Modulation of GM-CSF receptor beta-subunit and interleukin-6 receptor mRNA expression in a human megakaryocytic leukemia cell line.

Our present study was designed to clarify the mechanism by which the same megakaryocyte progenitor cells respond to various cytokines at different stages of megakaryocyte development. We examined the changes in mRNA expression of granulocyte macrophage colony-stimulating factor receptor beta-subunit (GM-CSFR beta-subunit), which was a common subunit of a high-affinity interleukin-3 receptor (IL-3R) and a high-affinity GM-CSFR, and interleukin-6 receptor (IL-6R) during megakaryocyte development in a human megakaryocytic leukemia cell line (CMK) which could proliferate and/or differentiate in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA), IL-3, GM-CSF, and IL-6. We found that GM-CSFR beta-subunit mRNA was expressed constitutively in CMK cells and was transiently down-regulated by TPA and IL-6, while the expression of IL-6R mRNA was increased by TPA in association with the differentiation of megakaryocytes. Furthermore, the TPA-induced down-regulation of GM-CSFR beta-subunit mRNA expression and its recovery were blocked by cycloheximide (CHX), a protein synthesis inhibitor, suggesting that these modulations required de novo protein synthesis. These findings imply that multi-lineage cytokines such as GM-CSF and IL-3 may contribute preferentially to the regulation of the earlier development of megakaryocyte progenitor cells with high densities of multi-lineage cytokine receptors, while IL-6 may be limited in its action to supporting the maturation of more differentiated megakaryocyte progenitor cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Obstructive solitary bronchial non-Hodgkin's lymphoma--a rare presentation of primary extranodal disease.

Endobronchial involvement by non-Hodgkin's lymphoma (NHL) is uncommon and usually occurs in the presence of more generalised disease. Solitary endobronchial lymphoma in the absence of disease elsewhere is extremely rare. In this report we describe a patient with an obstructing endobronchial mass which was the initial manifestation of NHL. The patient was treated initially with radiotherapy followed by cyclic combination chemotherapy with initial complete resolution of the endobronchial disease. However, he subsequently developed widespread lymphoma. This unusual presentation of NHL is discussed and the relevant literature is reviewed.


Granulocytic sarcomas of small intestine and brain are associated with acute myelomonocytic leukaemia with abnormal eosinophils and inversion of chromosome 16.

We report two cases of acute myelomonocytic leukaemia with abnormal eosinophils (M4Eo) in which the presenting feature was small bowel obstruction. We suggest there is a unique clinicopathological association between small intestine involvement with leukaemia and the M4Eo subtype. Central nervous system involvement by myeloblastoma occurred in one of the two cases which is a recognised feature of M4Eo and should necessitate prophylaxis with intrathecal therapy. Inversion of chromosome 16 which is a cytogenetic marker for M4Eo was demonstrable in one of the two cases.


Bone marrow necrosis and human parvovirus associated infection preceding an Ph1+ acute lymphoblastic leukemia.

A case of bone marrow necrosis associated with a serologically documented recent Parvovirus B 19 infection which preceded the development of PH1+ acute lymphoblastic leukemia is reported. No conclusions can be drawn on the basis of a single case but the question of the role of human Parvovirus B19 in the pathogenesis of bone marrow necrosis is discussed. It is suggested that the virus may act as a co-factor for the induction of bone marrow necrosis, in some cases.


The evolution of private geriatric care management. Guidelines for responsible practice.

Fueled by a series of demographic, occupational, and programmatic trends, private firms providing care management services to the elderly and their families have experienced rapid growth. A range of leadership challenges faces social workers entering this specialty. Exemplary ethical and professional behavior will ultimately determine the extent to which private geriatric care managers become a central component of the gerontological services network.


Private geriatric care management: how families are served.

This article provides a synopsis of a 1987 study of private care management and looks at the reasons why individuals and families seek out and use the services of private care managers. Among the most common reasons are: to negotiate the long-term-care system, to get help in filling out forms, to get objective assistance in assessing options, to mediate family conflict over what to do, and to plan and monitor the care of family members.


The business of for-profit case management.

The growing number of older Americans is creating a major market for home care and case management services. Large corporations are entering the field to cash in on the beckoning profits. Individuals who are moving from not-for-profit case management agencies to start their own for-profit businesses bring solid values and much needed experience. However, they must learn to respect their knowledge for its financial worth and to apply solid business skills if they are to succeed against the large diversified corporations entering the field.


Factors related to successful goal attainment in case management.

Goal planning is fundamental to the case management process. Manager and client identify specific long-term goals and then systematically break them down into the incremental behaviors needed to be accomplished to achieve each goal. The goal-setting process may fail as a result of four types of factors: (1) behavioral, (2) cognitive, (3) affective, and (4) environmental. To decrease the likelihood of failure, case managers must be knowledgeable about goal planning. They must help the client set goals that are expressed positively, that are client focused, realistic and achievable, measurable, and easily understood, with one observable outcome per goal. For both case manager and client, setting and attaining goals must be viewed as part of a continuing learning process, rather than a success-failure dichotomy.


Across the great divide. Integrating acute, post-acute and long-term care.

The importance of integrating acute, post-acute and long-term care is underscored by analyses of the extent of medical complexity among the SHMO enrollee population that qualified for a long-term care benefit on functional (not medical) criteria. In the SHMO programs, initiatives to promote integration of services at all levels of care have largely come from the care management units. Integrative practices will vary in different organizational settings. Examples from two very different SHMO sites illustrate how integrative practices are implemented. It is concluded that in order to serve adequately a medically complex and functionally impaired elder population a key function of care management is to develop cooperative relationships, and mechanisms for information exchange, with providers at all system levels, reaching across the great divide between the acute and long-term care systems.


[Urinary excretion of magnesium and calcium in patients with pulmonary squamous cell carcinoma treated by ionizing radiation].

Radiotherapy in patients with squamous cell lung cancer produced an increase of urinary magnesium and calcium secretion. This is probably due to the cytolysis of malignant cells and shifting of the acid--base balance toward acidity values. This interpretation is justified by the significant correlation between the regression of tumor and increased secretion of the intercellular cation--magnesium.


[Increased activity of erythrocyte superoxide dismutase as the result of adaptation to long-term oxygen therapy].

Activity of erythrocyte superoxide dismutase (SOD) was assessed in 17 patients with decompensated chronic cor pulmonale and in 15 healthy volunteers. Also the serum levels of uric acid and hematological parameters were analyzed. Recordings were made in ten day intervals during long term oxygen therapy. The authors have found the increase of SOD activity in the treated patients which can be interpreted as a generalized adaptation of the organism to long term oxygen therapy.


[Effect of TFX (Polfa) on the course of experimental bacterial infections in mice treated with ciprofloxacin, amikacin and cefoperazone].

The effect of TFX-Polfa administered alone or with three antibiotics on the survival time in mice infected with E.coli and S.aureus was estimated. TFX has been administered 48, 24, 2 hours before and 24, 48 and 72 hours after infection, in doses of 10 mg/kg of body weight. Ciprobay has been administered 2 hours after infection, and every 24 hours during the experiment in dose of 15, 7.5 and 3.75 mg/kg of body weight. Amikin has been used in doses of 7.5, 3.75 and 1.87 mg/kg of body weight, Cefobid in doses of 30, 15 and 7.5 mg/kg of body weight. The antibiotics have been administered 2 hours after infection and every 12 hours to the end of experiment. Animals have been observed 10 days after infection. The positive effect of TFX on the survival time in mice infected with E.coli has been observed, but this preparation has given a little effect on the tested parameter in mice infected with S.aureus. Best antibacterial effect of Ciprobay and Amikin has been observed after administration of clinical doses. However, simultaneous therapy with TFX and tested antibiotics resulted in various (positive and negative) effects on the survival time during 10 days of observation.


[Effect of ofloxacin, ciprofloxacin and nalidixic acid on serum theophylline level in patients treated with methylxanthine preparations].

The effect of two new quinolones, ofloxacin and ciprofloxacin and nalidixic acid on serum theophylline level in 17 asthma patients treated with methylxanthines was evaluated. Each quinolone was administered one tablet twice daily to all patients for three days. Blood samples were taken before quinolone administration and 1 h, 3h and 5 h after taking the drug on the first day. Last sample was taken 5 h after quinolone administration on the third day of therapy. The serum theophylline concentration was determined by 3 M Theo FAST test. It was found that in the patients treated with ciprofloxacin serum theophylline level in all samples was significantly higher as compared with the control sample. In the patients receiving ofloxacin the theophylline level only 1 h after receiving the drug the increase was statistical significant. Nalidixic acid had no influence on serum theophylline concentration. As these studies show, the new quinolones should be applied with great caution in patients treated with high doses of methylxanthines or in patients with decreased methylxanthines clearance. In patients with chronic obturative diseases and pulmonary infections the safer drug would appear to be ofloxacin.


[Natural history of pulmonary hypertension in chronic obstructive lung diseases and the usefulness of the simple noninvasive methods of its diagnosis].

In 47 patients with chronic obstructive pulmonary disease and 19 patients with idiopathic pulmonary fibrosis pulmonary haemodynamics were studied. Pulmonary arterial pressure was compared with non-invasive lung function tests, ecg and the chest X-ray. Correlations between pulmonary arterial pressure and some of those parameters were found. Investigations were repeated after 3 or 5 years depending on severity of pulmonary hypertension initially found. The progression of pulmonary hypertension was found. It was accompanied by intensification of non-invasive indices of pulmonary hypertension. More than half of patients with initially severe pulmonary hypertension (> or = 30 mmg Hg) did not survive 3 years.


[Prevalence of smoking among the staff of the Medical Academy and the State Clinical Hospital No. 1 in Gdańsk and among 5th-year medical students].

Addiction to smoking was assessed in workers of the Medical School, Central Clinical Hospital in Gdańsk and 50 medical students basing on the results of a simple questionnaire. 400 questionnaires were distributed. 287 were returned. 79% of the responders were females. 61.3% of the questioned subjects were smokers. 53.7% of the females were smokers, where as only 50% of the males smoked. The highest percentage of smokers was found in the group of orderlies, administrative workers and nurses (62-73%). Most of the health workers acknowledged knowing about the harmful effect of tobacco smoking. 95% believed that anti-smoking measures are inadequate in Poland.


[Castleman tumor].

A case of a mediastinal benign tumor in a 56 year old male is presented. The tumor was detected during a routine chest radiographic examination. The patient was asymptomatic. During thoracotomy two tumors were found. One was localized in the middle mediastinum the other in the posterior mediastinum. Both tumors were removed completely. Basing on the results of the clinical examination, of the laboratory data and the histopathological examination of the resected tumors a diagnosis of Castleman's tumor of the plasma-cell type was made.


[Sinus histiocytosis with generalized lymphadenopathy--Rosai-Dorfman disease].

