Effect of fiber supplementation of liquid diet on cecal bacteria and bacterial translocation in mice. Liquid Isosource was supplemented with soy fiber (Isosource-soy) or guar fiber (Isosource-guar) to study the effects of fiber supplementation of a liquid diet on the composition of mouse cecal flora and on the incidence of bacterial translocation, quantified as the number of mice with viable intestinal bacteria recovered from mesenteric lymph nodes. Mice fed chow, Isosource, Isosource-soy, or Isosource-guar had similar concentrations of cecal bacteria and similarly low incidences of bacterial translocation. In separate experiments, mice were given parenteral metronidazole or parenteral lipopolysaccharide (LPS) to induce cecal bacterial overgrowth and bacterial translocation. Compared with mice treated with diet alone, metronidazole- or LPS-treated mice had high numbers of cecal bacteria and high incidences of bacterial translocation. However, among the dietary groups of LPS-treated mice, the incidence of translocation was significantly less in mice fed Isosource-soy or Isosource-guar compared with mice fed unsupplemented Isosource. These results suggest that soy or guar supplementation of Isosource might be beneficial in some circumstances.


Hepatic resection for minute hepatocellular carcinoma. Twenty-three patients with minute hepatocellular carcinoma, defined as a solitary lesion less than or equal to 2 cm, underwent hepatectomy at our institute during the 10 years between January, 1979 and December, 1988. Hepatitis B surface antigen was positive in 4 patients and the preoperative serum alpha-fetoprotein level was within the normal range in 7 patients and slightly elevated (20-200 ng/mL) in 14 patients. Liver cirrhosis was present in 16 patients and chronic hepatitis in 6 patients. The diagnosis was first suspected from the results of periodic examinations, including echography and the measurement of alpha-fetoprotein, in all except one patient. Minor hepatic resection was performed in 22 patients, and lobectomy in one patient in whom the tumor was located centrally in the liver. Three patients died of hepatic failure in hospital following surgery, and the survival rates of the other 20 patients at 1, 3, and 5 years were 90, 79, and 61 percent, respectively. The prognostic factors that influenced long-term survival were investigated by comparing the survival curves. The only factor associated with a significant difference in survival was the severity of concomitant liver disease. Thus, severe cirrhosis is the main obstacle against the long-term survival of patients with minute hepatocellular carcinoma.


Clinicopathological features of advanced gastric cancer detected by periodic mass screening. We have attempted to clarify the clinicopathologic features of advanced gastric cancers that were detected in periodic mass screening by comparing them with those of cancers detected by an initial mass screening. The macroscopic type of the cancers detected by periodic mass screening included Borrmann's type 3 in 47% of cases but was an unclassifiable type because of its resemblance to early cancer in gross appearance in another 47% of cases. The predominant histologic type of the cancers was found to be poorly differentiated adenocarcinoma with scirrhous growth. From these results, advanced gastric cancers that resemble early cancers of the depressed type, in gross morphology, and which are histologically diagnosed as poorly differentiated scirrhous carcinoma, are assumed to be rapidly growing tumors. Therefore, some of the cases that have these characteristics may, in fact, represent early-phase scirrhous carcinoma, diffuse carcinoma classified as Borrmann's type 4, of the stomach.


Liver transplantation in tyrosinaemia type 1: the dilemma of timing the operation. Four children with tyrosinaemia type 1 received liver transplants. The metabolic disorder was corrected and all four had normal liver function on an unrestricted diet. Two children, transplanted at age five and seven years, proved to have occult hepatocellular carcinoma and both subsequently developed pulmonary metastases. One child was well 32 months after removal of a single pulmonary metastasis but the other child died with multiple metastases. The two younger children, transplanted at age 19 and 21 months, were well 28 and 44 months after operation, one after a second liver transplant. Our experience confirms the high risk of hepatocellular carcinoma in this disease and the potential value of early liver transplantation.


Recurrent nodular haemangiomas in Klippel-Trénaunay syndrome. A one-year-old child had hypertrophy of the left leg and an unusual constellation of a naevus flammeus and superficial enlarged veins of the trunk together with successive appearance and involution since birth of numerous nodular elements located in the naevus and in the surrounding normal skin. Microscopic examination of these elements showed haemangiomas with capillaries, cavernous channels and lymphangiomatous components. The benign nature of transient nodular elements located to the trunk and the lack of associated visceral vascular malformations in the Klippel-Trénaunay syndrome are documented.


Up-regulation of beta-adrenoceptors on circulating mononuclear cells after reduction of central sympathetic outflow by clonidine in normal subjects. Short term regulation of beta-adrenoceptors in peripheral blood mononuclear cells after sympathetic activation has been previously documented in normal individuals but changes after a central reduction in sympathetic activity are not known. We have studied beta-adrenoceptor number and affinity on peripheral blood mononuclear cells in normal subjects, before and after intravenous clonidine, an alpha 2-adrenoceptor agonist which lowers blood pressure predominantly by reducing central nervous system sympathetic outflow. After clonidine there was a decrease in plasma levels of noradrenaline and adrenaline, and an increase in growth hormone. There was up-regulation of beta-adrenergic receptors on peripheral blood mononuclear cells 30 and 60 min after clonidine which was related to the fall in blood pressure, noradrenaline and adrenaline levels and to the increase in growth hormone levels. The affinity of the receptors was decreased. Return to baseline values was observed after 2 h. Intracellular production of cAMP after isoproterenol stimulation demonstrated that the up-regulation was not functional. Our studies indicate short term up-regulation of beta-adrenoceptors in peripheral blood mononuclear cells after clonidine. These observations after a reduction in sympathetic activity may be of importance if they mirror the pattern of redistribution of adrenoceptors, which are present in a wide range of tissues.


Isolated failure of noradrenergic transmission in a case with orthostatic hypotension and hyperactivity of gastro-colic reflex. We describe a 37-year-old man with a long-standing history of impotence and urgency of defaecation. The latter invariably followed the ingestion of food. Studies of cardiovascular autonomic function disclosed asymptomatic orthostatic hypotension due to isolated sympathetic noradrenergic failure with intact cardiac vagal control. There were no other neurological abnormalities. Levels of plasma noradrenaline and urinary vanillylmandelic acid were very low but plasma dopamine and urinary homovanillic acid were normal. A low level of dopamine-beta-hydroxylase activity was detected in plasma. The patient's parents were first cousins. The parents, the brother and the sister were investigated and clinical and biochemical studies showed no abnormalities. We conclude that this is a further observation of orthostatic hypotension due to isolated adrenergic failure with clinical and biochemical features different from cases previously described.


Promoter fusions to the Activator transposase gene cause distinct patterns of Dissociation excision in tobacco cotyledons. To explore the effects of altering the level of Activator (Ac) transposase (TPase) expression, a series of plasmids was constructed in which heterologous promoters were fused to the TPase gene. Promoters for the cauliflower mosaic virus (CaMV) 35S transcript and the octopine synthase (ocs) and nopaline synthase (nos) genes were tested. These fusions, and constructs expressing TPase from the wild-type Ac promoter, were introduced into tobacco, and their activity was monitored by crossing to a line carrying Dissociation (Ds) in a streptomycin phosphotransferase gene (Ds::SPT). The SPT marker provides a record of somatic excisions of Ds that occur during embryo development. The patterns of somatic variegation that resulted from transactivation by each fusion were distinct and strikingly different from the pattern triggered by the wild-type Ac constructs. Unlike wild-type Ac, which caused transposition throughout embryo development, each fusion gave rise to sectors of discrete size. Sectors triggered by the CaMV 35S fusion were largest, ocs sectors were intermediate, and nos were smallest. These patterns appear to indicate differential timing of the activation of these promoters during embryogeny. Measurement of transcript abundance for each transformant indicated that the CaMV 35S-transformed plants accumulated approximately 1000-fold more TPase mRNA than plants containing wild-type Ac, whereas ocs- and nos-transformed lines accumulated about 100-fold and 20-fold higher levels, respectively. Measurements of germinal excision frequencies driven by the chimeric TPase fusions, however, indicated that increasing transcription does not necessarily result in an increase in germinal excision. These measurements showed that the ocs and nos fusions have very low rates of germinal excision. Only the CaMV 35S fusion transformants were found to have higher rates than the Ac constructs, although significant pod-to-pod variation was observed. Gel blot analysis of DNA from progeny carrying germinal excision events resulting from the CaMV 35S fusion showed that excision is associated with reinsertion and that siblings sometimes carry the same transposition events. These findings suggest that in tobacco there is no direct proportionality between TPase expression and Ac-Ds transposition activity. This possibility has important implications for understanding the regulation of Ac transposition and for designing efficient gene tagging systems.


Elevated levels of Activator transposase mRNA are associated with high frequencies of Dissociation excision in Arabidopsis. The Activator (Ac) element of maize is active at a low frequency in Arabidopsis. To determine whether this is due to poor expression of the Ac transposase gene, we obtained and studied 19 Arabidopsis transformants containing fusions of the octopine synthase (ocs), nopaline synthase (nos), cauliflower mosaic virus (CaMV) 35S, or Ac promoters to the transposase open reading frame. These transformants were examined both for their ability to drive excision of a Dissociation (Ds) element from a streptomycin resistance gene and for the abundance of the transposase mRNA. Most transformants containing the CaMV 35S fusion have high levels of transposase transcript and drive high frequencies of somatic and germinal excision. These results demonstrated that Arabidopsis contains all of the host functions required for high frequency excision of Ds. Moreover, transposase mRNA abundance varied about 1000-fold among our transformants; this variation enabled us to demonstrate that for the Ac, ocs, and CaMV 35S fusion, raising the mRNA level is closely correlated with increasing excision frequency. We discuss our data in relation to the behavior of Ac in Arabidopsis, maize, and tobacco.


L-baclofen-sensitive GABAB binding sites in the medial vestibular nucleus localized by immunocytochemistry. L-Baclofen-sensitive GABAB binding sites in the medial vestibular nucleus (MVN) were identified immunocytochemically and visualized ultrastructurally in L-baclofen-preinjected rats and monkeys, using a mouse monoclonal antibody with specificity for the p-chlorophenyl moiety of baclofen. Saline-preinjected animals showed no immunostain. In drug-injected animals, there was evidence for both pre- and postsynaptic GABAergic inhibition in MVN mediated by GABAB receptors. These neural elements could be utilized in control of velocity storage in the vestibulo-ocular reflex.


