[The pathways of the stabilization of the amplitude of postsynaptic potentials in the interneuronal synapses of vertebrates]. The influence of various factors on the degree of stabilization of postsynaptic potential amplitude has been studied by mathematical modelling. Increasing of transmitter release probability in single boutons, spatial non-uniformity of these probabilities, interaction of release sites, non-linear summation of potentials lead to amplitude stabilization. Temporal fluctuations of the release probability, failures of responses from contact groups and activation of a variable number of the fibers have the opposite influence. Comparison of synaptic transmission parameters in sensorimotor synapses of Cyclostomata, amphibians and mammals showed that during evolution of these synapses two different pathways of amplitude stabilization had been realized. Formation of highly effective chemical contacts is probably the most progressive pathway among them.


[Brain stem auditory evoked potentials in spinocerebellar degeneration]. A study was made of brainstem auditory evoked potentials (BAEP) in 66 patients from 56 families with different forms of spinocerebellar degenerations (SCD). 27 patients with olivopontocerebellar degeneration (OPCD), 13 patients suffering from Friedreich's disease (FD), 10 patients with Pierre Marie's familial ataxia (PMFA), 6 patients with late onset cerebellar atrophy (LOCA), and 10 patients with other forms of SCD were examined. The changes in BAEP turned out extremely diverse which can be regarded as a manifestation of marked phenotypic pleomorphism common to SCD. The most considerable changes in BAEP were discovered in FD and OPCD, whereas the least marked ones in PMFA and LOCA. The character and degree of BAEP disorders reflect the spreading and gravity of degenerative alterations in the brain stem in different forms of SCD. The authors discuss the possibility of the use of BAEP for objective estimation of the gravity and spreading of the pathological process as well as of the electrophysiological control over its course in SCD patients.


[Clinico-electrophysiological characteristics of women--heterozygote carriers of fragile X chromosome]. Overall 21 women, heterozygous carriers of fragile X-chromosome (7 cases of obligate carriership and 14 sporadic cases) were examined by electroencephalography and cytogenetically. In none of them the clinical examination revealed a typical somatic appearance characteristic of Martin-Bell syndrome. Certain somatic disorders, psychasthenic traits, tearfulness and uncommunicativeness were recorded in single cases. The level of intellectual development corresponded to the low norm. Analysis of the EEG has demonstrated that in 76% of the women examined, the character of alpha-rhythm differed from normal. According to its intensity, three variants of the EEG were distinguished. In the majority of women, the EEG manifested the signs of the dysfunction of the diencephalic structures and not gross focal alterations. The character of the EEG does not provide any data which may be of diagnostic importance in revealing heterozygous carriership of fragile X-chromosome.


[Comparative evaluation of various forms of parkinsonism in middle-aged and elderly patients (clinical, neuropsychological and computerized-tomographic studies)]. The clinical, neuropsychological and computed tomography data were compared in elderly and senile patients with trembling and akinetico-rigid forms of parkinsonism. 12 patients with trembling and 21 with akinetico-rigid forms were examined. The groups did not differ in age or sex. The results of computed brain tomography were estimated with the use of linear, volumetric and densitometric parameters. The akinetico-rigid form was associated with more diverse clinical and neuropsychological symptomatology, more noticeable cerebral atrophy. The gravity of the status of the patients with trembling parkinsonism was largely specified by the intensity of the hyperkinetic syndrome whereas that of the patients with the akinetico-rigid form not only by amyostatic disorders but also by the presence and severity of discoordination, pseudobulbar and mnemonic-intellectual disturbances. Disorders revealed in the patients with the akinetico-rigid form often attest to the dysfunction of the frontal parts of the brain. The nosological heterogeneity of these two forms of parkinsonism is under discussion.


[The pharmacological characteristics, immunochemical identification and study of the location of quisqualate receptors in the rat and human brains]. The kinetics of [3H]-L-glutamate binding to brain synaptic membranes (SM) and to glutamate-binding proteins (GBP) was determined with agonist and monoclonal antibodies (MAbs). It was revealed, that rat and human brain GBP have individual protein components with M(r) from 14 to 92 kDa. Quisqualate inhibited [3H]-L-glutamate binding to solubilized and to purified 68 kDa protein component. MAbs have the most activity, and NMDA was failure. It has been shown that 68 kDa component antigen determinants are similar to those of bovine, frog and rat brain synaptic membranes. Anti-GBP monoclonal antibodies blocked functional non-NMDA receptors in isolated frog spinal cord. Immunocytochemistry was done on rat and human brain sections. Distribution of quisqualate receptors was determined with light and electron microscopy. Some properties of vertebrate CNS non-NMDA receptors are discussed.