A case of sinus histiocytosis with generalized lymphadenopathy--Rosai-Dorfman disease--in a 54 year old male is presented. The diagnosis was made by biopsy of the lymph nodes and histopathological examination of the affected organ. Treatment with prednisone and cyclophosphamide induced clinical improvement. The diseases clinical advanced stage and organ localization and the immunosuppressive therapy resulted in a opportunistic infection--pneumocystidosis and death of the patient within half a year after starting of therapy.


[Immunological approach to the evaluation of the activity of the tuberculous process].

The aim of the study was to determine the immunological criteria of the activity of the lung tuberculosis. Some immunological tests based on cellular and humoral immunity were assessed in 3 groups: I--patients with active, bacteriologically confirmed tuberculosis, II--individuals with inactive minimal changes in the lung, III--healthy subjects. We found that among patients with active tuberculosis cellular immunity was depressed: both percentage of T-lymphocytes and their function, usually associated with cutaneous anergy to tuberculin. But the humoral immunity was enhanced. The authors have shown that the people with inactive minimal changes in the lung take place between the patients with active tuberculosis and healthy subjects.


[Lung cancer mortality rate in the Upper Silesia 1985-1990].

Mortality rates in the years 1985-1990 in males and females with lung cancer in the Katowice province were analysed. Partial rates according to age groups and "raw" rates were calculated. Basing on these, standardized rates were assessed (related to administrative units: 45 cities and 48 districts). The regions with the highest and lowest risk of mortality due to lung cancer were identified using the isarytmic method and standardized rates. The regions with the highest risk are those with a mortality rate for males of 85 per 100,000 and 13 per 100,000 for females. A statistically significant difference was seen between some regions (p = 0.01). The cohort analysis predicts that in the immediate future, an increase of mortality rates in rat es due to lung cancer can be predicted. Also a very dynamic increase in mortality rates in females can be foreseen.


[Ventilatory function of the lungs in galenite ore miners].

The study was carried out on a group of 54 galenite miners (composed of PbS slats) (age 37.9 +/- 7.5 years). The following parameters were assessed: VC, TLC, RV, RV%TLC, FEV1, FEV1%VC, Raw, Gaw, FEF50%, FEF75%. Also the ventilatory homogeneous index was calculated V2:V1 basing on the analysis of the flow volume curve. The control consisted of subjects living in the vicinity of the mine and smelting works. In 3.1% of the miners various stages of increased flow obstruction was found. In 8.3% signs of emphysematous changes, in 23.9% minor disturbances of bronchial resistance. In comparison with the control the miners demonstrated expiratory flow mainly in the small airways. Duration of exposure and co-existent chronic bronchitis increased the above mentioned observations and decreased the homogeneity index.


[Prospective epidemiological studies of chronic bronchitis in workers in a coke-processing plant].

In 1977 an epidemiological survey was carried out on incidence of chronic bronchitis in 652 workers (age 20-64) of a coking plant. The survey was repeated six years later in 300 of the original participants (56%). The aim of the study was to assess the effect of smoking, age and occupation on incidence of chronic bronchitis and dynamic change of FEV1 values during the six years. During the 6 years the incidence of chronic bronchitis rose from 22% to 43%. Mean annual decrease of FEV1 was higher in smokers as compared to nonsmokers (57.4 versus 52.6 ml per year). Higher mean falls of FEV1 values were noted in subjects with occupational risk factors present (68.4 ml) in comparison with those with low occupational exposure (51.2 ml). Susceptible subjects presented a thrice higher annual FEV1 decrease rate (142 ml).


[Chronic bronchitis and ventilatory capacity of the lungs in relation to the function of small airways in workers in a Coke-Processing Plants in Zabrze].

In 449 male workers of the Zabrze Coking Plant (age 18-62 years) a questionnaire analysis of presence of bronchitic symptoms was carried out. The following ventilatory parameters were also assessed: FVC, FEV1, PEF, MEF75, MEF50, MEF25. Chronic bronchitis was diagnosed in 30.5% of the analysed subjects. The incidence of chronic bronchitis was four-fold higher in smoking males (33.9%) in comparison with non-smokers (8.3%). The authors did not find any relation between the character of work in subject with occupational exposure to atmospheric pollutants and incidence of chronic bronchitis. Smoking produced a decrease in flow during forced expiration especially in males older than 40 years. Subjects with higher occupational exposure to atmospheric pollutants demonstrated higher incidence of airway obstruction (21.2%) and lower mean FEV1% and flow volumes in comparison to subjects with lower occupational exposure.


[Ofloxacin in the treatment of pulmonary changes caused by Mycobacterium avium and Mycobacterium xenopii].

Two cases of atypical mycobacterial infections are reported. The first caused by Mycobacterium avium in a patient with silicosis, the second by Mycobacterium xenopii in a subject with evidence of extensive post-tuberculosis pulmonary lesions. Both were treated with ofloxacin with good results.


National Advisory Committee on Immunization (NACI). Statement on Haemophilus influenzae type B conjugate vaccines for use in infants and children.

Currently three Haemophilus influenzae type b (Hib) conjugate vaccines are licensed for use in Canada in infants 2 months of age and older: HibTITER (Lederle Laboratories), PedvaxHIB (Merck Sharp & Dohme Canada) and Act-HIB (Pasteur-Mérieux, distributed in Canada by Connaught Laboratories Ltd.). A fourth vaccine, licensed both as a single product (ProHIBIT) or in combination with diphtheria toxoid-pertussis vaccine-tetanus toxoid (DPT-Hib) (Connaught), is limited to use in children 18 months of age or older. The following recommendations incorporate those previously published by the National Advisory Committee on Immunization(1), provide information on Act-HIB (which was licensed in March 1992) and summarize recently published data on the comparative immogenic effects and interchangeability of Hib conjugate vaccines (2-6).


Dental management of a child with anorexia nervosa who presents with severe tooth erosion.

Carbonated soft drinks can, when consumed regularly and in large quantities, cause severe dental erosion. In patients with anorexia nervosa the temptation to substitute these drinks for their regular diet has increased, since they are readily available, relatively cheap and their consumption is socially acceptable. The dental management of a young child suspected of having anorexia, who presented with severe dental erosion, is described.


Treatment options for the edentulous mandible.

Prosthodontic literature is replete with articles reporting longitudinal studies of, and clinical and laboratory procedures for, dental implants. There is no doubt that implant-borne prostheses are ideal treatment options for some patients; for other, alternatives must be considered. The purpose of this article is to present an overview of the aetiology of mandibular atrophy and to review a broad range of treatment options for patients who have atrophic mandibles. Whilst techniques are listed for completeness and not necessarily out of preference, with epidemiological and demographic trends in mind, the need for preventive prosthodontics is emphasised.


Problems associated with restoration of dentitions after orthodontic treatment using anterior tooth space.

Three case reports are presented to illustrate some problems associated with restoration of dentitions following orthodontic treatment using anterior tooth space. These include the projecting premaxilla, conversion of lateral incisors to resemble central incisors and excess residual space.


Evaluation of impression tray techniques in microstomic patients.

Three different methods of constructing impression trays for patients suffering from microstomia have been described. When sectional trays with connectors are designed, proper alignment of the sections is necessary for satisfactory results. Flexible trays, or even silicone putty without a tray, can be used for making preliminary impressions in these cases.


Long-term electromyography as a possible diagnostic aid for detecting craniomandibular dysfunctions.

As there is a positive correlation between oral parafunctional habits and craniomandibular disorders, objective electromyographic findings on parafunctional jaw activities could be very interesting for the diagnosis and management of craniomandibular disorders. The described pocket-sized and battery-operated electromyographic measuring device fulfils the essential requirements for monitoring and recording parafunctional activity in a chosen time period. It is easy to handle and can therefore be operated by a trained patient. At the end of the registration the collected data are transferred to a computer for statistical or graphical analysis. With this device, long-term electromyography on patients in their usual environment is possible. Further studies on parafunctional behaviours applying long-term electromyography could increase the understanding of craniomandibular disorders.


The replacement of missing maxillary central incisor teeth incorporating a midline diastema with a fixed-fixed resin-retained bridge.

The replacement of maxillary central incisor teeth with the maintenance of a midline diastema can create considerable problems for the restorative dental surgeon. This case report illustrates the treatment of such a case using a fixed-fixed resin-retained bridge with a modified palatal connector. Alternative treatment options are discussed.


In vitro microleakage of Class V composite resin restorations with and without light-cured glass-ionomer (poly alkenoate) cement lining.

The restoration of Class V cavities with composite resin when the cervical cavity margin extends onto the root surface poses a special problem with regard to marginal seal. The use of glass-ionomer (poly alkenoate) cement (GIC) linings has not overcome this problem completely. The efficacy of a new light-cured GIC lining was tested in vitro, as was the use of a modern dentine-bonding agent. The light-cured GIC lining afforded a reliable marginal seal but the efficacy of the dentine-bonding agent related to the time of finishing of the composite restoration.


Neurobiology and inflammatory arthritis.

As diagrammed in Figures 1 and 2, the central and peripheral nervous systems, the endocrine system, and immune and inflammatory systems are extensively integrated. The boundaries defining these systems are both anatomically and molecularly blurred. This realization has been driven by the ongoing revolution in molecular biology. Normally the deleterious effects of excessive or persistent inflammation are prevented by homeostatic neuroendocrine feedback loops. Conversely, dysfunction in these counter-regulatory responses may lead to important clinical phenomena such as autoimmunity and chronic inflammation in association with behavioral alterations such as fatigue, depressed mood, and loss of libido. Continued research into the neurobiology of inflammation appears likely to explain some of the fundamental abnormalities in diseases such as RA and lead to new, more effective therapies.


Peripheral and central administration of cocaine produce conditioned odor preferences in the infant rat.

To assess the development of the reinforcing properties of cocaine, 3- to 4-day-old rat pups were administered cocaine s.c (3.0-30.0 mg/kg), intracerebroventricularly (3.0-10.0 micrograms/injection), or directly to the nucleus accumbens (1-25 micrograms/injection) in the presence of a distinctive odor. Six to 8 h later, pups were allowed a choice between the paired odor and a non-paired odor. Compared to saline treated controls, pups demonstrated a preference for the odor paired with cocaine for each route of administration. Taken together with previous work demonstrating that cocaine increases responding for electrical self-stimulation of the basal forebrain, these results suggest that cocaine is rewarding in the infant animal and that mesolimbic structures may mediate, at least in part, those reinforcing properties.


Different thresholds of HSP70 and HSC70 heat shock mRNA induction in post-ischemic gerbil brain.