Autoradiographic distribution of [3H](+)-pentazocine and [3H]1,3-di-o-tolylguanidine (DTG) binding sites in guinea pig brain: a comparative study. Binding studies suggested the selectivity of (+)-pentazocine for sigma receptors, and subsequent synthesis and testing of [3H](+)-pentazocine confirmed its high potency and selectivity for sigma sites. Newer data have demonstrated the selectivity of (+)-pentazocine for a subtype of the sigma receptor called sigma-1. Based on these findings, the distribution of [3H](+)-pentazocine binding sites in the guinea pig brain was examined using in vitro autoradiography. [3H](+)-Pentazocine binding was high in the cingulate cortex, dorsal diagonal band, periaqueductal gray, cerebellum and cranial nerve nuclei. It was relatively low in the nucleus accumbens, neocortical areas and caudate nucleus. A significant correlation was found between the binding of [3H](+)-pentazocine and [3H]1,3-di-o-tolylguanidine, a selective sigma ligand across brain regions. However, certain nuclei exhibited markedly different ratios of binding of the two ligands. Since DTG is not selective for the sigma subtypes, while (+)-pentazocine is selective for the sigma-1 type, the data are suggestive of relative differences in the distributions of sigma-1 and sigma-2 sites.


Delta opiate receptors mediate tail-shock induced antinociception at supraspinal levels. Previous work has demonstrated that 3 pharmacologically and neuroanatomically distinct analgesia systems can be sequentially activated by increasing numbers of transcutaneous tail-shock. To date, the categorization of the early (after 2 tail-shocks) and late (after 80-100 tail-shocks) analgesias as opiate-mediated has been based on the ability of systemic naltrexone and morphine tolerance to block these effects. In contrast, the analgesia observed after 5-40 tail-shocks is unaffected by these manipulations, leading to its categorization as non-opiate. The preceding companion paper and the present work were aimed at identifying the neuroanatomical loci at which opiates exert their analgesic effects in this tail-shock paradigm and, further, to identify which opiate receptor subtypes are involved. The 8 experiments included in the present paper examined the effect of microinjecting either naltrexone (a relatively non-selective opiate receptor antagonist), binaltorphimine (kappa receptor antagonist), Cys2-Tyr3-Orn5-Pen7-amide (CTOP) (mu receptor antagonist), or naltrindole (delta receptor antagonist) either into the third ventricle or over the frontal cortex. Taken together, these experiments demonstrate that the late (80-100 shock) opiate analgesia is mediated by delta opiate receptors located within subcortical structures rostral to the 4th ventricle. No evidence for supraspinal opiate involvement in the early (2 shock) opiate analgesia was found.


Spino-bulbar neurons convey information to the brainstem about different phases of the locomotor cycle in the lamprey. Lamprey retriculospinal neurons show phasic oscillations of their membrane potential during fictive locomotion. This modulation originates from the spinal cord locomotor networks. The aim of the present study was to elucidate the pattern of discharge of the spino-bulbar axons responsible for this modulation. Experiments were performed on in vitro brainstem/spinal cord preparations. Two baths were formed in the recording chamber. The caudal one was perfused with 150 microM N-methyl-D-aspartate (NMDA) solution to induce fictive locomotion. The rostral bath containing the brain and the first 3-5 segments of the spinal cord was exposed to a 0 Ca2+ + 2.6 mM Mn2+ solution to block synaptic transmission and therefore to abolish any rhythmic descending activity. Spinobulbar axons were recorded intracellularly at the level of the brain/spinal cord junction. They exhibited phasic discharges correlated with the ongoing motor activity in the caudal pool. Some discharged in phase with either the ipsilateral or the contralateral ventral root bursts, others with either of the transition phases between these two bursts. These spinal cells with ascending axons, running in the ventrolateral spinal cord, may be important for modulating the activity of supraspinal neurons to match the ongoing locomotor activity.


Phasic modulation of transmission from vestibular inputs to reticulospinal neurons during fictive locomotion in lampreys. The aim of this study was to determine whether the transmission from sensory inputs to reticulospinal neurons is modulated during fictive locomotion in lampreys. Reticulospinal neurons play a key role in the control of locomotion; modulation of sensory transmission to these neurons might be of importance for the adaptation of the control they exert during locomotion. In this series of experiments, intracellular synaptic responses of reticulospinal neurons of the posterior rhombencephalic reticular nucleus elicited by electrical stimulation of vestibular nerves on each side were studied during fictive locomotion induced by 50 microM N-methyl-D-aspartate (NMDA). Interestingly, shortly after NMDA had reached the bath and much before locomotor discharges were apparent in the recorded ventral roots, there was a significant depression of the synaptic transmission from vestibular nerves. The effect was reversed by washing out the NMDA and persisted in the isolated brainstem after spinal transection at the first segmental level. As locomotor discharges appeared in the ventral roots, synaptic responses elicited by vestibular nerve stimulation showed a clear phasic modulation of their amplitude during the locomotor cycle. Responses to stimulation of the ipsilateral vestibular nerve were smaller during the ipsilateral burst discharge than during the contralateral activity, whilst responses to stimulation of the contralateral vestibular nerve were minimal during contralateral activity and maximal during ipsilateral activity. This opposite pattern of modulation observed in the same reticulospinal neuron suggests that the phasic modulation of vestibular transmission is not due to changes in the membrane properties of the reticulospinal cell but is produced at a pre-reticular level.(ABSTRACT TRUNCATED AT 250 WORDS)


Study on the stereoselectivity of inositol 1,4,5-trisphosphate recognition sites of bovine adrenal cortex. Inositol 1,4,5-trisphosphate (InsP3) is an intracellular messenger generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C in response to Ca(2+)-mobilizing stimuli. InsP3 interacts with a specific receptor responsible for the release of sequestered Ca2+ from an intracellular store. The purpose of the present study was to evaluate the relative affinities of the naturally occurring D-isomer of InsP3 and that of its L-stereoisomer for the InsP3 receptor and the InsP3 metabolizing enzymes from bovine adrenal cortex. The InsP3 receptor recognized D- and L-isomers with respective affinities of 4.8 nM and 7.3 microM. This high degree of selectivity was also reflected in the capacity of both isomers to mobilize Ca2+ from the microsomal preparation. The partially purified InsP3 kinase preparation was also able to discriminate between the two stereoisomers. The activity of the kinase was half-maximally inhibited in the presence of 11 microM L-InsP3, a value much higher than the Km of the kinase for D-InsP3 (0.4 microM). Both stereoisomers exhibited equipotent affinities (around 17 microM) for the particulate preparation of InsP3 phosphatase. The enzyme, however, appeared to hydrolyze L-InsP3 at a much slower rate. These results demonstrated that the different recognition sites for InsP3 were expressing distinct levels of stereoselectivity. This property, which is an important aspect of ligand-receptor interaction, could be exploited for the design of new selective drugs interfering with InsP3 action and metabolism.


Relationship between potency of L-isoprenaline and beta-adrenoceptor density estimated in single cells from tracheal smooth muscles of guinea pigs of different ages. An age-related change in potency of L-isoprenaline in the presence of ascorbic acid, desmethylimipramine, corticosterone, pargyline, and phentolamine was obtained in tracheal strips from guinea pigs of differing ages between 6 and 40 weeks. The potency in the strips from 100-week-old guinea pigs did not significantly differ from that in strips from 40-week-old animals. Single cells were prepared from the tracheal muscles of 6-, 10-, 40-, and 100-week-old guinea pigs. The specific binding of [3H]dihydroalprenolol to the single cells was saturable. The dissociation constants of [3H]dihydroalprenolol were in good agreement with those of the membrane fractions from the guinea-pig tracheal muscles, and did not change with age. An excellent relationship between the potency of L-isoprenaline and the maximum binding of [3H]dihydroalprenolol estimated in the preparations from 6- to 40-week-old guinea pigs was found, suggesting that the increase in the potency of L-isoprenaline is due to the increase in the maximum binding or receptor density. The value in the preparations from 100-week-old guinea pigs deviated significantly from the regression line. This suggests the possibility that the decrease in potency in the strips from 100-week-old animals is due to a change in post beta-receptor processes in responsiveness.


Differing significance of Na(+)-Ca2+ exchange in the regulation of cytosolic Ca2+ in rat exocrine gland acini and cardiac myocytes. In order to compare the importance of Na(+)-Ca2+ exchange in the regulation of cytosolic Ca2+ concentration (Ca2+i), acini obtained from rat pancreas and submandibular glands as well as cardiac myocytes were loaded with Na+ by inhibition of Na(+)-K+ ATPase activity then loaded with fura-2. In the exocrine tissues, incubation in K(+)-free buffer or with ouabain had no substantial effect on resting Ca2+i or on the changes in Ca2+i following exposure to carbachol as compared with acini incubated under control conditions. In contrast, rat cardiac myocytes, treated identically, showed marked changes in Ca2+i under resting and stimulated conditions as compared with controls. We conclude that the Na(+)-Ca2+ exchange systems of rat pancreatic and submandibular gland acini contribute little to the overall regulation of Ca2+i at rest during cholinergic stimulation.


Extraadrenal pheochromocytoma: dynamic demonstration at 0.5 tesla. We report a case of extraadrenal pheochromocytoma of Zuckerkandl imaged by dynamic magnetic resonance imaging (dynamic MRI) with a 0.5 tesla (T) machine. The dynamic MRI proved useful in disclosing the tumor clearly because of strong enhancement in its early phase. This case has the advantages of dynamic computed tomography (dynamic CT) as well as of T2-weighted spin-echo (SE) MR images.


Fast Na+ channels in smooth muscle from pregnant rat uterus. Smooth muscle cells normally do not possess fast Na+ channels, but inward current is carried through two types of Ca2+ channels: slow (L type) Ca2+ channels and fast (T type) Ca2+ channels. Whole-cell voltage clamp was done on single smooth muscle cells isolated from the longitudinal layer of the 18-day pregnant rat uterus. Depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow. The fast inward current decayed within 30 ms, depended on [Na]o, and was inhibited by tetrodotoxin (TTX) (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]o (or Ba2+), and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na+ channel current and that the slow inward current is a Ca2+ slow channel current. A fast-inactivating Ca2+ channel current was not evident. We conclude that the ion channels that generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells that possess fast Na+ channels. The Ca2+ channel current density was also higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and we suggest that the fast Na+ current may be involved in spread of excitation. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 = 12 mM) and nifedipine (K0.5 = 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) to a small extent. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions cannot be explained by a stimulation of ICa(s).


Thyroid cyst mistaken for carotid pseudoaneurysm by MR angiography. Case report. A normal volunteer for magnetic resonance angiography of the carotid arteries had an abnormal examination that suggested a common carotid pseudoaneurysm. High resolution sonography identified a normal carotid artery immediately adjacent to a thyroid lesion, part of which was cystic and which mimicked carotid aneurysmal disease.


