose of 5 micrograms/ml. Considering the relationship between gene amplification and tumorigenesis, it seems prudent to carry out a more exhaustive analysis of the carcinogenic potential of these agents.


The axonally secreted cell adhesion molecule, axonin-1. Primary structure, immunoglobulin-like and fibronectin-type-III-like domains and glycosyl-phosphatidylinositol anchorage.

Axonin-1 is an axon-associated cell adhesion molecule (AxCAM) of the chicken, which promotes neurite outgrowth by interaction with the AxCAM L1(G4) of the neuritic membrane. Here we report the cloning and sequence determination of a cDNA encoding axonin-1. Peptides generated by enzymatic cleavage showed similarity to the AxCAM F11. Degenerated polymerase chain reaction (PCR) primers were designed and an axonin-1 fragment was amplified from mRNA of embryonic retina. Screening of a cDNA library from embryonic brain resulted in the isolation of a 4.0-kb cDNA insert with an open reading frame of 3108 nucleotides. The deduced polypeptide of 1036 amino acids includes a putative hydrophobic N-terminal signal sequence of 23 or 25 amino acids and a C-terminal hydrophobic sequence of 29 amino acids which is suggestive of sequences serving as signal for the attachment of a glycosyl-phosphatidylinositol (glycosyl-PtdIns) anchor. The putative mature form of axonin-1 comprises six immunoglobulin-like repeats, followed by four fibronectin-type III repeats. Axonin-1 exhibits 75% amino acid identity with the AxCAM TAG-1 of the rat, suggesting that it is the chicken homologue of TAG-1. Like TAG-1, axonin-1 is glycosyl-PtdIns-anchored to the neuronal membrane; in contrast to TAG-1, it does not exhibit an Arg-Gly-Asp sequence.


Total synthesis of horse heart apocytochrome c by conformation-assisted condensation of two chemically synthesized fragments.

A fully synthetic peptide, corresponding to the entire 104-residue sequence of horse heart apocytochrome c with Met65 replaced by homoserine, has been obtained by an original conformation-assisted three-fragment condensation procedure. The method involves the selective joining of two synthetic fragments, namely residues 1-65 of the apopeptide with Met65 replaced by homoserine lactone and residues 66-104 of the protein in the presence of fragment 1-25 of the native heme-containing peptide. The joining conditions have been optimized with regard to solvent, pH and possible influence of additives. The presence of radical scavengers and the complete exclusion of oxygen were found essential in order to prevent oxidative side reactions. A sensitive method based on reverse-phase HPLC has been used to monitor the course of the reaction. Condensation yields up to 80% were obtained. The data obtained by this new three-fragment rejoining approach are discussed and compared to those of a similar two-fragment condensation procedure. Our data demonstrate how the folding properties of large synthetic peptide fragments, organized in a complex, can be utilized to extend the presently improved solid-phase peptide methods to the synthesis of a functioning protein with more than 100 residues.


Polyphasic motor unit action potentials in early radiculopathy: their presence and ephaptic transmission as an hypothesis.

Various investigators have reported the presence of polyphasic motor unit potentials in radiculopathy. Some of these potentials have been shown to occur the first few weeks after the onset of symptoms, before reinnervation has begun. We hypothesize that the polyphasic motor unit potential described in acute nerve root compression can be the result of ephaptic transmission between adjacent nerve axons in the region of the inflamed root. The historical, clinical and experimental evidence supporting this hypothesis is presented.


Transmission times from cutaneous and mixed nerves of lower limbs.

In each of 10 normal subjects, P28 and N31 far-field components as well as the cortical N37 and P40 waves were identified for both Sural (SN) and Posterior Tibial nerve (PTN) stimulation at the ankle. Peripheral conduction velocity along the popliteal fossa-L3 segment and spinal transmission velocity over the L3-Cv2 segment did not differ significantly for the two nerves. The average latencies of N37 and P40 to stimulation of PTN were 2.55 and 3.65 msec shorter than corresponding latencies from SN. If we consider the P28-N37 and P28-P40 intervals as central transmission times, only the former approaches upper limb CCT values closely enough to support the analogy between median nerve N20 and PTN N37. At the intracranial level, the P28-N37 inter-peak latency was significantly shorter for PTN than SN. The P28 component is generally attributed to lemniscal activity. Thus, the discrepancy in afferent transmission times from the leg's mixed and sensory nerves would appear to take place at a level that is supralemniscal but subcortical.