Thresholds of induction of heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 mRNAs after transient global ischemia in gerbil brain were investigated by in situ hybridization using cloned cDNA probes selective for each mRNA species. In sham control brain, HSP70 mRNA was little present, while HSC70 mRNA was present in most cell populations. A 0.5-min occlusion of bilateral common carotid arteries did not affect the amount of HSP70 and HSC70 mRNAs. The selective induction of HSC70 mRNA was observed in dentate granule cells at 1 h, and in most cells of hippocampus especially dentate gyrus at 3 h after 1 min of ischemia when induction of HSP70 mRNA was not evident in the identical brain. The selective induction diminished by 2 days. However, after 2 min of ischemia, HSP70 and HSC70 mRNAs were induced together in hippocampal cells from 1 h of the reperfusion, and the co-induction prolonged in CA1 cells until 2 days. Body temperatures monitored at rectum increased after the reperfusion with a peak at 30 min. The degree of increase of the body temperature was significantly higher in the case after 2-min ischemia than in the cases after 0.5- and 1-min ischemia. Although HSP70 and HSC70 mRNAs are generally co-induced in stressful conditions, our results suggest the different thresholds of the induction between HSP70 and HSC70 mRNAs after transient brain ischemia. The selective induction of HSC70 mRNA which is not accompanied by the induction of HSP70 mRNA may relate to the differences of the duration of ischemia and the degree of the increase of body temperature after ischemia.


Effect of inhibiting enkephalin catabolism in the VTA on motor activity and extracellular dopamine.

The mixed inhibitor of enkephalin catabolism, kelatorphan, was microinjected into the ventral tegmental area (VTA) of rats to determine if endogenous enkephalins can modulate dopamine transmission in the mesoaccumbens projection. The concentration of extracellular dopamine content in the nucleus accumbens was monitored using in vivo microdialysis simultaneously with measuring motor behavior. Kelatorphan microinjection into the VTA produced a dose-related increase in motor activity and extracellular dopamine in the nucleus accumbens. While the change in extracellular dopamine was modest as compared to exogenous stimulation by a mu agonist such as DAMGO, there was a marked increase in the extracellular content of dopamine and serotonin metabolites. This suggests that mesoaccumbens dopamine transmission is under tonic control of endogenous enkephalins at the ventral tegmental area level.


Expression of c-fos protein in rat brain after electrical stimulation of the aortic depressor nerve.

To reveal central nervous system (CNS) structures involved in the baroreceptor reflex we studied the distribution of Fos protein-like immunoreactivity in the rat brain after one hour of electrical stimulation of the aortic depressor nerve (ADN). In 13 male Wistar rats under urethane the ADN was cut on both sides and the central ends were placed on stimulating electrodes. Intermittent (11 s on, 6 s off) electrical stimulation at parameters set to elicit a drop in mean arterial pressure of 15-30 mmHg was applied to one, both or neither ADNs for 1 h. CNS sections were incubated for 48 h in anti-Fos antibody and prepared for visualization of the reaction product using the ABC immunoperoxidase technique. Label was found in several discrete brain nuclei primarily on the side ipsilateral to the side of stimulation. In the medulla labelled nuclei were found in the nucleus tractus solitarius, area postrema, rostral and caudal ventrolateral medulla, nucleus ambiguus and medullary reticular formation. In the pons labelled neurons were found in the lateral and ventrolateral parabrachial nucleus, locus coeruleus, pontine reticular field and A5 region. In the forebrain labelled nuclei were observed in the peri- and paraventricular hypothalamus, supraoptic nucleus, subfornical organ, preoptic area, central nucleus of the amygdala, median preoptic area, horizontal limb of the diagonal band, bed nucleus of the stria terminalis and islands of Calleja. In control animals moderate amounts of label were present in the supraoptic nucleus and periventricular hypothalamus bilaterally. These results define central pathways involved in mediating the baroreceptor reflex.


Characterization of a neurofilament-associated kinase that phosphorylates the middle molecular mass component of chicken neurofilaments.

We have examined the properties of a chicken neurofilament (NF) kinase partially purified from NF-enriched preparations. This kinase cosediments with NFs following extraction with Triton X-100 and can be separated in an active form from NFs by treatment with 0.8 M KCl. Sequential chromatography of the salt extract on DEAE-cellulose and phosphocellulose results in an approximately 500-fold increase in specific activity over endogenous NF preparations as measured by 32P-incorporation into the middle molecular mass component of NFs (NF-M). The kinase is Mg(2+)-dependent, second messenger-independent and inhibited by high concentrations of heparin. It shows selectivity for NF-M and evidence is presented that the kinase phosphorylates NF-M solely in the tail domain. The kinase can also phosphorylate the microtubule-associated proteins tau and MAP2 as well as mammalian NF-M, all of which share putative phosphorylation sequences with chicken NF-M.


Basic fibroblast growth factor rescues CNS neurons from cell death caused by high oxygen atmosphere in culture.

In the present study, we cultured rat CNS neurons and tested the neurotrophic support provided by basic fibroblast growth factor (bFGF) to prevent the oxygen-induced neuronal cell death. When rat basal forebrain (septum and vertical limb of diagonal band of Broca) cells of embryonic day 20 were cultured in a serum-free medium containing 5 microM cytosine arabinoside in a 50% oxygen atmosphere, the neuronal cells, which were immunostained by an anti-microtubule-associated protein 2 (MAP2) antibody, gradually died after 1 day in culture. After 3.5 days in culture, only 2-5% of neuronal cells survived. This oxygen-induced cell death of cultured basal forebrain neurons was reversed by the addition of bFGF at a concentration of 100 ng/ml. This cell-saving effect was dose-dependent, and the ED50 value was 12 ng/ml. Nerve growth factor (NGF) and insulin-like growth factor II could not prevent cell death. The activity of choline acetyltransferase was also maintained when bFGF was present in the basal forebrain culture. Viable astroglial cells, which were immunostained by an anti-glial fibrillary acidic protein, accounted for a few percent of the total number of cells after 3 days in culture both with and without 100 ng/ml of bFGF. The survival-enhancing effect of bFGF was observed not only in basal forebrain neurons but also in neocortical and hippocampal neurons. However, the sensitivity to oxygen toxicity of cultured neurons from the 3 CNS regions varied greatly. The neocortical neurons were the most sensitive to oxidative stress, while the hippocampal neurons were the most resistant. These results suggest that bFGF plays an important role in saving neuronal cells from oxidative stress during their long life without division.


Successful transmission of Creutzfeldt-Jakob disease from human to mouse verified by prion protein accumulation in mouse brains.

The accumulation of prion protein (PrP) was revealed in the brains of mice inoculated with the brain homogenate from seven patients with Creutzfeldt-Jakob disease (CJD) by immunohistochemistry using hydrolytic autoclaving. It was not found in the brains of mice inoculated with material from either two patients with Gerstmann-Sträussler syndrome or two with other dementing illnesses. PrP accumulation took the forms of diffuse neuropil accumulation in the gray matter and plaque-like accumulation in the white matter and was observed in particular areas in the supratentorial structure. Its distribution was narrower than that in the brains of mice infected with a mouse-adapted CJD strain. PrP accumulation was found not only in all histopathologically positive mice, but also in some histopathologically negative mice. In all groups of mice inoculated with the material from each CJD patient, the percentage of mice with PrP accumulation was equal to or exceeded that of mice with the histopathological findings. PrP immunohistochemistry using formic acid pretreatment stained such plaque-like accumulation less intensely than that using hydrolytic autoclaving and did not stain diffuse neuropil accumulation. Therefore, PrP accumulation which can be revealed in the brains of first-passage CJD mice by this new immunohistochemical method may be the most sensitive hallmark of successful transmission.


Histamine-caused itch induces Fos-like immunoreactivity in dorsal horn neurons: effect of morphine pretreatment.

It is well known that cutaneous pain causes c-fos gene expression in the dorsal horn neurons. The present study examined whether or not an itching sensation had the same effect on these neurons. In order to produce an itching sensation, cotton balls soaked in histamine solutions of different concentrations were applied to the lower limbs of rats and then the treated area was scratched with the tip of a pair of forceps. After 2 h, the number of neurons showing Fos-like immunoreactivity was significantly increased in a dose-dependent manner in the ipsilateral dorsal horn when compared to saline-treated and scratched controls. When scratching with the forceps was omitted, the number of these neurons was reduced to one-third, but was still significantly higher than in controls treated only with saline. Expression of Fos-like immunoreactivity in these animals was markedly reduced by morphine pretreatment (10 mg/kg, i.p.), suggesting that the transmission of both itch and pain is blocked by morphine.


Protein synthesis inhibition blocks maintenance but not induction of epileptogenesis in hippocampal slice.

We have been examining the role of protein synthesis in the development and maintenance of spontaneous bursting in the rat hippocampal slice. We used stimulus train induced bursting (STIB) as an in vitro model for epileptogenesis, to study the effects of 3 different protein synthesis inhibitors (cycloheximide, anisomycin, puromycin) on the development of bursting. We report here that none of these inhibitors blocked the induction of bursting, suggesting that protein synthesis is not essential for the development of electrically induced bursting. However, when established spontaneous bursting was examined in the presence of cycloheximide, the duration of the bursting phase was markedly reduced, suggesting that the maintenance of spontaneous bursting in the early hours requires ongoing protein synthesis.


Memory dysfunction following disruption of glutamergic systems in the temporal region of the rat: effects of agonistic amino acids.

It has previously been shown that disruptions of fiber connections between the temporal cortex (TC) and the lateral entorhinal cortex (LEC) in rats result in severely impaired retention of a simultaneous brightness discrimination task. This memory impairment is accompanied by reduced high affinity D-aspartate uptake in both TC and LEC. The purpose of this study was to investigate whether systemic administration of glutamergic agonists might ameliorate the mnemonic dysfunction seen to follow TC/LEC transections. The results from Experiment 1 show that agonists acting selectively at the NMDA receptors (NMDA and glycine) or the quisqualate receptors (AMPA) produced complete amelioration of the memory deficit. Injection of kainic acid only produced a slight improvement of memory. The results from Experiment 2 show that the positive effects of agonists are probably not attributable to peripheral adrenergic mechanisms, because blockade of sympathetic terminal release did not prevent mitigating effect of glycine. The results are discussed in terms of possible central nervous mechanisms interfered with by the various agonists.


Distribution of glycinergic terminals on lumbar motoneurons of the adult cat: an ultrastructural study.

The distribution of glycine-like immunoreactivity on cat lumbar motoneurons was examined in electron microscopy, using pre-embedding immunocytochemistry. In the dorsolateral portion of the ventral horn, numerous labeled axon terminals were presynaptic to somatic and dendritic profiles of alpha-motoneurons. Most of the glycinergic boutons contained pleomorphic vesicles and showed symmetrical contacts. On the somatic and proximal dendritic compartments, glycinergic terminals accounted for, respectively, 24.6 and 26.6% of the total number of terminals. There were very few glycinergic terminals on gamma-motoneurons. Immunoreactive axons, dendrites and cell bodies were also observed near the motoneurons. These results support the view that glycine plays a major role in the inhibition of alpha-motoneurons and suggest that inhibitory mechanisms occur on the soma as well as on dendrites.


[Pathology of the uterine cervix. Infectious lesions of the endocervix and exocervix].