Recent trends in the management of breast cancer. 1. Carcinoma in situ of the breast. Because of widespread screening for breast cancer, noninvasive (in-situ) cancer of the breast is diagnosed with increasing frequency. The two variants--lobular carcinoma in situ (LCIS) and ductal carcinoma in situ (DCIS)--can be cured with conservation of the breast. The diagnosis of LCIS indicates a high risk for future development of invasive carcinoma in either breast. Bilateral total mastectomy, with reconstruction if desired, is the only method of eliminating this risk, but local excision alone with close observation is acceptable. For DCIS, total mastectomy, with or without low axillary lymph-node dissection, offers near-complete cure. For selected cases, wide local excision, with or without dissection of the low axillary nodes, followed by breast irradiation provides survival rates comparable to those for mastectomy and a low risk of recurrence in the breast. Studies are in progress to determine if breast irradiation after local excision is necessary in all instances.


Comparison of partial and modified radical mastectomy in the community setting--"10 years later". Conservative surgery followed by postoperative radiation is considered equivalent to a modified radical mastectomy (MRM) for the treatment of early breast cancer. It cannot be assumed that results from selected academic centres are equivalent to those obtained in the general community setting, because there may be differences in patient selection or surgical or radiotherapy techniques that may adversely affect outcome. A quality-control study of women who were seen at the British Columbia Cancer Agency and were treated by partial mastectomy (PM) was begun in 1983. Eighty-four women who underwent conservative surgery between January 1979 and November 1982 and were referred to the British Columbia Cancer Agency were matched with 84 women who underwent MRM. The mean follow-up was 10.5 years. At 10 years disease-free survival in both groups was 63%. Survival overall for the PM group was 72.6% and for the MRM group was 69%. The survival rate decreased with increasing size of the tumour and increasing number of nodes. In women with lymph-node involvement there was a survival advantage for those treated by PM and radiation compared with those treated by MRM. The woman's age at diagnosis did not affect these findings. Recurrence and complication rates were similar in both groups, and treatment was considered equivalent.


Technetium 99m pentavalent dimercaptosuccinic acid and thallium 201 in detecting recurrent medullary carcinoma of the thyroid. To compare the effectiveness of thallium chloride 201 and technetium 99m pentavalent dimercaptosuccinic acid in evaluating medullary carcinoma of the thyroid (MCT), eight patients with a history of MCT underwent imaging with both radiopharmaceuticals. Thallium 201 consistently gave superior images, as well as providing one less false-negative scan. Positive scans were obtained in patients with elevation of basal calcitonin levels to more than 1,000 ng/L. All of the patients with positive scans had clinical evidence of local recurrence. Improved imaging with thallium 201 was obtained by early scanning.


Management of mesenteric desmoid tumours in familial adenomatous polyposis. Intra-abdominal desmoids are locally invasive fibrous tumours within the mesentery of the intestine. They occur in 4% to 13% of patients with familial adenomatous polyposis (FAP). Of 498 patients registered in the Steve Atanas Stavro FAP Registry at the Mount Sinai Hospital, Toronto, 53 (11%) had desmoids and 40 of them had mesenteric desmoids. There were 23 women and 17 men, and the mean age was 31 years. Most tumours were diagnosed after colectomy for FAP. Of the 40 patients 7 (18%) died of desmoid-related complications. Surgical excision of desmoids is hazardous and was associated with a recurrence rate of up to 85% in this study. Current research is directed at the role of antiestrogens in the fundamental mechanisms of cell growth and are now the subject of clinical trials.


Adenoviruses in the immunocompromised host. Adenoviruses are among the many pathogens and opportunistic agents that cause serious infection in the congenitally immunocompromised, in patients undergoing immunosuppressive treatment for organ and tissue transplants and for cancers, and in human immunodeficiency virus-infected patients. Adenovirus infections in these patients tend to become disseminated and severe, and the serotypes involved are clustered according to the age of the patient and the nature of the immunosuppression. Over 300 adenovirus infections in immunocompromised patients, with an overall case fatality rate of 48%, are reviewed in this paper. Children with severe combined immunodeficiency syndrome and other primary immunodeficiencies are exposed to the serotypes of subgroups B and C that commonly infect young children, and thus their infections are due to types 1 to 7 and 31 of subgenus A. Children with bone marrow and liver transplants often have lung and liver adenovirus infections that are due to an expanded set of subgenus A, B, C, and E serotypes. Adults with kidney transplants have viruses of subgenus B, mostly types 11, 34, and 35, which cause cystitis. This review indicates that 11% of transplant recipients become infected with adenoviruses, with case fatality rates from 60% for bone marrow transplant patients to 18% for renal transplant patients. Patients with AIDS become infected with a diversity of serotypes of all subgenera because their adult age and life-style expose them to many adenoviruses, possibly resulting in antigenically intermediate strains that are not found elsewhere. Interestingly, isolates from the urine of AIDS patients are generally of subgenus B and comprise types 11, 21, 34, 35, and intermediate strains of these types, whereas isolates from stool are of subgenus D and comprise many rare, new, and intermediate strains that are untypeable for practical purposes. It has been estimated that adenoviruses cause active infection in 12% of AIDS patients and that 45% of these infections terminate in death within 2 months. In all immunocompromised patients, generalized illness involving the central nervous system, respiratory system, hepatitis, and gastroenteritis usually have a fulminant course and result in death. Treatments for adenovirus infections are of little proven value, although certain purine and pyrimidine analogs have shown beneficial effects in vitro and may be promising drugs.


Hepatitis B virus infection and primary hepatocellular carcinoma. For many years, epidemiological studies have demonstrated a strong link between chronic hepatitis B virus (HBV) infection and the development of primary hepatocellular carcinoma (PHC). Other hepatocarcinogens such as hepatitis C virus and aflatoxin also contribute to hepatocarcinogenesis either in conjunction with HBV infection or alone. Cellular and molecular biological studies are providing explanations for the HBV-PHC relationship, and models are now being formulated to further test the relative importance of various factors such as viral DNA integration, activation of oncogenes, genetic instability, loss of tumor suppressor genes, and trans-activating properties of HBV to the pathogenesis of PHC. Further research will probably define more than a single mechanism whereby chronic HBV infection results in PHC.


Nerve growth factor receptor (NGFR) immunoreactivity in skeletal muscles of the rat. The peroxidase-antiperoxidase (PAP) method, and a specific monoclonal antibody (192-IgG) were used to determine the localization of nerve growth factor receptor (NGFr) in the skeletal muscles of the adult rats. The rectus femoris and the gastrocnemius (medialis and lateralis) muscles were analyzed. Occurrence of NGFr immunoreactivity was observed in: 1) a subpopulation of myelinated nerve fibers within muscle nerve trunks; 2) the vascular adventitia and nerve-like profiles around the blood vessels; 3) the outer capsule and the surface of the intrafusal muscle fibers of muscle spindles. Conversely, images, suggesting the presence of NGFr on muscle fibers or in motor end-plates, were not found. Our results suggest the presence of NGF-binding sites in sensory and sympathetic nerve fibers, and/or their target tissues localized on the skeletal muscles of the rat, whereas the motor nerve fibers lack of NGFr. The dependence of sympathetic neurons, proprioceptive primary sensory neurons, and motoneurons innervating the mammalian muscles upon NGF or other neurotrophic factors is discussed.


Migration of cranial neural crest cells to the pharyngeal arches and heart in rat embryos. The existence of a neural crest cell migration pathway from occipital levels of the hindbrain into the heart was suspected in mammalian embryos because it had previously been identified in avian embryos and because the Di George anomaly, an association between craniofacial and cardiac malformations, is most easily explained on the basis of abnormal neural crest cell migration to all of the affected structures. In order to demonstrate the existence of this pathway, neural crest cells were labelled in situ in rat embryos with the fluorescent dye DiI, and the embryos cultured for up to 48 h. Cells labelled between occipital somites 1 and 2 or 3 and 4 migrated within and dorsal to the third and fourth pharyngeal arches and into the outflow tract of the heart (conus cordis and truncus arteriosus). The cardiac labelling was in individually visible cells, in contrast to the mass of fluorescence seen in the pharyngeal and dorsal mesenchyme. Within the outflow tract wall, the labelled cells were enmeshed by strands of alcian blue-stained extracellular matrix. There was no labelling of cardiac cells following injections just rostral to, or just caudal to, somites one and four. This study establishes the existence and precise levels of origin of the 'cardiac' neural crest in a mammalian embryo.


Testosterone and FSH have independent, synergistic and stage-dependent effects upon spermatogenesis in the rat testis. Adult rats were hypophysectomized and treated with ethane dimethanesulphonate (EDS) selectively to eliminate the Leydig cells in the testis. By removing the source of endogenous gonadotrophins and androgens, the subsequent effects on the seminiferous epithelium were studied after 20 days of treatment with vehicle, or FSH (2 x 50 micrograms/day) or a low dose of testosterone (0.6 mg testosterone esters every 3rd day) alone or in combination. Compared to vehicle-treated hypophysectomized rats with Leydig cells, testis weight in saline-treated hypophysectomized rats treated with EDS declined by 50%, spermatogenesis was disrupted severely and only 18% of the tubules contained spermatids, these being confined to stages I-VI of the spermatogenic cycle. Treatment with either FSH or testosterone esters alone significantly (P less than 0.01) increased testis weight compared to vehicle-treated hypophysectomized rats treated with EDS and 40% of tubules contained spermatids either at stages I-VI after FSH, or at all stages I-XIV after testosterone treatment. Treatment with FSH and testosterone esters together maintained testis weights approximately 20% above vehicle-treated hypophysectomized controls; over 70% of the seminiferous tubules contained spermatids and there was a marked stimulation of spermatogenesis at all stages of the spermatogenic cycle. The results suggest, that in the absence of the pituitary gland and the Leydig cells, FSH alone partially supports spermatogenesis up to the development of round spermatids whereas testosterone is capable of maintaining spermatid development at all 14 stages of the cycle. When FSH and testosterone were administered in combination, the effects upon spermatogenesis were far greater than the response expected if their individual effects were simply additive. It is therefore concluded that FSH may play a role in normal spermatogenesis and that this role is essentially that of augmenting the response of the testis to testosterone. The biochemical mechanisms via which this might occur are discussed and hypophysectomized rats treated with EDS used in the present studies should provide a useful approach for their identification.


[Gene expression of NGFR, EGFR, CGA, NPY in 4 neuroblastoma cell lines]. Gene expression of nerve growth factor receptor (NGFR), epidermal growth factor receptor(EGFR), chromogranin A (CGA) and neuropeptide Y (NPY) in 4 neuroblastoma cell Lines without N-myc amplification was studied by using Northern blot technique. N type cells expressed more NGFR mRNA than S type cell's and have only little or no EGFR expression. S type cells had stronger expression of EGFR mRNA than that of N type cells accompanying with only less or even no NGFR expression. The results indicated that difference of gene expression of these growth factor receptors might be due to the various directions of tumor cell differentiation. Cells differentiating toward neurons gave more NGFR expression and cells prepared to be differentiating toward other direction might give more EGFR gene expression. Various gene expression of CGA and NPY in neuroblastoma cell lines might be due to the presence of different stages of tumor cell differentiation and NGF only induced differentiation of those neuroblastoma cells ready to be differentiating to neurons afterwards.