[Neuropsychological syndromes in hereditary neuromuscular pathology]. Clinical and experimental psychological studies (MMPI, Eisenks's questionnaire, methods by Luria and Kraepelin, types of attitude toward disease) carried out in 157 adults and children with progressive myodystrophies and amyotrophies revealed alterations in the neuropsychic sphere in 134 patients (85%). In the structure of borderline disorders, depressive disturbances prevailed (54.5%), and the asthenic and psychopathlike symptomatology could be seen. The use of the psychopharmacological and psychotherapeutic correction promoted regression of the psychopathological symptomatology in 76% of adults and in 84% of children.


[Various familial and demographic indicators in relation to the severity of mental retardation in the probands]. A comparative analysis was made of some familial and demographic characteristics in 3 groups of mentally retarded patients with varying intensity of deficiency. The hypothesis of different etiology of mild and severe mental retardation was confirmed. Appreciable differences were found in the microsocial characteristics in the intermediate group of patients with marked debility. In certain characteristics, this group was similar to the gravest patients whereas in some others, to the group with mild grades of intellectual underdevelopment. This fact can be accounted for by the heterogeneity of the intermediate group.


[Clinico-functional correlations in the evaluation of the development of children with perinatal lesions of the central nervous system during the first 6 months of life]. The purpose of the work was to design a model of the screening estimation of the psychomotor development of the child with perinatal CNS derangement seen in the first half-year of life since the neonatal period. Use was made of noninvasive diagnostic methods, available for examination both under in- and outpatient conditions. Complex analysis of the health status of the child with perinatal CNS pathology made every month of life according to the screening program allows one to decide the problem of the intensity of morphofunctional disorders of the brain and of the methods of choosing treatment and corrective measures.


[Neuropsychological studies of the gnostic processes in children with various forms of infantile cerebral palsy]. Psychometric and neuropsychological studies were carried out in 182 patients with three forms of infantile cerebral paralysis (ICP). Of these, 112 children presented with spastic diplegia, 50 with hemiparetic diplegia, and 20 with hyperkinetic diplegia. The children's age ranged from 8 to 14 years. Depending on the form of ICP, the structural characteristics of intellect were defined as were specific features of the development of higher cortical functions depending on the localization of the underdevelopment of different brain areas. The classification of the structure of the disorders with the aid of the methods used makes it possible to have a differentiated approach to the medical and pedagogical correction of those patients.


[Treatment of patients with neurocirculatory asthenia by intermittent hypobaric hypoxia]. Rheoencephalography and electroencephalography were employed during 25 days of hypobarotherapy of 62 patients suffering from vegetovascular dystonia with different types of brain +blood circulation. The efficacy of the treatment with the aid of periodic hypobaric hypoxia turned out inconclusive which was specified by the initial status of vegetative regulation of the vascular tone and initial dyscirculatory arteriovenous alterations determining in many respects specific features of cerebrovascular reactions. The positive response to intermittent hypoxia seen in the majority of patients allows recommending the given treatment schedule for hemodynamic disorders in patients suffering from vegetovascular dystonia with different types of brain +blood circulation.


[Possibility of using interferon in the treatment of alcoholism]. As many as 20 alcoholics were examined for the therapeutic efficacy of interferon. The control group was made up of 20 alcoholics who were treated by teturam. The clinical, neuroendocrine and immune data demonstrate the high efficacy of the use of interferon in alcoholics. The mechanisms of the drug action are under discussion.


[Late effects of psychogenic and radiation factors of the accident at the Chernobyl nuclear power plant on the functional state of human brain]. As many as 97 persons, victims to the Chernobyl accident, who developed vegetative dystonia were examined by clinical, ++patho-psychological and electroencephalography methods. 24 patients with vegetative dystonia who had not taken part in the liquidation of the consequences of the Chernobyl accident and not lived in the zones of alienation and radioactive contamination were examined too. In the victims to the Chernobyl accident, vegetovascular dystonia was marked by certain clinical and neurophysiological peculiarities in the form of combined vegetative disturbances and hypochondriac symptoms, signs of the schizoform organic syndrome with diffuse disorders of brain bioelectric activity and irritation of the subcortical structures. The contribution of psychogenic and prolonged ionizing radiation to the formation of changes in brain function in victims to the Chernobyl accident is under consideration.