Bioptic findings from the region of the endo- and exocervix of the uterus were analyzed in a retrospective study covering the years 1988-1990. The series studied consisted of 366 women. Infectious processes in the cervix were evaluated in this first part of the study. Over the period investigated, 155 cases of cervicitis were established. Of these 56 (36.2 %) were acute bacterial, 17 (10.9 %) were lympho-plasmocytic (chlamydial), and 4 (2.5 %) were herpetic. The largest group of 78 (50.3 %) patients had HPV induced lesions. Koilocytic dysplasia was grouped into three grades according to severity. Mild dysplasia occurred in 16 patients (22 %), intermediate in 19 patients (26 %) and severe dysplasia in 37 patients (52 %). (Fig. 6, Ref. 15.).


[Adverse effects of antidepressive agents in hospitalized geriatric patients].

The frequency, intensity and profile of adverse effects of antidepressants was studied in elderly patients. The series consisted of 102 patients with depression admitted to hospitals in Bratislava and Moscow. The adverse effects of amitriptyline (Amitriptylin Spofa) and maprotiline (Ludiomil Ciba-Geigy) were compared. The assessment done on days 0, 7, and 28 of treatment showed that xerostomia had the highest occurrence rate with both preparations studied. In patients treated with amitriptyline adverse effects were more severe and were recorded more frequently, requiring treatment withdrawal in 3 patients. The overall intensity of adverse effects was significantly higher with amitriptyline (p < 0.05). In the group of patients treated with amitriptyline the adverse effects were more marked in those with severe somatic pathology. The risk of amitriptyline treatment in elderly patients is being emphasized along with the need for monitoring and correcting adverse effects of the treatment. Although maprotiline exhibited a lower occurrence rate of adverse effects, cardiac functions should be regularly checked in patients with preexisting cardiac pathology. (Tab. 2, Fig. 3, Ref. 6.).


[A screening program for the diagnosis of early forms of prostatic carcinoma].

Carcinoma of the prostate is at present one of the most frequently occurring malignancies in the male population. The disease can be cured only at the early stage when it has not yet spread beyond the limits of the gland. Early, clinically asymptomatic forms of carcinoma of the prostate can be diagnosed only by screening examinations of men. The justification of screening has, however, not been conclusively established and the fear of damaging the patient on examination could not be dispelled. The project of an international co-operative study was accepted in Stockholm in 1990 with the aim to address this question. The Department of Urology of Derer's Hospital with Policlinic, along with several urological, oncological, and research departments, have decided to join the project in order to contribute to the improvement of diagnosis and treatment of carcinoma of the prostate. (Fig. 1, Ref. 12.).


[Comparison of the effect of nifedipine and verapamil on lipid metabolism in experimental atherosclerosis in rabbits].

The aim of the work was to study the effect of 8-week treatment with two calcium channel blockers on some parameters of lipid metabolism in rabbits. Experimental atherosclerosis was induced by 1% cholesterol diet. The calcium channel blockers were administered twice daily in the following doses: nifedipine 1.0 mg.kg-1 and verapamil 2.0 mg/kg-1 x day-1. The treatment with calcium channel blockers enhanced the level of total cholesterol as well as of triglycerides and decreased the HDL: total cholesterol ratio in all the experimental groups. The changes induced by nifedipine were, however, less significant compared to the effect of verapamil. The absolute amount of fatty acids determined by gas chromatography was lower after nifedipine administration in comparison to the verapamil treated group. Nifedipine induced a more favorable proportion in the composition of fatty acids. The increase in the level of monounsaturated fatty acids (palmitoleic and oleic acid) as well as the decrease in the level of polyunsaturated fatty acids (especially linoleic acid) was the lowest in the nifedipine treated group. (Fig. 6, Ref. 30.).


[The effect of verapamil and nifedipine on acid-base and electrolyte disorders induced by cholesterol in rabbits].

The influence of cholesterol atherogenic diet on the acid-base balance and electrolyte profile was investigated in rabbit blood and the effect of simultaneous administration of verapamil and nifedipine on the induced changes was evaluated. Cholesterol diet resulted in significant metabolic acidosis, hyperkalemia, and a slight decrease in plasma ionized calcium. On the other hand, longterm treatment with verapamil and nifedipine exhibited their protective effect, with the acid-base status and electrolyte profile maintained within the normal range. The protective effect of verapamil and nifedipine against cholesterol induced acid-base and electrolyte disturbances appears to be associated with their antiatherogenic activity. (Fig. 5, Ref. 10.).


Two-dimensional poly(acrylamide) electrophoresis of fluoresceinated glycopeptides. Resolution and structural characterization of ovalbumin glycans.

The microheterogeneous mixture of fluoresceinated glycopeptides (FGPs) obtained from the single site of glycosylation of chicken ovalbumin was resolved by a combination of discontinuous electrophoresis in a high-density poly(acrylamide) gel (PAGE) for sizing, in conjunction with borate-PAGE. Two FGPs of similar size but with different mobilities in borate-PAGE were purified and characterized by sequential exoglycosidase digestion and sizing on the discontinuous PAGE system, as well as by methylation analysis. The two FGPs of identical size are distinct and have structures beta-D-Glc pNAc-(1-->2)-alpha-D-Man p-(1-->3)-[beta-D-Glc pNAc-(1-->4)]-[beta-D-Glc pNAc-(1-->2)-alpha-D- Man p-(1-->6)]-beta-D-Man-p-(1-->4)-beta-D-Glc pNAc-(1-->4)-beta-D-Glc pNAc-1-->R and alpha-D-Man p-(1-->2)-alpha-D-Man p-(1-->3 or 6)-[alpha-D-Man p-(1-->3)-[alpha-D-Man p-(1-->6)]-alpha-D-Man p-(1-->6 or 3)]-beta-D-Man p-(1-->4)-beta-D-Glc pNAc-(1-->4)-beta-D-Glc pNAc-1-->R (R = Asn-(amino acids)-fluorescein). The results demonstrate that two-dimensional PAGE is applicable to the separation and characterization of complex mixtures of FGPs. The procedure is rapid, sensitive, and convenient for glycopeptide mapping, and for the purification and structural characterization of glycans. Furthermore, the FGPs can be characterized with affinity matrices, such as lectins, and by methylation analysis.


Regioselectivity in the sulfation of some chemically-modified heparins, and observations on their cation-binding characteristics.

Two modified forms of heparin, polymers A and B, have been prepared, one containing residues of nonsulfated alpha-L-idopyranosyluronic acid (3) and the other residues of alpha-L-galactopyranosyluronic acid (7), in place of the normal alpha-L-idopyranosyluronic acid 2-sulfate (1). In addition, both A and B contained 2-acetamido-2-deoxy-alpha-D-glucopyranosyl 6-sulfate residues (6) in place of the corresponding N-sulfated residues (2) of the original heparin. These polymers were subjected to sulfation under various conditions. Examination of the products by NMR spectroscopy showed that polymer A was sulfated initially at position-3 of residue 3, and that slower substitution occurred at position-3 of 6. By contrast, polymer B exhibited low regioselectivity, as sulfation occurred with about equal facility at positions-2 and -3 of 7, and -3 of 6. The sulfation products had no significant anti Xa activity. Based on the paramagnetic effects of Cu2+ and chemical shift displacements induced by Ca2+, NMR spectroscopy was used to compare cation-binding properties of A and B with those of heparin. In contrast to heparin, which forms a complex with Cu2+ detectable at a level of < 10(-3) mol per dimeric unit of the polymer, neither A nor B exhibited an interaction with the cation. However, polymer A was found to bind Ca2+, in this respect being distinct from the related modification, 1-->6, which contains a 2-sulfate group in 1, as well as from polymer B.


Structural analysis of the carbohydrate chains isolated from mistletoe (Viscum album) lectin I.

Two glycopeptide fractions prepared from mistletoe (Viscum album) lectin I by Pronase digestion were fractioned by affinity chromatography on a concanavalin A-Sepharose column. With 400-MHz 1H NMR spectroscopy, in conjunction with sugar analysis, the following oligosaccharide structures could be determined: two oligomannose-type glycans in the ratio 4:1, one containing six mannose and the other containing five mannose units, both with two 2-acetamido-2-deoxyglucose units. In addition, a mannotriosyl-->N,N'-diacetylchitobiose glycan containing a xylosyl group and an alpha-fucosyl group (1-->3)-linked to the 2-acetamido-2-deoxyglycosyl-1 residue, a common core element of many plant glycoproteins, was also observed.


The structure of the sialic acid-containing Escherichia coli O104 O-specific polysaccharide and its linkage to the core region in lipopolysaccharide.

Mild acid hydrolysis of Escherichia coli O104 lipopolysaccharide released an O-specific polysaccharide, a tetrasaccharide repeating unit, the corresponding dimer, and a disaccharide fragment of the repeating unit. Complete and incomplete cores, and oligosaccharides comprising fragments of the repeating unit and the core region, were also obtained. On the basis of sugar and methylation analysis, FAB-mass spectrometry and NMR spectroscopy of the hydrolysis products, the repeating unit of the O-specific polysaccharide was shown to be the tetrasaccharide:-->4)-alpha-D-Galp-(1-->4)-alpha-Neup5,7,9Ac3++ +-(2-->3)-beta-D- Galp-(1-->3)-beta-D-GalpNAc (1-->. The linkage between the O-specific polysaccharide chain and the core region, which appeared to be of the R2 type, was established. These results indicate that N-acetylneuraminic acid, located in the O-specific polysaccharide, is an inherent lipopolysaccharide component.


In vitro biosynthesis of GbOse4Cer (globoside) and GM2 ganglioside by the (1-->3) and (1-->4)-N-acetyl beta-D-galactosaminyltransferases from embryonic chicken brain. Solubilization, purification, and characterization of the transferases.

(1-->4)-N-Acetyl-beta-D-galactosaminyltransferase (GalNAcT-1) and (1-->3)-N-acetyl-beta-D-galactosaminyltransferase (GalNAcT-2), which are involved in the in vitro biosynthesis of GM2 and GbOse4Cer glycosphingolipids, respectively, have been solubilized and separated by differential detergent extraction from a membrane preparation of 19-day-old embryonic chicken brain. The separated GalNAcT-1 activity had a pH optima of 7.8-8.0, and the separated GalNAcT-2 activity a single pH optimum of 7.2. Furthermore, the partially purified GalNAcT-2 preparation catalyzed the transfer of N-acetylgalactosamine from UDP-D-[3H]GalNAc to only GbOse3Cer and nLcOse5Cer. Both GalNAcT-1 and GalNAcT-2 activities were purified to approximately 316- and 428-fold, respectively, by use of UDP-hexanolamine-Sepharose 4B affinity-column chromatography. However, the partially purified GalNAcT-1 preparation appeared to be active only with GM3, lactosylceramide, and lactotriaosylceramide. The proposed linkage of the N-acetylgalactosamine unit incorporated into GM3 is beta-D-GalpNAc-(1-->4)-GM3 from the isolation of [3H]threitol after hydrolysis of the desialylated, lead tetraacetate-treated, enzymic product, beta-D-GalpNAc-(1-->4)-beta-D-[6-3H]Galp-(1-->4)-beta-D-Glcp-(1-->1)-Cer . In addition, beta-D-GalpNAc-(1-->3)-GbOse3Cer was produced, as shown by the identification of 2,4,6-tri-O-methyl-galactose after permethylation and hydrolysis of the GalNAcT-2 enzymic product, GalpNAc-[6-3H]Galp--->Gal-->Glc-->Cer.