[An immunohistochemical study of ferritin in ovarian epithelial tumours]. Ferritin in 93 ovarian epithelial tumors, 10 normal adult ovaries and 4 fetal ovaries were studied with double-PAP technique. Ferritin was demonstrated in 38.89% of the benign ovarian tumors, 46.67% of the borderline and 70.0% of the malignant tumors (P less than 0.05). The positive distribution of ferritin in the serous tumors was more than that in the mucinous and endometrioid tumor (P less than 0.05) and the positive grading of ferritin starring was corresponding to the pathohistopathologic grading of the ovarian tumors. It is considered that ferritin might be used as an immunohistochemical marker in the study of ovarian tumors.


[Flow cytometric analysis of trophoblastic tumors]. 19 cases of malignant trophoblastic tumors were studied by flow cytometry in comparison with 9 cases of benign hydatidiform mole that had been followed up for 1.5 years with no history of malignancy. The results showed that there were significant differences in DNA index (DI), S phase of cells and the pattern of ploidies between the malignant trophoblastic tumor and benign hydatidiform mole. It is considered that flow cytometry is a useful parameter in reference to the diagnosis, chemotherapy and assessing prognosis of malignant trophoblastic tumor.


Human mesothelioma cells and asbestos-exposed mesothelial cells are selectively resistant to amosite toxicity: a possible mechanism for tumor promotion by asbestos. To determine if asbestos exposure could contribute to mesothelial cell carcinogenesis by selection and/or expansion of an initiated cell population, we compared normal human pleural mesothelial cells to either human mesothelioma cell lines or mesothelial cells transfected with cancer-related genes for sensitivity to amosite fibers in vitro. Neither normal nor mesothelioma cells were directly stimulated to replicate or increase DNA synthesis by any of the asbestos exposure conditions tested. The potential selective effect of asbestos exposure was demonstrated by a differential sensitivity of normal mesothelial cells and mesothelioma cells to amosite: for example, up to 20-fold higher concentrations of amosite fibers were required to inhibit replication of mesothelioma cell lines than normal mesothelial cells. In addition, a significant resistance (4-fold) to amosite toxicity was observed for SV40 immortalized mesothelial cell lines that had previously been selected in vitro for resistance to asbestos. SV40 immortalized cells that have become tumorigenic after transfection with either Ha-ras or PDGF A-chain genes were not significantly more resistant to the cytotoxic effects of amosite than primary normal cells, and the primary cells were equally sensitive to amosite as mesothelial cells that were only immortalized by SV40. The sensitivity of normal mesothelial cells to asbestos does not appear to be simply a result of general fragility of the mesothelial cells, since similar levels of hydrogen peroxide and silica were cytotoxic for normal mesothelial cells and mesothelioma cell lines. Because mesothelioma cells have a greater resistance to asbestos cytotoxicity than normal mesothelial cells, we hypothesize that a differential resistance to cell killing by asbestos fibers in vivo may result in a selective expansion of an initiated or transformed cell population and thus contribute to the carcinogenesis process. Since tumorigenicity and asbestos resistance occur independently of one another in genetically altered mesothelial cell lines, genotypic and phenotypic alterations that lead to tumorigenic conversion may not be the same changes that provide resistance to cell killing by asbestos.


Effect of topoisomerase poisoning by antitumor drugs VM 26, fostriecin and camptothecin on DNA repair replication by mammalian cell extracts. A recently developed in vitro excision-repair system was used to investigate the effect of the topoisomerase poisons VM 26, fostriecin and camptothecin on DNA repair replication carried out by Chinese hamster ovary cell extracts. VM 26 and camptothecin partially inhibit topoisomerases II and I respectively, which are present in the repair-competent extracts, but have only slight effects on the repair efficiency. On the contrary, the antitumor drug fostriecin markedly affects repair replication but, in contrast to a previous report, does not seem to have, under the experimental conditions used, any inhibitory effect on topoisomerase II. This lack of correlation between the ability to inhibit DNA topoisomerases and the effect on DNA repair replication suggests that topoisomerases should not play a primary role in mammalian excision repair. The use of cleavable-complex stabilizing poisons to investigate the role of eukaryotic topoisomerases in DNA excision repair is discussed.


Effects of caffeine, nicotine, ethanol and sodium selenite on pancreatic carcinogenesis in hamsters after initiation with N-nitrosobis(2-oxopropyl)amine. The modulating effects of caffeine, nicotine, ethanol and sodium selenite on development of N-nitrosobis(2-oxopropyl)-amine (BOP)-initiated pancreatic tumors were investigated. Female Syrian golden hamsters were given s.c. injections of BOP (10 mg/kg body weight) or saline alone once a week for 3 weeks and then administered 2000 p.p.m. caffeine, 25 p.p.m. nicotine, 20% ethanol or 4 p.p.m. sodium selenite in their drinking water for the next 37 weeks. Control animals were given tap water alone after BOP initiation. Only the BOP-treated groups developed pancreatic adenocarcinomas and dysplasias. The multiplicity of pancreatic carcinomas was significantly higher (P less than 0.05) in animals receiving caffeine than in the controls. In addition, caffeine treatment slightly increased the incidence of carcinomas. Nicotine and ethanol also showed tendencies to enhance pancreatic carcinogenesis, although there were statistically no significant differences regarding lesion development. In contrast, sodium selenite administration was associated with a tendency for a decrease in the number of carcinomas and dysplasias. Thus, among these chemicals of obvious significance to human life-style, caffeine enhanced the development of pancreatic tumors when administered during the post-initiation phase in this hamster model.


Inositol phosphates and phosphoinositides in rat liver nodules. Total amounts and turnover rates of phosphoinositides and inositol phosphates in normal rat liver and hepatocyte nodules were investigated. Male Wistar rats were injected i.p. with [3H]inositol 18-20 h before killing. The amount of phosphatidylinositol in a homogenate preparation was roughly doubled in the nodules, though levels of polyphosphoinositides were approximately the same. Basal levels of inositol phosphates were the same in nodules and in normal liver. Turnover rates of inositol tris- and tetrakisphosphates were studied after stimulation of intact cells with vasopressin for different periods of time (0-5 min). The initial rate of formation of inositol trisphosphate after agonist exposure was fast in both nodular and normal cells. Nodular cells reached peak amount of inositol trisphosphate at 2.5-fold basal levels after 20 s, while normal cells peaked after 40 s at 4.5 times the basal amount. The level of inositol tetrakisphosphate was enhanced very quickly in normal cells, but in the nodular cells there was no increase of this inositol phosphate after vasopressin stimulation. To investigate the mechanism of this difference, the activities of inositol 1,4,5-trisphosphate kinase and of inositol 1,4,5-trisphosphate phosphatase were studied. Both activities were rapid and equal in nodules and normal liver. The amount of cell surface receptors for vasopressin was shown to be one-third in the nodules, as compared to normal cells. This quantitative decrease in receptor number was reflected in lower formation of inositol trisphosphate when stimulated with vasopressin, but could not explain the loss of inositol tetrakisphosphate response in nodules. The significance of the reported alterations in second messenger traffic for the growth regulation of nodular cells and for their progression to carcinoma is not yet known, but could add to the nodules being less dependent on growth regulating signals.


Immunohistochemical and biochemical evidence for a cardiovascular mineralocorticoid receptor. The presence of mineralocorticoid receptors (MRs) and their physicochemical characteristics were investigated in the heart and blood vessels of rabbits. Immunohistochemical methods using the monoclonal anti-idiotypic antibody H10E, which interacts with the steroid binding domain of MRs, revealed the presence of immunoreactive material in the heart and large blood vessels. In the heart, a positive staining was observed in myocytes and endothelial cells of atria and ventricles. In vessels, MRs were detected in the aorta and pulmonary artery. They were localized in endothelial and vascular smooth muscle cells. No staining was present in the small vascular bed, arterioles, and capillaries. In all these studies, the mineralocorticoid specificity of the staining was assessed by in situ competition experiments with aldosterone and RU486, a glucocorticoid antagonist. The presence of MRs in the heart and vessels was further demonstrated by specific aldosterone binding to one class of high affinity binding sites in the cytosol of the adrenalectomized rabbit heart (Kd, 0.25 nM; maximum MR concentration, 15-20 fmol/mg protein), whose mineralocorticoid specificity has been clearly established by competition studies. Sedimentation gradient analyses revealed that the cardiovascular MR is an 8.5S hetero-oligomer that includes the heat shock protein 90. The physicochemical characteristics of the cardiovascular MRs are virtually identical to those of the renal MRs. Altogether, our results clearly demonstrate the presence of MRs in the cardiovascular system. This supports the possibility of direct aldosterone actions in the heart and blood vessels.


Positive chronotropic responses induced by alpha 1-adrenergic stimulation of normal and "ischemic" Purkinje fibers have different receptor-effector coupling mechanisms. We studied the mechanisms underlying the increase in automaticity induced by alpha 1-adrenergic stimulation of normal and "ischemic" canine Purkinje fibers. Fibers were superfused with a control Tyrode's solution, followed by an ischemic superfusate that included 10 mM KCl, 5 mM NaHCO3, Po2 of 10-25 mm Hg, and pH 6.7. To exclude beta-adrenergic actions, propranolol was added to all solutions. In the presence of phenylephrine, normal automaticity at high membrane potentials usually decreased, whereas the incidence of abnormal automaticity during ischemia was increased from a control value of 10% to 30%. Block of an alpha 1-receptor subtype with chloroethylclonidine in the presence of phenylephrine caused normal automaticity to increase in all fibers studied and significantly increased abnormal automaticity to 70%. The alpha-adrenergic-induced increase in automaticity did not occur in ischemic fibers from animals pretreated with pertussis toxin (PTX), which ADP-ribosylated and functionally inactivated the 41-kd family of GTP regulatory proteins. In contrast, the use of PTX enhanced the increase in automaticity induced by phenylephrine in normally polarized Purkinje fibers. Ryanodine, which blocks sarcoplasmic reticulum Ca2+ release, attenuated the increase in normal automaticity in nonischemic fibers but had no effect on abnormal automaticity in ischemic fibers. The increase in abnormal automaticity was, however, blocked by the alpha 1 subtype blocker WB 4101, which also blocks the increase in automaticity in normal fibers. In conclusion, the increase in abnormal automaticity in ischemic Purkinje fibers depends on a WB 4101-sensitive alpha 1-adrenergic receptor subtype whose actions are transduced by a PTX-sensitive 41-kd G protein and are not blocked by ryanodine.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of the sodium current in single human atrial myocytes. Patch-clamp recording techniques have permitted measurement of the fast Na+ current (INa) in isolated cardiac cells from a number of species in recent years. However, there is still only very little information concerning human cardiac INa. The purpose of this study was to describe the kinetics of INa in normal-appearing, Ca(2+)-tolerant, enzymatically isolated human atrial myocytes using whole-cell voltage-clamp techniques. Atrial specimens were obtained from 46 patients undergoing open heart surgery. Cs+ was substituted for K+ in both pipette and external solutions and F- was added to the former. The reversal potential of the rapid inward current varied approximately 57 mV at 17 +/- 1 degrees C with a 10-fold change in [Na+]o, and the current was completely blocked by 100 microM tetrodotoxin, findings typical of the fast cardiac Na+ current. The tetrodotoxin dose-response curve was best fitted by an equation describing binding to high- and low-affinity sites. INa was activated at a voltage threshold of -70 to -60 mV, and peak inward current was obtained at approximately -30 mV (holding potential, -140 mV). The inactivation time course was voltage dependent and was fitted best by the sum of two exponentials. The relation between voltage and steady-state availability (h infinity) was sigmoidal with the half-inactivation at -95.8 +/- 0.9 mV and a slope factor of 5.3 +/- 0.1 mV (n = 46), and we did not observe a significant difference with disease and age. The overlap of the h infinity and activation curves suggested the presence of a Na+ "window" current. Recovery from inactivation also was voltage dependent and best fitted by a model describing the sum of two exponentials. Recovery occurred after an initial delay at potentials positive to -140 mV, suggesting that inactivation of human atrial INa is a multistate process. We conclude that INa of normal-appearing, Ca(2+)-tolerant human atrial myocytes is similar to that of other mammalian cardiac cells with the possible exception of having two tetrodotoxin binding sites.