[Effect of chronic pathology of the upper cervical sympathetic ganglion on the cerebral cortex (experimental study)]. A study was made of the character of disorders of glycogen metabolism and dynamics of the glycogen synthetic properties of sensorimotor cortical neurons at different stages of dystrophic processes under chronic irritation of the anterior cervical ganglion. The authors demonstrate the local glycogen synthesis in synapse ultrastructures, which is of paramount importance for local homeostasis, ensuring high plasticity and dynamism of cortical synapses in information transmission. Decimetric radiotherapy revealed that the sclerosed sympathetic ganglion exerts a permanent tonic effect on the regulation of intracerebral vessels. It may be assumed that control of sympathetic fibers from the cervical ganglion is aimed to a definite measure at specialized regulation of energy brain supply.


[Pathological status of catecholamines in the corpus striatum in hepatocerebral dystrophy (Wilson-Konovalov disease)]. The content of catecholamines in the striatum was measured in 2 patients suffering from hepatocerebral dystrophy (Wilson-Konovalov disease). It is noted that in different clinical manifestations of the disease, the changes in the content of noradrenaline in the striatum varied. A male patient with marked tremor spreading manifested a considerable rise of the content of catecholamines, primarily in the n. caudatus. At the same time in a female patient with a grave akinetic -rigid syndrome and the signs of liver failure, the content of catecholamines, particularly dopamine, in the putamen was low. In view of this fact it is assumed that disorders of cerebral metabolism of catecholamines, dopamine in particular, evidently related to deposition of excess copper, leading to the changes of their content in basal ganglia underline the clinical pleomorphism of the disease and play the key role in the development of extrapyramidal motor disturbances characteristic of hepatocerebral dystrophy.


[Ultrastructural basis of the effect of haloperidol on the brain]. It has been established in rat experiments that haloperidol administration gives rise to the formation of a great number of coated vesicles in the cytoplasm of the sensorimotor cortex, thalamus, hippocamp, and central grey matter as well as to lysosomal activation and appearance of subsurface cisternae. Besides, the movement of mitochondria to the neuronal processes is intensified. The karyotropic action of haloperidol manifests in an increase of the size and amount of nuclear pores and in activation of the nucleoli. The drug action is also characterized by the lowering of the number of microtubules in the dendrites within the first hours after haloperidol administration, followed by its recovery in a day. The synaptic apparatus remains unchanged.


Familial amyloidosis of the Finnish type (FAF). A clinical study of 30 patients. The clinical findings of familial amyloidosis of the Finnish type (FAF) were recorded in a series of 30 patients. The onset was in the 3rd or 4th decade with slow progression so that the majority was in good health still in the 7th decade. Decreased vision and corneal lattice dystrophy together with blepharochalasis were common. Signs of cranial neuropathy especially affecting the facial nerve were found in all and peripheral polyneuropathy mainly affecting the vibration and touch senses in 26 patients. Hypotrichosis, tongue and skin changes were also characteristic. Amyloid was found in all skin, sural nerve and muscle biopsies. FAF thus shows a triad of typical neurological, ophthalmological and dermatological manifestations distinct from other amyloidoses.


Hydrocephalus, corneal opacities, deafness, valvular heart disease, deformed toes and leptomeningeal fibrous thickening in adult siblings: a new syndrome associated with beta-glucocerebrosidase deficiency and a mosaic population of storage cells. We describe three adult siblings with communicating hydrocephalus, corneal opacities, deafness, valvular heart disease, and deformed toes associated with glucosylceramide (glc-cer)-beta-glucosidase deficiency. The common manifestations of Gaucher disease were not evident. Supranuclear gaze palsies characteristic of type 3 were noted from early childhood, although the major signs were undeveloped until early adult life. Autopsy disclosed thickened leptomeninges with perivascular fibrosis, non-rheumatic calcified aortic and mitral stenosis with marked fibrosis, and mild infiltration of Gaucher cells in the reticuloendothelial organs. In contrast to the slight accumulation of glc-cer in the liver and spleen, the activity of glc-cer-beta-glucosidase was markedly decreased in the tissues, as much as in a patient with type 2 Gaucher disease. Common mutations were not found in the glucocerebrosidase gene.