Immunochemical studies on the combining site of the A + N blood type specific Moluccella laevis lectin.

The specificity of the anti A+N lectin of Moluccella laevis (MLL) was examined by hemagglutination experiments with enzyme-modified human erythrocytes and by inhibition of hemagglutination. In addition, binding to various glycoproteins and inhibition by different sugars and glycoproteins were examined by enzyme immunoassay with antibodies to the lectin. Treatment of AMM erythrocytes with proteolytic enzymes increased their agglutinability by MLL 4-16-fold; similar treatment of ONN cells decreased their agglutinability 8-16-fold. This is in line with the known location and enzyme sensitivity of A and N specificity determinants. Treatment of the erythrocytes with sialidase increased their agglutinability and abolished the distinction between N and M cells. Hapten inhibition of hemagglutination of AMM and ONN erythrocytes by the lectin, and its binding to glycoproteins measured by enzyme immunoassay, confirmed the high specificity of MLL for N-acetyl-D-galactosamine (200-500 times more than for D-galactose) and suggested the presence of hydrophobic interactions around HO-2 of the D-galactose unit. The methyl alpha-glycosides of D-galactose and of N-acetyl-D-galactosamine were better inhibitors than the corresponding beta-glycosides; this preference was abolished, and sometimes reversed, when the p-nitrophenyl glycosides of the same monosaccharides were tested, stressing again the importance of hydrophobic interactions in the binding of carbohydrates to MLL. The lectin reacted well with ONN substance and with glycophorin A of the N phenotype (GPAN), but did not react with OMM substance or GPAM. The strongest inhibitor was asialo ovine submaxillary mucin, which contains many unsubstituted alpha-D-GalpNAc-(1-->3)-Ser/Thr residues; calculated per N-acetyl-D-galactosamine residue, it was 1500 stronger than free N-acetyl-D-galactosamine. In accordance with this result, it was found that the lectin strongly agglutinates Tn cells. The specificity of MLL can, thus, be defined as anti-Tn, crossreactive with blood types A and N, and with sialosyl-Tn. The N-specificity can best be explained by assuming that GPAN contains a small number of unsubstituted or partially sialylated alpha-D-GalpNAc-(1-->3)-Ser/Thr residues, which are present in smaller proportions, if at all, in GPAM.


Patterns of alpha-L-fucosidase in acute myeloid leukemia cells. Comparison with promyelocytic HL-60 cell line.

Changes were observed in alpha-L-fucosidase forms in cells from acute myelocytic leukemias (AML). Total alpha-L-fucosidase activity was not significantly different for normal granulocytes and leukemic cells, but enzymic profiles obtained by chromatofocusing are quite different. In granulocyte profile, two main peaks are present (B and more acidic A) which were eluted at pH 5.2 with a shoulder at pH 4.6. In AMLs the B form is present but weakly expressed, whereas the more acidic forms are the major ones. This pattern may be related either to the malignancy character or to the stage at which the differentiation is stopped. Experiments on an HL-60 cell line (promyelocytic cells corresponding to the AML 3 type) showed that differentiation induced by dimethyl sulfoxide leads to the appearance of the B form present in normal mature cells. Thus the repartition of the enzyme forms seems to be related to the stage of differentiation of the myelocytic cells.


Control of glycoprotein synthesis. Characterization of (1-->4)-N-acetyl-beta-D-glucosaminyltransferases acting on the alpha-D-(1-->3)- and alpha-D-(1-->6)-linked arms of N-linked oligosaccharides.

Hen oviduct membranes contain at least three N-acetyl-beta-D-glucosaminyltransferases (GlcNAc-T) that attach a beta GlcNAc residue in (1-4)-linkage to a D-Man p residue of the N-linked oligosaccharide core, i.e., (1-->4)-beta-D-GlcNAc-T III which adds a "bisecting" GlcNAc group to form the beta-D-GlcpNAc-(1-->4)-beta-D-Man p-(1-->4)-D-GlcNAc moiety; (1-->2)-beta-D-GlcNAc-T IV which adds a GlcNAc group to the (1-->3)-alpha-D-Man arm to form the beta-D-GlcpNAc-(1-->4)-[beta-D- GlcpNAc-(1-->2)]-alpha-D-Man p-(1-->3)-beta-D-Man p-(1-->4)-D-GlcpNAc component; and (1-->4)-beta-D-GlcNAc-T VI which adds a GlcNAc group to the alpha-D-Man p residue of beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpNAc- (1-->2)]-alpha-D-Man p-R to form beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpNAc-(1-->4)]-[beta-D-GlcpNAc- (1-->2)]-alpha-D-Man p-R. We now report a novel (1-->4)-beta-D-GlcNAc-T activity (GlcNAc-T VI') in hen oviduct membranes that transfers GlcNAc to beta-D-GlcpNAc-(1-->2)-alpha-D-Man p-(1-->6)-beta-D-Man p-R to form beta-D-GlcpNAc-(1-->4)-[beta-D-GlcpNAc-(1-->2)]-alpha-D-Man p-(1-->6)- beta-D-Man p-R. The structure of the enzyme product was confirmed by 1H NMR spectroscopy, FAB-mass spectrometry and methylation analysis. Previous work with GlcNAc-T IV was carried out with biantennary substrates; we now show that hen oviduct membrane GlcNAc-T IV can also transfer GlcNAc to monoantennary beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->3)-beta-D-Man p-R to form beta-D-GlcpNAc-(1-->4)-[beta-D-GlcpNAc-(1-->2)]-alpha-D-Man p- (1-->3)-beta-D-Man p-R. The findings that GlcNAc-T VI' and IV have similar kinetic characteristics and that hen oviduct membranes can convert methyl beta-D-GlcpNAc-(1-->2)-alpha-D-Man p to methyl beta-D-GlcpNAc-(1-->4)-[beta-D-GlcpNAc-(1-->2)]-alpha-D-Man p suggest that these two activities may be due to the same enzyme. The R-group of the beta-D-GlcpNAc-(1-->2)-alpha-D-Man p-(1-->6)-beta-D-Man p (or Glcp)-R substrate has an important influence on GlcNAc-T VI' enzyme activity.(ABSTRACT TRUNCATED AT 400 WORDS)


Use of compounds naturally labeled with stable isotopes for the study of the metabolism of glycoprotein neutral sugars by gas-liquid chromatography-isotope-ratio mass spectrometry. Technical validation in the rat.

In order to develop an alternative method to radioactive labeling for the study of the glycoprotein sugar metabolism in man, the possible use of stable isotopes provided by naturally, 13C-enriched dietary compounds has been explored in rat intestine and serum. Rats were fed a semisynthetic diet containing 67% wheat starch (containing 1.08692 13C atom/100 carbon atoms) for a week, and then the same diet containing corn starch (1.10042% 13C) for a week. Neutral sugars were prepared from delipidated, trichloroacetic acid-precipitable material from 200-400 mg of intestinal mucosa or 1 mL of serum, separated, and analyzed as alditol acetates by gas-liquid chromatography coupled to isotope-ratio mass spectrometry. This technique allowed the determination, in a single experiment, of the amount and 13C abundance of six neutral sugars (including xylose used as internal standard). Despite the low abundance of 13C in natural products, the sensitivity of the technique was found to be sufficient to detect isotopic enrichment as low as 0.001% with good accuracy and reproducibility in 2 micrograms of each glycoprotein neutral sugar. As an example, the pattern of labeling by dietary D-glucose from corn starch appears to be very different for fucose, ribose, mannose, galactose, and glucose of intestine or serum macromolecules.


Monoclonal antibody to amphomycin. A tool to study the topography of dolichol monophosphate in the membrane.

Understanding the topographical orientation of dolichol monophosphate (Dol-P) in the membrane of the endoplasmic reticulum (ER) is of utmost importance for studying the regulation of asparagine-linked protein glycosylation in eukaryotic cells. This was practically impossible due to the nonavailability of a suitable probe. Recent studies on the specific interaction between a lipopeptide, amphomycin, and Dol-P, provided an insight to develop a monospecific antibody to amphomycin which could recognize the amphomycin-Dol-P complex in order to detect Dol-P immunocytochemically in the ER membrane. We report herein the successful production of a monoclonal antibody to amphomycin. The antibody belongs to the IgG+IgM subclasses and is specific for amphomycin when analyzed by the enzyme-linked immunoassay and immunoblot procedures. The antibody recognizes with equal potency both the native amphomycin and also mild acid-hydrolyzed amphomycin from which N-terminal fatty acylated aspartic acid has been removed. Preincubation of amphomycin with the antibody partially reduced the inhibitory action of amphomycin on dolichol phosphate mannosyltransferase (EC 2.4.1.83). Furthermore, exposure of capillary endothelial cells to amphomycin, followed by the monoclonal antibody to amphomycin, followed sequentially by staining with FITC-conjugated goat anti-mouse IgG and examination under a fluorescent microscope gives intense fluorescence at the perinuclear region of the cell with a structure reminiscent of the ER.


[Synthesis of modified oligosaccharides of the N-glycoprotein as substrate for N-acetylglucosaminyltransferase I].

In the synthesis of modified derivatives of octyl O-(alpha-D-mannopyranosyl)-(1-->3)-O-[(alpha-D-mannopyranosyl)-(1-->6)]- beta-D-mannopyranoside, 4.,5-epoxypentyl, a 4-diazirinopentyl, and a 5-(iodoacetamido)pentyl group were attached to the 3''-OH of the trisaccharide. The diazirino derivative may be especially suitable for photolabeling of the active site of N-acetylglucosaminyltransferase I (GlcNAcT-I). In addition, the 2'-OH group of the above-mentioned trisaccharide was reduced to a 2'-deoxy group and substituted 2'-O-methyl group.


Glycosylation using a one-electron-transfer, homogeneous reagent. Application to an efficient synthesis of the trimannosyl core of N-glycosylproteins.

Double glycosylation of methyl 2,4-di-O-benzyl-beta-D-mannopyranoside with ethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-1-thio-alpha-D-mannopyranoside using as promoter tris(4-bromophenyl)ammoniumyl hexachloroantimonate, a stable, commercial, and crystalline radical cation, afforded after debenzoylation methyl 2,4-di-O-benzyl-3,6-di-O-(3,4,6-tri-O-benzyl-alpha-D-mannopyranoside in excellent yield. Other mannosyl donors were also investigated.


Relationship between the content of [14C]glucose-derived monosaccharides in glycoprotein oligosaccharide chains and the state of enterocytic differentiation of HT-29 cells.