Dependence of hypoxic cellular calcium loading on Na(+)-Ca2+ exchange. Na(+)-Ca2+ exchange has been shown to contribute to reperfusion- and reoxygenation-induced cellular Ca2+ loading and damage in the heart. Despite the fact that both [Na+]i and [Ca2+]i have been documented to rise during ischemia and hypoxia, it remains unclear whether the rise in [Ca2+]i occurring during hypoxia is linked to the rise in [Na+]i via Na(+)-Ca2+ exchange before reoxygenation and how this relates to cellular injury. Single electrically stimulated (0.2 Hz) adult rat cardiac myocytes loaded with Na(+)-sensitive benzofuran isophthalate (SBFI), the new fluorescent probe, were exposed to glucose-free hypoxia (PO2 less than 0.02 mm Hg), and SBFI fluorescence was monitored to index changes in [Na+]i. Parallel experiments were performed with indo-1-loaded cells to index [Ca2+]i. The SBFI fluorescence ratio (excitation, 350/380 nm) rose significantly during hypoxia after the onset of ATP-depletion contracture, consistent with a rise in [Na+]i. At reoxygenation, the ratio fell rapidly toward baseline levels. The indo-1 fluorescence ratio (emission, 410/490 nm) also rose only after the onset of rigor contracture and then often showed a secondary rise early after reoxygenation at a time when [Na+]i fell. The increase in both [Na+]i and [Ca2+]i, seen during hypoxia, could be markedly reduced by performing experiments in Na(+)-free buffer. These experiments suggested that hypoxic Ca2+ loading is linked to a rise in Na+i via Na(+)-Ca2+ exchange. To show that Na(+)-Ca2+ exchange activity was not fully inhibited by profound intracellular ATP depletion, cells were exposed to cyanide, and then buffer Na+ was abruptly removed after contracture occurred. The sudden removal of buffer Na+ would be expected to stimulate cell Ca2+ entry via Na(+)-Ca2+ exchange. A large rapid rise in the indo-1 fluorescence ratio ensued, which was consistent with abrupt cell Ca2+ loading via the exchanger. The effect of reducing hypoxic buffer [Na+] on cell morphology after reoxygenation was examined. Ninety-five percent of cells studied in a normal Na(+)-containing buffer (144 mM NaCl, n = 38) and reoxygenated 30 minutes after the onset of hypoxic rigor underwent hypercontracture. Only 12% of cells studied in Na(+)-free buffer (144 mM choline chloride, n = 17) hypercontracted at reoxygenation (p less than 0.05). Myocytes were also exposed to hypoxia in the presence of R 56865, a compound that blocks noninactivating components of the Na+ current. R 56865 blunted the rise in [Na+]i typically seen after the onset of rigor, suggesting that Na+ entry may occur, in part, through voltage-gated Na+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)


Cardiac sarcoplasmic reticulum chloride channels regulated by protein kinase A. In heart cells, several plasma membrane ion channels are targets for phosphorylation. However, it is not known whether sarcoplasmic reticulum (SR) ion channels, which are also essential in the regulation of cardiac function, are regulated by second-messenger systems. Here, we show that a Cl- channel in the cardiac SR membrane is activated by the catalytic subunit of protein kinase A (PKA). Purified cardiac heavy SR vesicles were incorporated into planar lipid bilayers. This channel spontaneously inactivated within a few minutes after the channel was incorporated into the bilayer. Mg-ATP (2-5 mM), but not the nonhydrolyzable ATP analogue 5'-adenylylimidodiphosphate, added to the cis solution prevented this spontaneous channel inactivation. After the inactivation process occurred, the catalytic subunit of PKA (with 0.05 mM Mg-ATP) reactivated this channel. These effects of Mg-ATP and PKA on the Cl- channel were prevented by an inhibitor of PKA. Thus, these results suggest that this SR Cl- channel is a novel target of PKA-dependent phosphorylation in cardiac muscle regulation.


Angiotensin II stimulates two myelin basic protein/microtubule-associated protein 2 kinases in cultured vascular smooth muscle cells. In cultured vascular smooth muscle cells, angiotensin II (Ang II) stimulated a cytosolic protein kinase activity toward myelin basic protein (MBP) in a time- and dose-dependent manner. Phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate also increased the MBP kinase activity. Downregulation of protein kinase C by prolonged treatment of the cells with phorbol 12,13-dibutyrate markedly attenuated the Ang II- and PMA-induced MBP kinase activation. The Ang II- and PMA-stimulated MBP kinase activities were resolved almost equally into two distinct fractions on Mono-Q HR5/5 column chromatography (kinase 1 and kinase 2). The kinase assay in polyacrylamide gel revealed that apparent molecular masses of kinase 1 and kinase 2 were 40 and 45 kd, respectively. Microtubule-associated protein 2 also served as a substrate for both the kinases. Immunoblot analysis with an antiphosphotyrosine antibody suggested that both the kinases were tyrosine-phosphorylated during the action of Ang II. Phosphoamino acid analysis revealed that Ang II and PMA induced phosphorylation of both the kinases on serine/threonine as well as tyrosine residues. Phosphopeptide mapping patterns of kinase 1 and kinase 2 isolated from Ang II-stimulated cells were almost identical with those from PMA-stimulated cells. These results indicate that in vascular smooth muscle cells Ang II activates two species of MBP/microtubule-associated protein 2 kinases mainly through the protein kinase C-signaling pathway and suggest that tyrosine and serine/threonine phosphorylation may be involved in this process.


Endothelin reverses the effects of acidosis on the intracellular Ca2+ transient and contractility in ferret myocardium. Endothelin may play an important role in modulating myocardial contractility under certain pathophysiological conditions. To determine whether endothelin beneficially modulates myocardial contractility in the common clinical condition of acidosis, we compared the effects of endothelin-1 on intracellular Ca2+ transients and isometric contractions under normal (extracellular pH [pH(o)] 7.4) and acidotic (pH(o) 6.4) conditions in ferret papillary muscles (n = 33) loaded with the Ca(2+)-regulated bioluminescent indicator aequorin. A pH(o) of 6.4 was induced by replacing 92% of HCO3- with Cl- in the bathing medium. The effects of endothelin at pH(o) 6.4 differed from the effects at pH(o) 7.4 in that 1) the minimally effective concentration of endothelin was 30-fold lower (1 x 10(-10) M at pH(o) 6.4; 3 x 10(-9) M at pH(o) 7.4) and the concentration-response curve of endothelin was significantly shifted to the left with a decrease in log EC50 from -7.83 +/- 0.13 to -8.92 +/- 0.10 (p less than 0.001), indicating an increased sensitivity of myocardium to endothelin; 2) endothelin produced an increase of approximately 375% in tension development at pH(o) 6.4 (approximately 62% at pH(o) 7.4) (p less than 0.001) without increasing peak [Ca2+]i (approximately 13% increase at pH(o) 7.4, p less than 0.001), indicating an increase in myofilament Ca2+ responsiveness; and 3) endothelin significantly abbreviated (approximately -19%, p less than 0.001) the prolonged intracellular Ca2+ transient induced by acidosis (pH(o) 6.4). In addition, pretreatment with 10 microM of the Na(+)-H+ exchange inhibitor 5-(N-methyl-N-isobutyl)-amiloride significantly attenuated endothelin-induced effects on the intracellular Ca2+ transient and contraction during acidosis.(ABSTRACT TRUNCATED AT 250 WORDS)