Specific binding sites of platelet activating factor on the intact bovine cerebral microvascular endothelial cells and antagonism of drugs. [3H]Triazolodiazepine ([3H]WEB 2086), an antagonist of platelet activating factor (PAF) receptor, was studied as radioligand on intact cerebral microvascular endothelial cells (CMEC). The results showed that the binding of [3H]triazolodiazepine reached and maintained at an equilibrium after 15-120 min of incubation and that it was saturable with increasing concentration of radioligand. Scatchard analysis indicated that there were 2 specific binding sites on CMEC, its Kd1, Bmax1, Kd2, and Bmax2 were 3.13 nmol.L-1, 1.50 pmol/3 x 10(5) cells, 83.96 nmol.L-1, and 12.96 pmol/3 x 10(5) cells, respectively. The binding of [3H]triazolodiazepine to CMEC was displaced by C16-PAF and 1,5-bis-(3,4-dimethoxyphenyl)-tetrahydro-(4H)-pyran (SZ-1), which IC50 were 0.43 nmol.L-1 and 0.125 mumol.L-1, respectively. These data suggested the existence of PAF specific binding sites on CMEC.


Noradrenergic neuronal dysregulation in panic disorder: the effects of intravenous yohimbine and clonidine in panic disorder patients. In order to evaluate possible abnormal noradrenergic neuronal functional regulation in patients with panic disorder, the behavioral, biochemical and cardiovascular effects of intravenous yohimbine (0.4 mg/kg) and clonidine (2 micrograms/kg) were determined in 15 healthy subjects and 38 patients with panic disorder. A subgroup of 24 panic disorder patients were observed to experience yohimbine-induced panic attacks and had larger yohimbine-induced increases in plasma 3-methoxy-4-hydroxyphenylglycol (MHPG) than healthy subjects and other panic disorder patients. A blunted growth hormone response to clonidine and a significant clonidine-induced decrease in plasma MHPG was also observed in this subgroup of panic disorder patients. These data replicate and extend previous investigations, which are consistent with a large body of preclinical and human data relating increased noradrenergic neuronal function to human anxiety and fear states.


Effects of endothelin-1 on signal transduction in UMR-106 osteoblastic cells. Endothelin-1 is now recognized to affect the functions of a number of tissues and to activate calcium/phospholipid second messenger pathways in target cells. In the present study, we characterized its effects on signal transduction in UMR-106 cells. To study calcium transients elicited by endothelin-1, cells were loaded either with fluo-3 (for the measurement of cytosolic free calcium) or chlortetracycline (for the measurement of intracellularly stored calcium) as fluorescent probes. Intracellular production of inositol phosphates and cyclic AMP was also measured. Endothelin-1 elicited dose-dependent cytosolic calcium transients with an ED50 of 20 nM. This effect was also seen in EGTA-containing or calcium-free medium; however, the signals were reduced in magnitude. The dihydropyridine calcium channel antagonist nifedipine did not affect the response. Repeated administration of endothelin-1 resulted in homologous desensitization of the response. A 4 minute pretreatment with phorbol ester reduced the initial response to endothelin-1 in both calcium-containing and calcium-free media. A 24 h pretreatment with indomethacin had no effect on response. Using chlortetracycline as an indicator, a significant reduction in intracellularly stored calcium by endothelin-1 was observed. This was prevented by 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate, a blocker of calcium release from internal stores. Endothelin-1 also stimulated the dose-dependent production of inositol phosphates by UMR-106 cells. Indomethacin was also without effect on this process. The increase in inositol trisphosphates was seen within the same time frame as the increase in cytosolic calcium. Endothelin-1 did not influence cyclic AMP production over 5 minutes in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Local blood flow changes in malignant brain tumours under induced hypertension. Changes in tumour blood flow under an induced hypertensive state were examined in malignant brain tumours to know if the precondition for the effectiveness of induced hypertensive chemotherapy--relative increase in tumour blood flow--are fulfilled. Tumour blood flow was measured under both a resting and an induced hypertensive state in 12 patients with various malignant brain tumours (6 gliomas, 6 metastatic brain tumours) using xenon-enhanced computed tomography. The blood pressure was elevated 40% above the systemic blood pressure of the resting state by the infusion of angiotensin II. Tumour blood flow increased 30% on average above the normal brain tissue blood flow after the induction of an induced hypertensive state (p < 0.05). The tumour blood flow increased in 11 cases of malignant tumours, but decreased in one case with massive brain oedema after induced hypertension. The increase in blood flow was higher in hypervascular tumours and less in hypovascular tumours. Therefore, induced hypertensive chemotherapy probably will be more effective in hypervascular malignant brain tumours with small mass effects.