The HT-29 cell line derived from a human colon adenocarcinoma has a glucose-dependent state of differentiation which is negatively correlated with the presence of D-glucose in the culture medium. The contribution of glucose to the biosynthesis of N-glycan chains, as a function of the differentiation state of HT-29 cells, was shown by: (a) [14C]glucose incorporation by undifferentiated HT-29 cells being lower after 2 h and higher after 19 h of metabolic labeling than that by differentiated cells; (b) a lack of glucose in the culture medium of undifferentiated HT-29 cells diminishing [14C]glucose incorporation into glycan chains, but not changing the glucose distribution between lipid- and protein-linked saccharides; (c) glucose behavior in undifferentiated HT-29 cells being not related to mannose-glycan metabolism, as the high-mannose compounds labeled with glucose and observed by HPLC showing a different distribution associated with the duration of glucose labeling; and (d) glucose being interconverted into other monosaccharide-glycan constituents in proportions different in differentiated and undifferentiated cell populations.


Role of ATP dependent potassium channels in myocardial ischaemia.

Recently, a class of potassium (K) channels has been discovered which are regulated by the intracellular level of ATP. These channels have been termed ATP dependent K channels (KATP) and have been found to exist in the heart, skeletal muscle, pancreatic beta cells, brain, and smooth muscle. In this article, we discuss the function of the KATP channel in the ischaemic myocardium and present evidence to suggest that activation of these channels may, on the one hand, result in a marked cardioprotective effect from reversible or irreversible electrical, functional or biochemical damage or, on the other hand, have the potential to produce electrical instability and a proarrhythmic effect. The therapeutic potential of potassium channel modulators is also discussed.


Role of potassium channels in cycle length dependent regulation of action potential duration in mammalian cardiac Purkinje and ventricular muscle fibres.

This review examines the putative role played by three repolarising potassium currents, namely the transient outward current (ito), the inward rectifying current (iK1), and the late outward rectifying current (iK), in the regulation of action potential duration in cardiac Purkinje and ventricular muscle fibres under normal physiological conditions. The role of other potassium currents, including the ATP activated current (iK,ATP) under these conditions is uncertain. Personal experiences and work of others are reviewed to summarise: (1) regulation of normal cycle length dependent action potential duration: (2) the characteristics of ito, iK1, and iK pertinent to repolarisation; and (3) the effects of potassium channel blockers and activators on cycle length dependent action potential duration. The presence of ito creates a notch after depolarisation and limits action potential duration at long cycles. Block of iK1 prolongs action potential duration predominantly by slowing phase 3 of the action potential. Block of iK prolongs the duration predominantly by lengthening phase 2 of the action potential, and the lengthening becomes more pronounced at longer cycles. Activation of iK,ATP shortens the duration, and the shortening becomes more pronounced at longer cycles. Each of the three major repolarising potassium currents appears to play a different role in modulating the action potential duration. Ito creates a notch which resets the early course of plateau, and also limits the duration at long cycles. IK1 contributes to maintenance of plateau and controls repolarisation course during phase 3 of the action potential. IK plays major role in controlling action potential duration within a wide range of cycle lengths in Purkinje fibres, and when present, also in ventricular muscle fibres.


Influence of KATP channel modulation on net potassium efflux from ischaemic mammalian cardiac tissue.

The aim was to investigate the effect of pre-exposure to inhibitors or activators of the ATP regulated K+ (KATP) channels on the ischaemia induced early changes of the extracellular K+ concentration, the extracellular pH, and the action potential in mammalian cardiac tissue. An in vitro model simulating ischaemia was applied to isolated guinea pig papillary muscles while simultaneous microelectrode measurements of the transmembrane potentials and of the pH and K+ activity at the surface of the preparation (pHs and asK respectively) were made. During conditions of superfusion arrest, when accumulation of metabolic acids causes acidification of pHs, the simultaneous development of a true hypoxic state of the muscle is required in order to induce shortening of the action potential duration and accumulation of extracellular K+ with associated membrane depolarisation. Glibenclamide (10-50 microM) slowed the decrease of the action potential duration in 50% of the muscles, while the increase of asK was only moderately (approximately 20%) influenced. In the presence of 200 microM of the KATP channel inhibitor, the mean K+ accumulation was not significantly different from the control. The presence of tolbutamide (1 mM) had no effect on the decrease of action potential duration, but moderately slowed the increase of asK. Superfusion with lemakalim (BRL 38227) decreased action potential duration dose dependently. In papillary muscles in which action potential duration was shortened to approximately 60% of the control duration by presuperfusion with lemakalim, a subsequent ischaemic episode did not increase but rather delayed the rise in asK. During simulated ischaemia in the presence of Ba2+ ions (1 mM), asK showed a transient decrease followed by a rise at a rate similar to that in the absence of Ba2+. Early ischaemic K+ accumulation and surface acidification are relatively insensitive to KATP channel inhibition or activation prior to the ischaemic insult. The rather loose coupling of the increase of asK and the decrease of action potential duration, as well as the limited effect of sulphonylureas on the K+ increase, suggests that mechanisms other than KATP channel opening could possibly contribute to the initial phase of ischaemic K+ accumulation.


Effects of diltiazem and noradrenaline on extracellular potassium changes in the globally ischaemic rat heart.

The aim was to investigate the effects of a calcium antagonist (diltiazem) and a catecholamine (noradrenaline) on extracellular potassium accumulation during global ischaemia. Extracellular potassium concentration ([K+]e) was measured during 30 min global ischaemia in the isolated rat heart using a valinomycin potassium sensitive electrode. Contracture development during ischaemia was measured throughout with an intraventricular balloon inserted into the left ventricle and myocardial adenine nucleotides were measured in separate series of hearts. In control hearts, [K+]e showed a characteristic triphasic change during 30 min global ischaemia. This consisted of an early rising phase followed by a transient falling phase after the initial peak of [K+]e, and then a late rising phase. Diltiazem suppressed the rate of rise of [K+]e during early ischaemia, but extended the time course of the early [K+]e rise with the higher dose, abolishing the transient falling phase of [K+]e. During late ischaemia, the rise in [K+]e was attenuated by diltiazem. Noradrenaline also suppressed the early extracellular potassium accumulation, but in contrast to diltiazem, hastened the time course of the late [K+]e rise. Although diltiazem suppresses the early potassium loss during ischaemia as previously described, the drug also decreases the [K+]e fall by some as yet unknown mechanism, so that the [K+]e level becomes higher than control during the falling phase.


Effects of intracoronary cromakalim on postischaemic contractile function and action potential duration.

The effects of intracoronary cromakalim (1 microgram.kg-1 x min-1) on postischaemic contractile function and monophasic action potential duration at the 95% repolarisation level (APD95) were assessed in a canine model of stunned myocardium. Animals (n = 24) were anaesthetised, subjected to a left thoracotomy, and the left anterior descending coronary artery was isolated. Measurements of segmental shortening and monophasic action potential were taken before drug, after drug, during a 15 min occlusion, and at times following reperfusion. Cromakalim significantly improved reperfusion recovery of function. Both preischaemic and postischaemic myocardial blood flows were increased with cromakalim, although there was no significant change in collateral blood flow during ischaemia relative to vehicle. In the absence of ischaemia, cromakalim reduced APD95 by 8%. There was no change in APD95 with vehicle alone. During coronary occlusion, cromakalim significantly reduced APD95 by 27% as compared with 8% in the vehicle group. APD95 values returned to preocclusion levels within minutes of reperfusion and remained there throughout reperfusion (30 min). Glyburide (3.0 mg.kg-1 intravenously), a blocker of ATP sensitive potassium channels, abolished the APD95 effects of cromakalim. One out of six animals given glyburide survived through reperfusion. In contrast, four out of five of the glyburide+cromakalim treated animals survived through reperfusion. Cardioprotection with cromakalim is likely to be mediated through activation of ATP sensitive potassium channels, measured as a decrease in action potential duration. Shortening of action potential duration with cromakalim was shown to be augmented in the presence of ischaemia. It is suggested that a possible interrelationship between cardioprotection and action potential duration shortening may exist such that the cardioprotective effects of cromakalim may be selective for ischaemic conditions.


Blockade of ischaemic preconditioning in dogs by the novel ATP dependent potassium channel antagonist sodium 5-hydroxydecanoate.

The aims were: (1) to determine if a new ischaemia selective ATP dependent potassium (KATP) channel antagonist, sodium 5-hydroxydecanoate (5-HD), blocks ischaemic preconditioning in dogs; (2) to determine whether a small intracoronary dose of glibenclamide, a classical sulphonylurea KATP channel antagonist, could block ischaemic preconditioning independent of systemic metabolic effects. Barbitone anaesthetised dogs were subjected to 60 min of left circumflex coronary artery occlusion followed by 5 h of reperfusion. Preconditioning was produced by a single 5 min left circumflex occlusion followed by 10 min of reperfusion prior to the 60 min occlusion period. 5-HD (150 micrograms.kg-1 x min-1) or vehicle was given by intracoronary infusion into the ischaemic region over 20 min, beginning 15 min prior to the 60 min occlusion period in the presence or absence of preconditioning. Glibenclamide (3 micrograms.kg-1 x min-1) was given by intracoronary infusion into the left circumflex artery during the 5 min preconditioning period or during the first 5 min of occlusion in preconditioned or non-preconditioned dogs. Transmural myocardial blood flow was measured by radioactive microspheres and infarct size determined by triphenyltetrazolium staining and expressed as a percent of the area at risk. There were no differences in haemodynamic variables, myocardial blood flow, area at risk, or blood glucose between groups. Infarct size was markedly reduced in preconditioned dogs compared to control animals, at 7(SEM 2)% v 29(4)%, p < 0.05 The reduction in infarct size by preconditioning was blocked completely by intracoronary 5-HD, or by intracoronary glibenclamide given during preconditioning or during the first 5 min of the prolonged occlusion period. Neither 5-HD nor glibenclamide affected infarct size in the absence of preconditioning at the doses studied. These results further strengthen the hypothesis that activation of myocardial KATP channels is involved in the mechanism of ischaemic preconditioning in dogs.


Effects of potassium channel modulation during global ischaemia in isolated rat heart with and without cardioplegia.