In vitro effects of a recombinant toxin targeted to the fibroblast growth factor receptor on rat vascular smooth muscle and endothelial cells. The dominant mechanism responsible for restenosis after angioplasty is believed to be the activation of medial smooth muscle cells (SMCs), leading to their proliferation, migration to the subintima, and further proliferation. To develop novel strategies that might inhibit or prevent restenosis, we previously used a chimeric toxin composed of transforming growth factor-alpha (which targets the epidermal growth factor receptor) and mutated Pseudomonas exotoxin to preferentially recognize and kill rapidly proliferating, versus quiescent, vascular SMCs. We have recently cloned and expressed a recombinant gene encoding Pseudomonas exotoxin with a mutated (nonfunctional) cell recognition domain fused with the ligand acidic fibroblast growth factor, termed aFGF-PE66(4Glu)KDEL; thus, this recombinant toxin targets the fibroblast growth factor receptor. In the present study, we evaluated the relative effects of this chimeric toxin on quiescent versus rapidly proliferating vascular SMCs and also determined whether aFGF-PE66(4Glu)KDEL exerted different effects on SMCs versus endothelial cells. Rapidly proliferating SMCs (grown in 10% fetal bovine serum) were very sensitive to the cytotoxic effects of aFGF-PE66(4Glu)KDEL, whereas cytotoxicity was significantly less when the SMCs were in a quiescent state (grown in medium supplemented with 0.5% fetal bovine serum). The chimeric toxin was also significantly less cytotoxic against endothelial cells. Competition studies using excess acidic fibroblast growth factor indicated that the cytotoxic effects are specifically mediated by the fibroblast growth factor receptor. Thus, the present studies suggest a potentially expanded role of recombinant toxin therapy in restenosis: multiple receptors can be targeted, and cytotoxic effects, at least in vitro, can be preferentially directed to rapidly proliferating vascular SMCs, with relative sparing of vascular endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Mechanisms for the positive inotropic effect of alpha 1-adrenoceptor stimulation in rat cardiac myocytes. alpha 1-Adrenoceptor activation can enhance myocardial contractility, and two possible inotropic mechanisms are an increase in myofilament Ca2+ sensitivity and action potential prolongation, which can increase net Ca2+ entry into cells. In adult rat ventricular myocytes (bath Ca2+, 1 mM; stimulated at 0.2-0.5 Hz), the drug 4-aminopyridine and the whole-cell voltage clamp have been used to control Ca2+ entry and differentiate between the two mechanisms. At 22-23 degrees C the specific alpha 1-adrenoceptor agonist methoxamine (100 microM) prolonged action potential duration at 50% repolarization from 55 +/- 2 to 81 +/- 5 msec, delayed time to peak contraction, and increased shortening amplitude from 5.3 +/- 0.6 to 7.8 +/- 1 microns (n = 18). Reduction of the transient outward current and other K+ currents by methoxamine was the major cause of action potential prolongation in rat myocytes with little change in the L-type calcium current. Block of the transient outward current with 2 mM 4-aminopyridine prolonged action potential duration from 52 +/- 6 to 98 +/- 12 msec and increased unloaded cell shortening from 2.9 +/- 0.4 to 6.6 +/- 0.6 microns (n = 4). Subsequently, methoxamine no longer increased cell shortening, although significant potentiation of twitch amplitude was still seen after a brief rest interval. In voltage-clamp experiments, with 70-500-msec pulses, although membrane currents were reduced, methoxamine had no positive inotropic effect and reduced cell shortening from 5.3 +/- 0.7 to 4.97 +/- 0.8 microns at pulse potentials positive to -40 mV. Similar alpha 1-adrenoceptor responses were observed at 35 degrees C during action potential and voltage-clamp experiments, which could be blocked by 10 microM prazosin. In myocytes loaded with the Ca2+ indicator indo-1, alpha 1-adrenoceptor stimulation or 4-aminopyridine both increased cell contraction and intracellular Ca2+ transients by similar amounts. As in unloaded cells, prior exposure to 4-aminopyridine prevented any inotropic effect of methoxamine without changing the systolic intracellular Ca2+ transient. The results indicated that under our experimental conditions positive inotropy in rat cardiomyocytes on exposure to alpha 1-adrenoceptor agonists was strongly correlated with the action potential prolongation that accompanied K+ current reduction. In addition, modulation of K+ channels could occur independent of changes in contractility and/or [Ca2+]i.


The effect of graded haemorrhage on erythropoietin production in the immature ovine foetus. 1. Basal and haemorrhage-stimulated erythropoietin (Epo) and ACTH levels were measured in the chronically cannulated immature ovine foetus (less than 125 days) by radio immunoassay (RIA). 2. Basal erythropoietin levels were found to be higher than those previously reported in the late gestation (greater than 130 days) ovine foetus, but were lower than those observed in the neonatal lamb. 3. In control foetuses (Protocol 1) the small degree of haemorrhage associated with the sampling procedure increased the plasma Epo values from 11.4 +/- 3.0 (n = 5) mU/mL to 23.8 +/- 4.3 mU/mL at 24 h (mean +/- s.e.m.). There was a significant monotonic increase with time (F = 16.4; d.f. 1,19; P = 0.001). An initial haemorrhage of approximately 10% blood volume (Protocol 2) increased plasma Epo values from 7.3 +/- 2.3 to 24.2 +/- 7.1 mU/mL (n = 3). 4. Haemorrhage of 20% fetal blood volume (Protocol 3) produced an increase in plasma Epo from 9.3 +/- 1.7 to 54.7 +/- 15.5 mU/mL at 6 h and to 57.6 +/- 7.3 mU/mL at 24 h (n = 5). By repeated measures ANOVA, the effect of the 20% haemorrhage was significant when compared with the control group (F = 7.32, d.f. 2,16, P = 0.006). There was a significantly greater decrease in haematocrit (F = 6.7, d.f. 2,20, P = 0.004) and haemoglobin (F = 5.0, d.f. 2,20, P = 0.013) in animals of Protocol 3 than in those of Protocol 1. 5. Fetal blood gases and plasma adrenocorticotropic hormone (ACTH) did not alter with haemorrhage, indicating the tolerance of the foetus to this degree of haemorrhage.


Presynaptic alpha 2-adrenoceptor-mediated modulation of norepinephrine release from vascular adrenergic neurons in reduced renal mass salt hypertensive rats. The present study was designed to investigate the presynaptic alpha 2-adrenoceptor function to inhibit norepinephrine (NE) release in blood vessels of reduced renal mass salt hypertensive rats (Na-loaded HT). Isolated perfused mesenteric vasculatures were prepared from Na-loaded HT and normotensive control rats (NT-control), and the NE release and vascular responsiveness were examined. Periarterial nerve stimulation caused a significantly greater release of NE and pressor responses in Na-loaded HT than in NT-control. Yohimbine, a potent alpha 2-adrenoceptor antagonist, demonstrated the facilitatory effects on NE release during nerve stimulation. The effects were significantly attenuated in Na-loaded HT compared with NT-control. These results demonstrate that vascular sympathetic nervous activity might be enhanced in Na-loaded HT. Furthermore, the increased NE release from vascular adrenergic neurons in Na-loaded HT could partially depend on impaired presynaptic alpha 2-adrenoceptor-mediated modulation, which might contribute to the pathogenesis and maintenance of this form of salt-dependent hypertension.


Mineral-matrix interactions in bone and cartilage. Mineral-matrix interactions regulate the process of hydroxyapatite formation in bones and teeth. In mineralizing tissues, many anionic macromolecules bind to mineral. By means of this binding, such molecules are able to regulate the size and shape of the mineral crystals, determine the site of initial crystal deposition, and determine the type of mineral crystals deposited. Collagen, which provides a template for hydroxyapatite deposition; extracellular matrix vesicles, which provide a protected environment for crystal deposition; and noncollagenous matrix proteins that have high affinities for hydroxyapatite have all been shown to affect mineralization in vitro. Some of the noncollagenous proteins have been shown to be capable of promoting and inhibiting mineral formation and growth, depending on their concentration and whether they are immobilized or free in solution. This review surveys the current understanding of mineral-matrix relationships involved in endochondral, intramembranous, and appositional bone formation, outlining the way in which mineral deposition is controlled in mammalian calcified tissues. The structural basis for the interaction of the matrix molecules with hydroxyapatite is presented, as is the in vitro and in situ data implicating the matrix molecules that interact with hydroxyapatite to control mineralization.


Inhibition and affinity chromatography of chicken lung angiotensin I-converting enzyme with captopril. 1. Angiotensin I-converting enzyme (EC 3.4.15.1) has been purified to electrophoretic homogeneity from chicken lung by using a facile two-step protocol which included affinity chromatography on Sepharose-bound captopril. 2. Captopril was a potent inhibitor of chicken lung angiotensin I-converting enzyme with Ki values of 2.0 nmol/l and 1.6 nmol/l for detergent-extracted and trypsin-extracted angiotensin I-converting enzymes, respectively. 3. Molecular weight comparison of trypsin-extracted (M(r)270,000) and detergent-extracted (M(r)690,000) angiotensin I-converting enzyme indicated that membrane-binding sequence contributed to a large extent to the enzyme molecule. 4. Kinetic properties of both forms of the enzyme suggested that the membrane-bound sequence contributed to an increase of the enzyme-substrate affinity.


Polyphosphoinositide synthesis and protein phosphorylation in the plasma membrane from full-grown Bufo arenarum oocytes. 1. Polyphosphoinositide content and phosphorylation of lipids and proteins were analyzed in oocytes of the toad Bufo arenarum Hensel. 2. Plasma membrane-enriched fractions obtained from full-grown, prophase-arrested oocytes incorporated 32P into both phospholipids and proteins after incubation with [gamma-32P]ATP in an Mg(2+)-containing medium. Phosphatidylinositol 4-phosphate (PIP), phosphatidate (PA) and phosphatidylinositol-4,5-bisphosphate (PIP2) were the only labelled lipids. The 32P incorporation depended on incubation time, the amount of protein, and the ATP concentration. 3. Autoradiography of polyacrylamide gel electropherograms and scintillation counting showed that the radioactivity was mainly associated with a group of membrane proteins having an M(r) of 87,000. 4. This paper provides evidence for the capacity of prophase-arrested oocytes from Bufo arenarum to synthesize polyphosphoinositides and to phosphorylate distinct membrane proteins.


Isolation and partial characterization of binding proteins for immobilized delta endotoxin from solubilized brush border membrane vesicles of the silkworm, Bombyx mori, and the common cutworm, Spodoptera litura. 1. Brush border membrane vesicles (BBMV) were prepared from the Bombyx mori, and Spodoptera litura, midguts. BBMV was solubilized. 2. Activated delta endotoxin from Bacillus thuringiensis were immobilized. 3. Solubilized BBMV proteins were applied to the toxin column and the proteins bound were analyzed by SDS-PAGE. 4. In the case of B. mori M(r) = 220,000, 150,000 and 130,000 and in the case of S. litura 160,000 bands were detected. 5. The bindings were inhibited by N-acetyl galactosamine and mannose. 6. The binding proteins applied onto a Con A column and eluted by 0.1 M methyl-alpha-glucose, suggesting that hybrid type sugar sidechain may involve in the interaction.


Changes in public concern about transmission of AIDS from dentist to patient after CDC report. This study was conducted to examine the impact on the public of a report from the Centers for Disease Control of the possible transmission of AIDS from a Florida dentist to his patient. Differences in the findings between two identical telephone surveys conducted approximately five months apart are reported. The second survey occurred approximately three months following the CDC report. The response rates to spring and fall 1990 surveys were 68.9 percent and 69.9 percent, respectively. Approximately 60 percent of the respondents to the fall survey reported that they had read or heard of the incident. Public concern about the transmission of AIDS in the dental office increased significantly following the CDC report. This concern was expressed in a decreased willingness to remain in the practice of dentists infected with AIDS or those who treated infected patients. In addition, respondents to the fall survey were less respectful of provider and patient confidentiality.


[Preoperative regional radiofrequency thermochemotherapy of colorectal cancer: clinical and pathological studies]. Thirty patients with advanced colorectal cancer were treated preoperatively by combined regional radiofrequency hyperthermia and 5-FU. After the treatment, the gross tumor regressive rate was 76.7%, and the tumor integral resection rate of colorectal cancer was 92.3%. Nine patients with advanced colorectal cancer, considered unoperable and treated with thermochemotherapy, underwent a successful en bloc resection of their tumors. Marked destruction of cancer cells was observed after thermochemotherapy. Moderate-severe damage of cancer cells was observed in 76.9% of patients in the thermochemotherapy group, 33.3% in the chemotherapy group, and 42.3% in the operation group (P less than 0.005). The results indicate that the treatment is more effective in the treatment of colorectal cancer than 5-FU chemotherapy alone.