Structural diversity among sulfated alpha-L-galactans from ascidians (tunicates). Studies on the species Ciona intestinalis and Herdmania monus.

Sulfated polysaccharides occurring in the tunic of different species of ascidians differ markedly in electrophoretic pattern and chemical composition. A purified sulfated alpha-L-galactan from Herdmania monus was studied using methylation analysis and NMR spectroscopy; it is composed mainly of 3-sulfated 4-linked alpha-L-galactopyranoyl units. This is the first description of a homo-polymer of sulfated alpha-L-galactose. In contrast, the sulfated L-galactan from Ciona intestinalis shows marked structural heterogeneity and a low sulfate content. These data indicate unusual structural diversity among sulfated L-galactans from different species of ascidians.


Identification and molecular characterization of the calmodulin-binding subunit gene (CMP1) of protein phosphatase 2B from Saccharomyces cerevisiae. An alpha-factor inducible gene.

A method has been developed for the rapid purification of yeast calmodulin in high yield. Using a 125I-labeled calmodulin SDS/PAGE gel overlay procedure with either yeast or bovine calmodulin, we show that the bovine and yeast proteins recognize the same proteins in total yeast extracts. However, yeast calmodulin does not bind to many of the proteins in vertebrate cells identified using bovine calmodulin. A lambda gt11 yeast genomic expression library was screened with yeast or bovine brain 125I-calmodulin to identify sequences derived from calmodulin binding proteins. Twelve clones were recovered, all containing a common DNA insert; all bound calmodulin in a Ca(2+)-dependent manner. The complete coding sequence was recovered and sequenced. The predicted protein sequence show greater than 50% identity to the A subunit of vertebrate protein phosphatase 2B. The gene was designated CMP1 and shown to reside on chromosome IV. Disruption or over-expression of CMP1 have no obvious phenotype; yeast appears to contain one or more CMP1-related genes. The protein product of the CMP1 gene is elevated by alpha-factor treatment, suggesting an involvement of protein phosphatase 2B in the mating response.


Mössbauer study of the native, reduced and substrate-reacted Desulfovibrio gigas aldehyde oxido-reductase.

The Desulfovibrio gigas aldehyde-oxido-reductase contains molybdenum and iron-sulfur clusters. Mössbauer spectroscopy was used to characterize the iron-sulfur clusters. Spectra of the enzyme in its oxidized, partially reduced and benzaldehyde-reacted states were recorded at different temperatures and applied magnetic fields. All the iron atoms in D. gigas aldehyde oxido-reductase are organized as [2Fe-2S] clusters. In the oxidized enzyme, the clusters are diamagnetic and exhibit a single quadrupole doublet with parameters (delta EQ = 0.62 +/- 0.02 mm/s and delta = 0.27 +/- 0.01 mm/s) typical for the [2Fe-2S]2+ state. Mössbauer spectra of the reduced clusters also show the characteristics of a [2Fe-2S]1+ cluster and can be explained by a spin-coupling model proposed for the [2Fe-2S] cluster where a high-spin ferrous ion (S = 2) is antiferromagnetically coupled to a high-spin ferric ion (S = 5/2) to form a S = 1/2 system. Two ferrous sites with different delta EQ values (3.42 mm/s and 2.93 mm/s at 85 K) are observed for the reduced enzyme, indicating the presence of two types of [2Fe-2S] clusters in the D. gigas enzyme. Taking this observation together with the re-evaluated value of iron content (3.5 +/- 0.1 Fe/molecule), it is concluded that, similar to other Mo-hydroxylases, the D. gigas aldehyde oxido-reductase also contains two spectroscopically distinguishable [2Fe-2S] clusters.


Mössbauer characterization of the tetraheme cytochrome c3 from Desulfovibrio baculatus (DSM 1743). Spectral deconvolution of the heme components.

Mössbauer spectroscopy was used to study the tetraheme cytochrome c3 from Desulfovibrio baculatus (DSM 1743). Samples with different degrees of reduction were prepared using a redoxtitration technique. In the reduced cytochrome c3, all four hemes are reduced and exhibit diamagnetic Mössbauer spectra typical for low-spin ferrous hemes (S = 0). In the oxidized protein, the hemes are low-spin ferric (S = 1/2) and exhibit overlapping magnetic Mössbauer spectra. A method of differential spectroscopy was applied to deconvolute the four overlapping heme spectra and a crystal-field model was used for data analysis. Characteristic Mössbauer spectral components for each heme group are obtained. Hyperfine and crystal-field parameters for all four hemes are determined from these deconvoluted spectra.