The opening of potassium (K+) channels during regional ischaemia may, by inducing rapid contractile arrest, be an intrinsic energy sparing mechanism. Thus K+ channel openers (for example, lemakalim) exert significant anti-ischaemic effects, whereas glibenclamide exacerbates ischaemic contracture and limits postischaemic functional recovery. The aim of the study was to investigate the ability of these compounds to influence ischaemic injury when used either alone or in combination with rapid arrest induced by a high K+ cardioplegic solution. Contractile function of isolated Langendorff perfused rat hearts was assessed using an intraventricular balloon. Hearts were subjected to normothermic global ischaemia (20 min) or cardioplegic arrest (35 min) with and without glibenclamide or lemakalim. Lemakalim (10 mumol.litre-1) or glibenclamide (10 mumol.litre-1) was given, in the presence or absence of cardioplegia, for 2 min immediately prior to the onset of ischaemia. The rate of ischaemia induced contractile failure, the severity of ischaemic contracture, and the degree of postischaemic functional recovery were all measured. In the absence of cardioplegia, the time to contractile arrest in control hearts was 133 (SEM 4) s. This was increased by glibenclamide, to 145(6) s, and decreased by lemakalim, to 112(6) s. The time to onset of ischaemic contracture [8(1) min] was accelerated by glibenclamide [4(1) min] and delayed by lemakalim [14(1) min]. Lemakalim significantly improved the recovery of left ventricular developed pressure from 49(7)% in control hearts to 65(3)%, and left ventricular end diastolic pressure from 41(3) to 21(4) mm Hg. Hearts pretreated with glibenclamide showed similar recoveries to control hearts. During reperfusion, lemakalim exerted a transient vasodilator effect whereas glibenclamide caused a transient vasoconstriction. When either glibenclamide or lemakalim was added to a high K+ cardioplegic solution and hearts rendered ischaemic for 35 min, the ability of both compounds to influence ischaemic contracture and postischaemic functional recovery was lost. In additional studies the effect of lemakalim on the relative times to ischaemia induced mechanical failure and electrical arrest was assessed. In control hearts the time to contractile failure was 128(5) s and the time to electrical arrest was 241(30) s, while in the lemakalim treated hearts the values were 103(2) s and 509(161) s, respectively. In the lemakalim group all the hearts showed sustained ventricular arrhythmias; in the control group there were no arrhythmias. Lemakalim can exert a significant anti-ischaemic effect when given alone. This effect is lost when it is used in combination with high K+ cardioplegia. The anti-ischaemic properties of lemakalim may thus be limited to its ability to accelerate contractile arrest.


Effects of glibenclamide on ventricular arrhythmias and cardiac function in ischaemia and reperfusion in isolated rat heart.

The aim was to investigate the effects of glibenclamide, a specific blocker of the ATP sensitive potassium channel, on the incidence of ventricular arrhythmias and the functional changes occurring during myocardial ischaemia and reperfusion. Hearts (n = 10 per group) were obtained from male Wistar rats, weight 250-300 g. The study was performed in isolated Langendorff perfused rat hearts subjected to ligation of the left coronary artery and reperfusion. Because of the occurrence of arrhythmias, cardiac function was not evaluated during reperfusion. Glibenclamide (1 or 10 microM) was added to the perfusion solution before the coronary artery occlusion, during ischaemia or after reperfusion. In some experiments the incidence of various durations of ischaemia (5, 10, 15, and 30 min) was evaluated. During the preischaemic period, glibenclamide induced a marked reduction in coronary flow, with a slight decrease in heart rate and left ventricular pressure. The ischaemia induced decrease in left ventricular pressure was markedly attenuated when glibenclamide was given before ischaemia. Thus the isovolumetric left ventricular pressure measured after 15 min ischaemia, which represents 59(SEM 6)% of the preischaemic value in the control group, was increased to 82(9) and 94(8)% in presence of glibenclamide (1 and 10 microM, p < 0.05 respectively). The effect was less pronounced when glibenclamide was added to the perfusion fluid during the ischaemic period. None of the hearts showed ventricular fibrillation during the ischaemic period. Glibenclamide (1 and 10 microM) did not reduce the incidence of reperfusion induced ventricular fibrillation. However, a defibrillatory action was observed since glibenclamide reduced the duration of ventricular fibrillation during reperfusion. Glibenclamide may increase the probability of spontaneous termination of ventricular fibrillation and facilitate the restoration of the myocardial function during regional ischaemia.


Regulation of ATP sensitive potassium channel of isolated guinea pig ventricular myocytes by sarcolemmal monocarboxylate transport.

The aim was to describe the effects of extracellular application of monocarboxylates (pyruvate, lactate, or acetate) on current through KATP channels (iK,ATP) in isolated guinea pig ventricular myocytes. The iK,ATP was elicited during whole cell voltage clamping by application of metabolic poisons, 2,4-dinitrophenol (150 microM) or glucose free cyanide (1 mM) and could be blocked by glibenclamide (3 microM). Extracellular application of monocarboxylates, pyruvate (0.1-10 mM), L-lactate (0.1-10 mM), and acetate (10 mM) led to a rapid inhibition of iK,ATP--an effect which was fully reversible upon washout. Substances without any effect on iK,ATP were (10 mM each) gluconate, citrate, glutamate, creatine, succinate, and glycine. The mechanism underlying the effects of monocarboxylates on iK,ATP was unlikely to be related to an increased ATP production, since D-lactate (10 mM) essentially had the same effect on iK,ATP as the L-isomer of lactate. Furthermore, with intracellular dialysis of alpha-cyano-4-hydroxycinnamate (0.1-0.5 mM), which inhibits pyruvate uptake into mitochondria, extracellular pyruvate exerted the same inhibitory effect on iK,ATP. High concentrations of extracellular alpha-cyano-4-hydroxycinnamate (4 mM), which blocks the sarcolemmal monocarboxylate carrier, prevented the effects on iK,ATP by pyruvate, L-lactate, D-lactate, and acetate. Furthermore, intracellular dialysis with D-lactate (10 mM) led to a more rapid onset of iK,ATP when activated by ATP free dialysis. Activity of isolated KATP channels, measured in isolated membrane patches in the inside out or outside out configuration, typically had a single channel conductance of around 80 pS and was blocked by glibenclamide (3-9 microM). No significant effect of pyruvate was observed in either patch configuration. In cardiac tissue there may be some modulatory role involving monocarboxylate transport on KATP channel activity, the nature of which is unclear at present but which may involve cytosolic pH changes. Physiological and pathophysiological implications of these findings are discussed.


Comparative studies of ATP sensitive potassium channels in heart and pancreatic beta cells using Vaughan-Williams class Ia antiarrhythmics.

Actions of cibenzoline and disopyramide, agents with Vaughan-Williams class Ia antiarrhythmic action, on ATP sensitive K+ (KATP) channels were examined in heart and pancreatic beta cells. Single ventricular myocytes and beta cells were prepared enzymatically from adult Wistar rat hearts and pancreatic islets. Using patch clamp techniques, KATP channel activities were recorded in whole cell and single channel modes. In whole cell experiments, myocytes were bathed with Tyrode's medium (34 degrees C); inside out patches were bathed with internal solutions (22-24 degrees C) containing 1 microM ATP and varying concentrations of cibenzoline or disopyramide. Myocytes were voltage clamped at -40 mV and glibenclamide blockade conductance was produced by cromakalim. Micromolar concentrations of both cibenzoline and disopyramide suppressed cromakalim induced conductance. When applied to the cytosolic surface of the cell membrane in inside out configuration, both drugs reversibly inhibited single KATP channel activities. Neither unitary conductance nor intraburst fast kinetics was affected by the compounds. At a holding potential of -40 mV under symmetrical approximately 150 mM K+ conditions, half maximum doses (IC50) were 0.9 microM [Hill coefficient (h) = 1.3] for cibenzoline induced block of cardiac KATP channels and 1.8 microM (h = 1.0) for disopyramide block. At +40 mV, IC50 for cibenzoline block was 1.4 microM (h = 0.9). Thus there was little voltage dependence in cibenzoline induced channel block. A similar IC50 value of 2.5 microM (h = 1.2 at -60 mV under symmetrical approximately 150 mM K+) was observed for cibenzoline induced block of KATP channels. Near therapeutic concentrations of cibenzoline and disopyramide inhibit KATP channel activities in both heart and pancreatic beta cells. This may be causally related to the fasting hypoglycaemia which is sometimes reported in patients receiving the drugs. These antiarrhythmic agents may also modulate myocardial electrical properties during hypoxia or ischaemia.


Blockade of 2,4-dinitrophenol induced ATP sensitive potassium current in guinea pig ventricular myocytes by class I antiarrhythmic drugs.

The aim was to assess the effects of various antiarrhythmic drugs on 2,4-dinitrophenol (DNP) induced outward current (IDNP), presumably the ATP sensitive K+ current (IK,ATP) of isolated cardiac cells and to discuss mechanisms involved in the hypoglycaemia which occurs in patients on these drugs. The quasi-steady state current-voltage relationship from the isolated guinea pig ventricular cells was measured using whole cell voltage clamp techniques with a ramp pulse programme. The effects of seven different antiarrhythmic drugs on IDNP were examined. Action potentials were elicited at a rate of 0.2 Hz by an intracellular current injection. DNP (50 mumol.litre-1) increased the quasi-steady state outward current at potentials positive to about -60 mV. This current (IDNP) was completely inhibited by the subsequent application of glibenclamide (1 mumol.litre-1), thereby suggesting that the IDNP is probably IK,ATP. Cibenzoline (10 mumol.litre-1, class Ia), disopyramide (30 mumol.litre-1, class Ia), and procainamide (100 mumol.litre-1, class Ia) significantly inhibited the IDNP by 95.5(SD 11.3)%, 77.8(21.2)%, and 76.4(23.9)% respectively. Flecainide (class 1c) inhibited the IDNP by 66.9(23.9)% at 10 mumol.litre-1 but not at 2 mumol.litre-1. Mexiletine (30 mumol.litre-1, class Ib), pilsicainide (50 mumol.litre-1, class Ic), and E4031 (10 mumol.litre-1, class III) at concentrations as high as approximately fivefold the clinically effective blood levels, did not suppress IDNP. Except for 10 mumol.litre-1 flecainide, all the concentrations listed above which blocked IDNP were within twofold of the clinical blood concentrations documented to be effective for suppression of arrhythmias. Cibenzoline, disopyramide, and procainamide, but not flecainide, belong to class Ia antiarrhythmic drugs. All these class Ia antiarrhythmic drugs "shortened" the action potential duration of guinea pig ventricular cells, an opposite change to that noted for multicellular preparations, eg, guinea pig papillary muscles. Class Ia antiarrhythmic drugs (cibenzoline, disopyramide, and procainamide) inhibit IDNP (presumably IK,ATP) in guinea pig ventricular cells within a range of therapeutic concentrations. This inhibitory effect of IK,ATP can probably explain the hypoglycaemia which occurs in some patients receiving these drugs, and the prolongation of the action potential duration alleged to occur in "superfused" papillary muscles.


Dofetilide, a new class III antiarrhythmic agent, reduces pacing induced heterogeneity of repolarisation in vivo.