[Total gastrectomy via thoracotomy for cancer of gastric cardia and fundus. Report of 90 cases]. From 1978 through 1990, 90 total gastrectomy with esophagojejunostomy via thoracotomy were performed for the treatment of cancer of cardia or fundus of stomach. 85/90 patients were at TNM III-stage and 5/90-at IV-stage. 30-day post-resectional mortality was 1.1%. Five-year survival rate was 13.8%. 14CO2 respiratory test and clinical evaluation of 34 post-operative patients showed that total gastrectomy may decrease the incidence of positive residual cancer along the incision lines. It may also spare the patient from small-stomach syndrome. There was no statistical difference in postoperative fat absorption and digestive function between ordinary proximal subtotal gastrectomy and total gastrectomy.


Cytomegalovirus peritonitis in a patient with the acquired immunodeficiency syndrome. Peritonitis has been reported infrequently in patients with the acquired immunodeficiency syndrome (AIDS). Intestinal or colonic perforation resulting from cytomegalovirus (CMV) enteritis is the most common cause of peritonitis in these patients. We report a patient with CMV peritonitis occurring in the absence of perforation (primary peritonitis) to alert physicians to this potentially treatable disorder.


Abortion of virologically negative foetuses following experimental challenge of pregnant pony mares with equid herpesvirus 1. From 1988 to 1991, 51 pregnant pony mares were challenged intranasally or by aerosol with an isolate of EHV-1 (AB4) originally recovered from a quadriplegic mare. This resulted in 32 abortions, occurring from 9 to 29 days after infection. In 14 of the early abortions (Days 9-14), EHV-1 was not demonstrated in the foetal tissues by virus isolation or immunostaining despite no other non-viral cause for the abortion being evident. Application of the polymerase chain reaction to foetal tissues from 9 of these cases also proved negative. One of the 14 mares was destroyed immediately after abortion, and post-mortem examination revealed severe and widespread vasculitis, thrombosis and secondary ischaemic damage in the endometrium with replication of EHV-1 in endothelial cells. These findings suggest that EHV-1 abortion can occur due to endometrial damage without the establishment of a foetal infection.


An intracellular ATP-dependent calcium pump within the yeast Schizosaccharomyces pombe, encoded by the gene cta3. We have permeabilized the plasma membranes of Schizosaccharomyces pombe cell with nystatin and measured ATP-dependent Ca2+ uptake in the presence of KNO3 and a protonophore in order to inhibit Ca2+ uptake into the vacuole. ATP-dependent Ca2+ accumulation into non-vacuolar Ca(2+)-storing organelles was detected. This Ca2+ uptake activity was maximal at pH 6 and inhibited by vanadate, the inhibitor of P-type ATPases. The null mutation of cta3, a putative Ca2+ gene, [Ghislain, M., Goffeau, A., Halachmi, D. and Eilam, Y. (1990) J. Biol. Chem. 265, 18400-18407] strongly reduced the level of ATP-dependent Ca2+ uptake into non-vacuolar intracellular storing organelles. This result suggests that cta3 encodes an intracellular ATP-dependent Ca2+ pump. The residual ATP-dependent Ca2+ uptake in the mutant strain indicated the presence of a second nonvacuolar, intracellular Ca(2+)-ATPase encoded by a different gene.


Acetylcholinesterases of the nematode Steinernema carpocapsae. Characterization of two types of amphiphilic forms differing in their mode of membrane association. We analyzed the molecular forms of acetylcholinesterase (AChE) in the nematode Steinernema carpocapsae. Two major AChEs are involved in acetylcholine hydrolysis. The first class of AChE is highly sensitive to eserine (IC50 = 0.05 microM). The corresponding molecular forms are: an amphiphilic 14S form converted into a hydrophilic 14.5S form by mild proteolysis and two hydrophilic 12S and 7S forms. Reduction of the amphiphilic 14S form with 10 mM dithiothreitol produces hydrophilic 7S and 4S forms, indicating that it is an oligomer of hydrophilic catalytic subunits linked by disulfide bond(s) to a hydrophobic structural element that confers the amphiphilicity to the complex. Sedimentation coefficients suggest that 4S, 7S, 12S forms correspond to hydrophilic monomer, dimer, tetramer and that the 14S form is also a tetramer linked to one structural element. The second class of AChE is less sensitive to eserine (IC50 = 0.1 mM). Corresponding molecular forms are hydrophilic and amphiphilic 4S forms (monomers) and a major amphiphilic 7S form converted into a hydrophilic dimer by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C. This amphiphilic 7S form thus possesses a glycolipid anchor. It appears that Steinernema (a very primitive invertebrate) presents AChEs with two types of membrane association that closely resemble those described for amphiphilic G2 and G4 forms of AChE in more evolved animals.


A major oligomeric fibroblast proteoglycan identified as a novel large form of type-XII collagen. Cultured chick embryo skin fibroblasts release a major component with a native molecular mass of about 1 MDa, which resolves into three polypeptide bands of about 300, 350 and 600 kDa upon reduction. We report here the purification of this oligomeric protein and show, by means of polyclonal and monoclonal antibodies, that its three polypeptide constituents are closely related. The 600-kDa polypeptide is likely to be a dimer of two smaller subunits which are cross-linked by non-reducible bonds. By electron microscopy, isolated oligomeric molecules exhibit a novel cruciform structure with a large central globular domain. One arm has the shape of a thin rod about 70 nm in length. The three other arms are thicker, longer (90 nm) and flexible, and carry a prominent double globule at their distal ends. Collagenase treatment of the oligomeric fibroblast protein yields two resistant fragments of about 270 kDa and 320 kDa. The intact 350-kDa and 600-kDa (but not the 300-kDa) polypeptides are chondroitinase sensitive and labeled by metabolic incorporation of [35S]sulfate; collagenase treatment does not remove any [35S] sulfate. Hence, the intact fibroblast protein has glycosaminoglycan chains attached to its non-collagenous domain. Three amino acid sequences obtained from chymotryptic fragments of the fibroblast protein correspond to sequences predicted for chick type-XII collagen from its full-length cDNA [Yamagata, M., Yamada, K. M., Yamada, S. S., Shinomura, T., Tanaka, H., Nishida, Y., Obara, M. & Kimata, K. (1991) J. Cell Biol. 115, 209-221]. However, the novel fibroblast protein described here differs significantly from previously isolated forms of type-XII collagen: its subunits are larger by one third, and it is a proteoglycan.


The oxygen dependence of the mitochondrial respiration rate in ascites tumor cells. The effect of the oxygen concentration on the rate of oxygen consumption by 786 and TA3 ascites tumor cell lines has been determined under steady-flow conditions with a membraneless fast-responding O2 electrode and using ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine as electron donors. The reaction was initiated by rapid injection of O2 into anaerobically incubated test system. The time-dependence of the intact cell respiration showed three distinct phases; an early very fast but short duration phase, a subsequent slow phase that prevailed for most of the reaction period and a third phase which preceded the reestablishment of anaerobiosis. Kinetic analysis of the reaction indicated a linkage between the catalytic efficiency and the transmembrane electrochemical potential. The rates of O2 uptake, obtained in the presence of both protonophores and ionophores, were monotonic and pseudo-first order over 90% of the course of O2 consumption. Extrapolation of the observed rates to zero time, at which zero delta mu H+ and thus constant flow prevails, was used to calculate the oxygen concentration for the half-maximal respiratory rate, which was found to be in the range 1.55-2.10 microM O2. No noticeable variation in the value of this kinetic parameter was found between the two cell lines used. Possible reasons for discrepancies in published reports on the oxygen dependence of the cytochrome c oxidase activity in various mitochondrial and reconstituted systems are discussed.


Inactivation of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by phenylglyoxal. Evidence for essential arginine residues. Treatment of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with the arginine-specific reagent, phenylglyoxal, irreversibly inactivated both 6-phosphofructo-2-kinase and fructose-6-bisphosphatase in a time-dependent and dose-dependent manner. Fructose 6-phosphate protected against 2,6-phosphofructo-2-kinase inactivation, whereas MgGTP protected against fructose-2,6-bisphosphatase inactivation. Semi-logarithmic plots of the time course of inactivation by different phenylglyoxal concentrations were non-linear, suggesting that more than one arginine residue was modified. The stoichiometry of phenylglyoxal incorporation indicated that at least 2 mol/mol enzyme subunit were incorporated. Enzyme which had been phosphorylated by cyclic-AMP-dependent protein kinase was inactivated to a lesser degree by phenylglyoxal, suggesting that the serine residue (Ser32) phosphorylated by cyclic-AMP-dependent protein kinase interacts with a modified arginine residue. Chymotryptic cleavage of the modified protein and microsequencing showed that Arg225, in the 6-phosphofructo-2-kinase domain, was one of the residues modified by phenylglyoxal. The protection by fructose 6-phosphate against the labelling of chymotryptic fragments containing Arg225, suggests that this residue is involved in fructose 6-phosphate binding in the 6-phosphofructo-2-kinase domain of the bifunctional enzyme.


Characterisation by mass spectrometry and 1H-NMR of novel hexasaccharides among the acidic O-linked carbohydrate chains of bovine submaxillary mucin. The acidic oligosaccharide alditols released from bovine submaxillary-gland mucin by Carlson degradation were investigated by a combination of liquid secondary-ion mass spectrometry, methylation analysis and 1H-NMR. Among the largest structures identified were four branched hexasaccharides, three of them novel, comprising two separate pairs of structures. One pair contained the sequence Fuc(alpha 1-2)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-) (Fuc, L-fucose), at C3 of N-acetylgalactosaminitol and differed only by substitution at C6 by N-acetylneuraminic or N-glycolylneuraminic acid. The other pair also differed in substitution of the sialic acid linked at C6 and contained the GalNAc-(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)GlcNAc(beta 1-), sequence at C3 of N-acetylgalactosaminitol. The Lewis(y) and blood-group-A determinants of these sequences have not been found previously in the acidic oligosaccharides of bovine submaxillary-gland mucin, although they have recently been characterised in the neutral chains of bovine submaxillary-gland mucin.


Cytochrome b positive X-linked chronic granulomatous disease: a normal cell surface expression of cytochrome b. The polymorphonuclear (PMN) cells from a patient with cytochrome b positive X-linked chronic granulomatous disease (Xb+ CGD) were studied using flow cytometry. Both the cell surface expression of monoclonal antibody defined cytochrome b and the superoxide production (intracellular 2',7'-Dichlorofluorescin Diacetate oxidation) were investigated at a single cell level. Flow cytometry clearly demonstrated the complete absence of superoxide production in the patient's PMN cells, the mosaicism in his mother's PMN cells and also indicated the normal cell surface expression of cytochrome b. The results obtained by Western blot analysis and reduced-minus-oxidized spectra confirmed the presence of functional and normal amounts of cytochrome b. We concluded that this is a case of Xb+ CGD with a normal cell surface expression of cytochrome b.