The aim was to determine whether dofetilide, a new class III antiarrhythmic agent, could reduce the heterogeneity of repolarisation produced by rapid cardiac pacing and to compare it with quinidine. Increased heterogeneity of repolarisation times may be causally linked to malignant cardiac re-entrant arrhythmias. Studies were performed in open chest, artificially ventilated, pentobarbitone anaesthetised beagle dogs of 13 to 15 kg body weight. Myocardial electrocardiograms were simultaneously recorded from 31 electrodes located on the left and right ventricular surfaces of the in situ canine heart. Recordings were obtained over a single cardiac cycle during rapid ventricular pacing at the maximum following frequency. Computer assisted measurements of activation time, activation-repolarisation interval, and repolarisation time were performed and the heterogeneity of these measurements calculated. Measurements were performed before treatment and after each incremental intravenous dose of dofetilide (3, 10, 30, and 100 micrograms.kg-1), quinidine (1, 3, and 10 mg.kg-1), or saline vehicle. Dofetilide increased repolarisation time via a selective prolongation of activation-repolarisation interval, activation time being unchanged. It reduced the heterogeneity of repolarisation time and activation-repolarisation interval, heterogeneity of activation time being unchanged. Quinidine increased repolarisation time by a non-selective increase of both activation-repolarisation interval and activation time. It increased the heterogeneity of activation times which offset a decrease in heterogeneity of activation-repolarisation intervals to produce no change in heterogeneity of repolarisation times. Dofetilide, but not quinidine, reduces pacing induced asynchrony of repolarisation in the canine heart and this effect may contribute to its antiarrhythmic activity.


Class III antiarrhythmic action by potassium channel blockade: dofetilide attenuates hypoxia induced electromechanical changes.

The aim was to examine the electromechanical effects of dofetilide, a new class III antiarrhythmic agent, in isolated guinea pig ventricular muscle during hypoxia. Hypoxia was induced by superfusing guinea pig right ventricular papillary, muscles with Tyrode's solution gassed with 95% N2 + 5% CO2 [PO2 = 5.3(SEM 1.3) kPa]. Prior to hypoxia, the preparations were either pretreated for 30 min with 0.1 microM dofetilide (n = 6) or with 100 microM glibenclamide (a blocker of ATP sensitive K+ channels, n = 6), or not pretreated (n = 6). Sixteen additional preparations were exposed to 1 mM nicorandil (an activator of ATP sensitive K+ channels) in the absence (n = 6) and presence of dofetilide (n = 6) or glibenclamide (n = 4). Transmembrane action potentials and developed force were recorded using conventional microelectrode techniques and a force transducer. During normoxia, dofetilide markedly increased APD90 from 236(SEM 6) ms to 298(7) ms (p < 0.05) and the effective refractory period (ERP) from 248(5) ms to 315(6) ms (p < 0.05). In the drug free group, 60 min hypoxia decreased APD90 by 47(5)% (p < 0.05), ERP by 48(4)% (p < 0.05) and developed force by 71(6)% (p < 0.05) of baseline, respectively. These hypoxia induced effects were significantly attenuated after pretreatment with dofetilide or glibenclamide. Nicorandil decreased APD90 by 45(5)% (p < 0.05), ERP by 44(6)% (p < 0.05), and developed force by 69(10)% (p < 0.05) of baseline, respectively. Pretreatment with dofetilide or glibenclamide also significantly attenuated the nicorandil induced decreases in APD90, ERP, and developed force. Dofetilide, like glibenclamide, effectively attenuates hypoxia and nicorandil induced action potential shortening and the associated reduction in contractile force. Thus dofetidile would be expected to retain its antiarrhythmic efficacy during myocardial hypoxia or ischaemia.


Antiarrhythmic and electrophysiological effects of amiodarone, lignocaine, and penticainide in anaesthetised rats.

The aim was to correlate the relative abilities of amiodarone, lignocaine, and penticainide in suppressing ventricular tachyarrhythmias in response to coronary artery occlusion with their relative abilities to prolong ventricular functional refractory periods and to reduce intraventricular conduction velocities in anaesthetised open chest rats. Functional refractory period was measured with a paired electrical stimulation technique. Intraventricular conduction velocity was monitored using the QRS duration of the ECG. Low doses of these three drugs were selected which protected against ventricular tachyarrhythmias induced by coronary occlusion. These doses prolonged the functional refractory period without increasing QRS duration. High doses of both amiodarone and lignocaine were more effective than low doses in suppressing coronary occlusion induced ventricular tachyarrhythmias. Such high doses greatly prolonged the functional refractory period but prolonged the QRS duration only moderately. In contrast, higher doses of penticainide failed to inhibit coronary occlusion induced ventricular tachyarrhythmias. These higher doses of penticainide prolonged the functional refractory period but prolonged the QRS duration even more markedly. Among these three drugs an increase in functional refractory period appears to be antiarrhythmic. In contrast, widening of the QRS complex without a corresponding increase in functional refractory period, as produced by penticainide, appears to counteract the antiarrhythmic effect.


Differential block of cardiac delayed rectifier current by class Ic antiarrhythmic drugs: evidence for open channel block and unblock.

The aim was to compare the effects of the class Ic antiarrhythmic drugs flecainide, encainide, and recainam on the delayed rectifier current, IK. Membrane currents were studied using the single suction pipette voltage clamp technique in freshly dissociated cat ventricular myocytes bathed in HEPES buffered physiological saline at 32 degrees C. Flecainide and encainide decreased IK with IC50 values of 2.1 microM and 6 microM, respectively. Recainam (100 microM) reduced IK by only 7 (SEM 3)% after 20-30 min exposure and by 19% after an 80 min exposure (IC50 > 400 microM). None of the compounds blocked the inward rectifier, IK1. Block of IK by flecainide and encainide increased with depolarisation following a voltage dependence similar to that describing channel activation. Flecainide and encainide also slowed the time course of the IK tail currents, consistent with drug dissociating from open channels. The observed voltage dependence for IK block by flecainide and encainide resembles the interaction reported between these agents and the excitatory sodium channel, ie, depolarisation enhances block while repolarisation leads to removal of block. The results further suggest that the electrophysiological profile of class Ic agents can have a markedly different ionic basis, ie, K+ channel block by flecainide and encainide is balanced by a potent block of sodium channels, while recainam appears to be a weak but relatively specific blocker of sodium channels only. These differences are not readily accommodated by the current Harrison-Vaughan-Williams classification scheme, and suggest the possibility that potentially important drug specific differences can exist within the same antiarrhythmic drug class.


Differential inhibition of potassium currents in rat ventricular myocytes by capsaicin.

Capsaicin is a pungent irritant present in peppers of the Capsicum family. Its major target of action is believed to be sensory neurones. Capsaicin has also been shown to prolong cardiac action potential in atrial muscle, perhaps by local release of calcitonin gene related peptide which in turn enhances inward calcium currents. However, capsaicin has been shown to inhibit K+ current in neurones. Since such an action could contribute to action potential prolonging activity of capsaicin in heart, the aim of the study was to examine the effects of capsaicin on cardiac K+ currents. Ionic currents and action potentials were examined in isolated adult rat ventricular myocytes using the whole cell variant of the patch clamp technique at 25 degrees C. Capsaicin (10 microM) increased the action potential duration (APD50) from 45 ms to 166 ms. This effect was associated with an inhibition of three distinct K+ currents. The decreasing rank order of potency was: transient outward K+ current (ITO, IC50 = 6.4 microM), a voltage dependent non-inactivating outward current (IK, IC = 11.5 microM), and the inward rectifier K+ current (IK1, IC50 = 46.9 microM). Capsaicin induced block of ITO was characterised by a decrease in the peak current amplitude and an increase in the rate of inactivation. The inactivation of ITO in the absence of capsaicin was well described by a single exponential [tau = 77 (SEM 2) ms at +40 mV, n = 10]. However, in the presence of 10 microM capsaicin inactivation was best described by the sum of two exponentials [tau FAST = 4.4(0.5) ms; tau SLOW = 92.4(3.0) ms, n = 10] with the fast component contributing 46(2)% of the total decay. A small but consistent hyperpolarising shift (approximately 3 mV) in the steady state voltage dependence of inactivation of ITO was induced by 10 microM capsaicin. Capsaicin had no effect on the rate of ITO recovery from inactivation (tau = 49 ms and 48 ms for control and drug respectively). The capsaicin analogue, resiniferatoxin, which as an irritant is up to 10(4)-fold more potent than capsaicin, had no effect on any of the K+ currents when present at concentrations of up to 10 microM. In contrast another capsaicin analogue, zingerone (30 microM) blocked ITO by 52(12)% and IK by 35%. Capsaicin produces a prolongation of the rat ventricular action potential, an effect which is associated with inhibition of potassium currents.


Differential effects of the new class III antiarrhythmic agents almokalant, E-4031 and D-sotalol, and of quinidine, on delayed rectifier currents in guinea pig ventricular myocytes.

The effects of almokalant (4-[3-ethyl[3-(propylsulphinyl)propyl]-amino]-2-hydroxy-propoxy]- benzonitrile), E-4031 (1-[2-(6-methyl-2-pyridyl)-ethyl]-4-(4-methylsulphonyl-amino- benzoyl)piperidine), d-sotalol, and quinidine were investigated on the delayed K+ rectifier current IK. The aim of the study was to compare the drug action on the two components of this current. Membrane currents were measured in ventricular myocytes from guinea pig hearts with the whole cell voltage clamp technique. IK was activated during clamp steps from a holding potential of -40 mV to test potentials -30 and +50 mV. The tail current Itail was measured upon stepping back to holding potential. In control experiments. IK and Itail declined spontaneously ("run down"). With 300 ms long test pulses to +50 mV, only d-sotalol (10(-4) M) caused a significant further decrease in IK, whereas all four agents significantly reduced Itail (almokalant 10(-6) M, E-4031 10(-7) M, quinidine 10(-5) M). When tested with 1 s long clamp steps at various potentials almokalant (3 x 10(-6) M), E-4031 (10(-6) M), quinidine (10(-5) M), and d-sotalol (10(-4) M) reduced IK in the potential range between -20 and +40 mV, yielding a bell shaped inward rectifying drug sensitive current. Itail was reduced by almokalant and E-4031 over the whole voltage range with saturation of block positive to +20 mV. Similar reductions with quinidine but not with d-sotalol were also significant. With rest pulses to +50 mV of increasing duration (25 ms-4000 ms), Itail developed with a faster time course than IK and therefore the ratio of Itail/IK declined with pulse duration. With almokalant and E-4031, this ratio became independent of test pulse duration. For 250 ms pulses, Itail/IK was also significantly reduced by d-sotalol and quinidine. Inhibition of the rapidly activating inwardly rectifying component of IK is prominent with almokalant and E-4031 and less pronounced with d-sotalol and quinidine. Since inhibition of this component prolongs the cardiac action potential, it should contribute to the antiarrhythmic properties of the agents.


Impact of overbite on indicators of temporomandibular joint dysfunction.

Epidemiological studies have suggested that deep overbite is associated with symptoms of temporomandibular dysfunction (TMD). This finding was directly tested by deliberately constituted groups of deep and normal overbite subjects matched for age and sex. Eighty-one subjects participated. Dependent measures included the TMJ Scale test; measures of muscle activity in the right and left frontalis, temporalis, and masseter muscles; and pressure threshold meter readings for the same sites. The results showed no differences between deep and normal overbite subjects for all the dependent measures studied. The role of overbite alone in producing TMD symptoms is questioned.