Defect of epidermal 12(S)-hydroxyeicosatetraenoic acid receptors in psoriasis. 12-hydroxyeicosatetraenoic acid (12-HETE) is assumed to play a central role in the pathophysiology of psoriasis. Since its effects in skin are mediated by specific high-affinity receptors, we studied the receptor characteristics in cultured epidermal cells from involved and apparently healthy skin of psoriasis patients by radioligand binding assay. Involved and uninvolved psoriatic epidermal cells showed a fourfold decrease in the number of 12-HETE binding sites as compared with normal healthy individuals and patients with atopic dermatitis, while receptor affinity remained unchanged. The decrease in receptor number was evident in psoriatic cells even in long-term culture and was not due to receptor down-regulation, defective response to interferon gamma or to protease degradation of receptor protein. The decrease in the number of 12-HETE receptors detectable even in clinically normal psoriatic skin functionally leads to diminished 12-HETE uptake and may thus represent a primary central molecular defect in the pathophysiology of the disease.


Erythrocyte Li+/Na+ and Na+/H+ exchange, cardiac anatomy and function in insulin-dependent diabetics. It has been proposed that an increased activity of cell membrane Na+/H+ exchange, mirrored by increased erythrocyte Li+/Na+ exchange, may facilitate cell hypertrophy and hyperplasia. Patients with insulin-dependent diabetes mellitus may develop a specific cardiomyopathy with systolic and diastolic abnormalities and increased thickness of the left ventricle. Therefore, we have investigated the relationships between erythrocyte Li+/Na+ and Na+/H+ exchange and echocardiographic parameters in 31 male insulin-dependent diabetics (aged 17-68), in good metabolic control. Three had untreated mild hypertension. In all patients the urinary albumin excretion rate was less than 200 micrograms min-1. Ten patients had a Li+/Na+ countertransport higher than 0.37 mmol l-1 cell h-1, the upper normal limit for our laboratory (0.49 +/- 0.10, mean +/- SD). In comparison with the patients with normal countertransport, they had increased interventricular septum thickness and relative wall thickness (h/r). End diastolic volume and cardiac index were reduced while blood pressure and urinary albumin excretion rate were similar. In the whole study group, interventricular septum thickness was significantly correlated to Li+/Na+ exchange (r = 0.61, P less than 0.001) and Na+/H+ exchange (r = 0.35, P less than 0.05), independently of the effect of age and blood pressure. Posterior wall thickness was correlated to Li+/Na+ exchange (r = 0.38, P less than 0.05) and h/r to Li+/Na+ exchange (r = 0.41, P less than 0.05) and to Na+/H+ exchange (r = 0.44, P less than 0.05). Li+/Na+ exchange was negatively correlated to cardiac index (r = -0.37, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Some pharmacokinetic and pharmacodynamic interactions between digoxin and gentamicin. Some pharmacokinetic and pharmacodynamic interactions between digoxin and gentamicin were studied in experiments on rabbits, guinea-pigs and cats. An increase of digoxin serum levels and changes in some basic pharmacokinetic parameters of digoxin (t1/2 alpha t1/2 beta, AUC, C1) were found in gentamicin-pretreated rabbits, the changes being dependent on the dose and schedule of administration. The most pronounced changes were those in digoxin kinetics during simultaneous 5-day treatment with digoxin (0.035 mg/kg i.v.) and nontoxic (10 and 2 mg/kg) doses of gentamicin. The toxicity of digoxin in guinea-pigs, assessed by administration of lethal doses of digoxin, was increased only after the highest dose of gentamicin (100 mg/kg), while after nontoxic or close to therapeutic doses (10 and 2 mg/kg) of gentamicin, the digoxin toxicity was either unchanged or even decreased. Digoxin decreased the nerve-muscle blocking effect of gentamicin on cat ischiadicus-gastrocnemius preparation. The possible mechanisms involved are discussed.


Central benzodiazepine receptors in human brain: estimation of regional Bmax and KD values with positron emission tomography. Studies of central benzodiazepine receptors in the human brain in vivo are now possible using positron emission tomography (PET) and [11C]flumazenil. With the aim of measuring Bmax and Kd in brain regions, we used a two-injection [11C]flumazenil (at high and low specific radioactivity, respectively) pseudo-equilibrium paradigm to evaluate, in seven unmedicated healthy volunteers, the relative merits of three 'reference' structures (pons, hemispheric white matter and corpus callosum) in which the free radioligand concentration in brain tissue was estimated 15-40 min after i.v. injection of the radioligand. By means of high-resolution PET, the Bmax and Kd were calculated for each subject in 18 gray matter structures, based on a two-point Scatchard plot. We found that the use of the corpus callosum as reference often resulted in spurious Bmax and Kd values. The pons was the best reference structure because it provided satisfactory Bmax values (closest to in vitro data) and most consistent Kd values, and was the region easiest to sample on PET images. The pattern of regional Bmax was consistent with that expected from in vitro studies, with values highest in the cerebral cortex, intermediate in the cerebellum, and lowest in the striatum and the thalamus. The Kd values were uniform among regions and were consistent with earlier in vitro and in vivo data. This work documents the feasibility of estimating Bmax and Kd of central benzodiazepine receptors in multiple brain regions for clinical research.


High performance liquid chromatography and preliminary pharmacokinetics of rufloxacin and its metabolites N-desmethylrufloxacin and rufloxacinsulfoxide in plasma and urine of humans. An HPLC analysis was developed for the measurement of rufloxacin and two of its possible metabolites N-desmethylrufloxacin and rufloxacinsulfoxide. Humans are unable to form these two metabolites, while no other metabolites could be detected in the HPLC chromatogram. The half-life of elimination of rufloxacin in two human subjects was 27 and 38 h respectively, while a constant percentage of 25-26% of the dose is excreted unchanged in the urine.


Release of neuropeptide Y and noradrenaline during afferent nerve stimulation. The present study was carried out to investigate the possibility that noradrenaline (NA) and neuropeptide Y (NPY) are co-released after afferent vagal or saphenous stimulation (1, 5, 10 and 20 Hz) in chloralose-anaesthetized dogs. Electrical stimulation of the vagus elicited an increase in plasma NA levels for the 5, 10 and 20 (but not 1) Hz frequencies. Blood pressure only increased after a 20-Hz stimulation. In contrast, no change in plasma NPY levels was observed whatever the frequency of stimulation. Electrical stimulation of the saphenous nerve failed to change plasma NA and NPY levels. The present data suggest that (1) the release of NA varies according to the frequency of stimulation of nociceptive fibres, (2) NPY release does not seem to be involved in the pressor effect elicited by the stimulation of nociceptive-sensitive fibres, and (3) NPY and NA release are not necessarily linked.


Effects of pharmacological manipulation of dopaminergic and cholinergic neurotransmission in genetically dystonic hamsters. In an inbred line of Syrian hamsters, attacks of sustained dystonic postures of the limbs and trunk can be initiated by handling or mild environmental stimuli (e.g. new cage). The severity of the dystonic syndrome in these mutant hamsters (gene symbol dtSZ) is age-dependent, with a peak at about 30-40 days of age. A scoring system for grading the type and severity of the dystonic attacks can be used to study the activity of drugs against dystonic movements with individual pre- and post-drug vehicle trials as control. The effects of drugs which alter dopaminergic or cholinergic functions in the brain were studied in selectively bred dystonic hamsters and age-matched non-dystonic controls. The dopamine precursor levodopa (injected together with carbidopa) and the dopamine receptor agonist apomorphine increased the severity of dystonia in hamsters when administered prior to the age of maximum severity of dystonia. A very similar effect was observed with the cholinomimetic pilocarpine. In contrast, the dopamine receptor antagonist haloperidol caused a marked overall reduction in dystonic movements. Anticholinergic drugs, i.e. trihexyphenidyl and biperiden, increased the latency to onset of the dystonic attack, but did not reduce its severity. No differences were observed between dystonic and non-dystonic hamsters with respect to extent and duration of stereotypies induced by dopaminergic and cholinergic drugs or hypolocomotion and catalepsy produced by haloperidol. The data suggest that dopaminergic hyperactivity might be involved in the pathophysiology of dystonia in dtSZ mutant hamsters.


Immune-complex alveolitis in the rat: evidence for platelet activating factor and leukotrienes as mediators of the vascular lesions. In the present study we investigated the involvement of lipid mediators in an experimental model of immune-complex alveolitis induced in rat lungs by intrabronchial instillation of rabbit antibodies to ovalbumin followed by i.v. injection of the antigen. It was found that the reaction did not induce detectable oedema, as measured by the dry:wet weight ratio. A marked influx of neutrophils was observed in the bronchoalveolar lavage fluid, progressing from 6 to 24 h in parallel with the development of haemorrhagic lesions in lung parenchyma. The intensity of these lesions, evaluated by the concentration of extravascular haemoglobin, was not significantly affected by pretreatment of the animals with a cyclo-oxygenase inhibitor (indomethacin), a thromboxane inhibitor (econazole) or a thromboxane antagonist (L-655,240). However, the antagonists of platelet activating factor (PAF), WEB-2086 and BN-52021, and the lipoxygenase inhibitors, nor-dihydroguaiaretic acid and L-663,536, all significantly inhibited the haemorrhagic lesions. A peptide leukotriene antagonist (L-660,711) had no effect. Furthermore, the PAF antagonists inhibited the levels of LTB4, but not of PGE2 and thromboxane, released into the bronchoalveolar space 1 h after induction of the reaction. These results suggest that the haemorrhagic lesions in this model of immune-complex alveolitis are mediated by PAF and leukotrienes, possibly LTB4.


Effects of thromboxane agonists on cardiac adrenergic neurotransmission. The effects of thromboxane B2 (TxB2) and of two thromboxane mimetics, dl-(9,11), (11,12)-dimethano-TxA2 (ONO 11006) and 9,11-dideoxy-11,9-epoxymethano prostaglandin F2 alpha (U46619) on the cardiac response to adrenergic nerve stimulation in isolated guinea-pig atria were evaluated. All the agonists dose dependently reduced the positive inotropic effect induced by field stimulation, U46619 being the most active. The inhibitory effect of U46619 was reduced by the thromboxane receptor antagonists, sulotroban and AH 23848B. U46619 did not significantly reduce the positive inotropic effect induced by exogenous noradrenaline. However U46619 was unable to modify the tritium overflow induced by field stimulation in preparations preloaded with [3H]noradrenaline. In addition to this influence on adrenergic neurotransmission, U46619 also had a direct positive inotropic effect on cardiac contractility, which was antagonized by AH 23848B. These results indicate that U46619 reduces the cardiac response to sympathetic nerve stimulation and that is also has a direct stimulatory effect on cardiac muscle.