Modulation of human monocyte superoxide production by recombinant interleukin-3. We have examined the generation of superoxide by human monocytes isolated from peripheral blood cultured in the presence of recombinant human interleukin-3 in comparison to tumor necrosis factor-alpha and interferon-gamma. The rate of superoxide production of unstimulated and stimulated monocytes [by formyl-methionyl-leucyl-phenylalanine (0.1 microM) and by phorbol-myristate-acetate (2 ng/ml and 200 ng/ml)] decreased during the culture period in the absence of interleukin-3, whereas cells treated with interleukin-3 maintained or surpassed their initial superoxide-producing capacity. An increase of phorbol-myristate-acetate- and formyl-methionyl-leucyl-phenylalanine-stimulated superoxide production of monocytes cultured with interleukin-3 compared to control cells was detected first after 24 h of monocyte culture. It was maximal after 96 h of monocyte culture. At this time the stimulated superoxide production of monocytes cultured in the presence of interleukin-3 surpassed that of interferon-gamma and tumor necrosis factor-alpha treated cells. Suboptimal concentrations of the stimulus phorbol-myristate-acetate (2 ng/ml) resulted in higher priming than 200 ng/ml phorbol-myristate-acetate. A dose dependence of the effect of interleukin-3 on the superoxide production was observed. Our results demonstrate that interleukin-3 primes cultured human peripheral blood monocytes for enhanced stimulated respiratory burst activity to a higher extent than interferon-gamma and tumor necrosis factor-alpha.


Bradykinin induces a B2 receptor-mediated calcium signal linked to prostanoid formation in human gingival fibroblasts in vitro. The aim of the study was to determine the effect of bradykinin (BK) on the level of cytoplasmic-free Ca2+, [Ca2+]i, in human gingival fibroblasts and its relation to BK-induced prostanoid formation. BK, but not des-Arg9-BK, induced a significant rapid (within seconds) and transient increase in [Ca2+]i, that was not dependent on extracellular Ca2+. The stimulatory effect of BK was seen in concentrations at or above 10(-8) M, with the most pronounced effect at 10(-6) M. D-Arg0-Hyp3-Thi5,8-DPhe7-BK, a BK B2 receptor antagonist, but not des-Arg9-Leu8-BK, a BK B1 receptor antagonist, blocked BK-induced rise in [Ca2+]i. The BK B2 receptor antagonist also significantly reduced BK-induced PGE2 formation. When extracellular Ca2+ in the incubation medium was depleted, either by addition of EGTA or by omission of Ca2+ addition, BK still caused a significant stimulation of PGE2 formation. The calcium ionophores A23187 and ionomycin, similar to BK, caused a burst of PGE2 formation. The two phorbol esters phorbol 12,13-dibutyrate and 4-beta-phorbol-didecanoate positively amplified calcium ionophore A23187-induced PGE2 formation. The results indicate that BK-induced PGE2 formation in gingival fibroblasts is coupled to an increase in [Ca2+]i mediated by the BK B2 receptor, and which is independent of extracellular Ca2+.


Effects of SC-41930 on leukocyte adherence and emigration in rat mesenteric venules. This study demonstrates that SC-41930, 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8-p ropyl-2H-1-benzopyran-2-carboxylic acid, an orally active LTB4 receptor antagonist, reduces LTB4-induced leukocyte adhesion and emigration in rat mesenteric venules. The mesentery of Sprague-Dawley rats was prepared for intravital microscopic examination and venules of 25-35 microns were chosen for evaluation. In control animals, LTB4 (20 nM) was superfused over the mesentery for 30 min. In the treatment group SC-41930 (5 microM) was superfused for 30 min, followed by a 30 min superfusion with SC-41930 and LTB4. The LTB4-induced increase in leukocyte adherence and emigration in postcapillary venules was significantly attenuated by pretreatment with SC-41930. Other experiments demonstrated that platelet-activating-factor-induced leukocyte adherence was not affected by SC-41930. These results indicate that SC-41930 is a potent inhibitor of LTB4-induced leukocyte-endothelial cell adhesive interactions in postcapillary venules.


Orthodontic microabrasive reproximation. Reproximation (enamel stripping) is described in the literature as a clinical procedure for correction of tooth size deviations. The objective of this study was to qualitatively assess, by means of scanning electron microscopy, (1) the differences exhibited on enamel with mechanical and chemical methods of stripping, and (2) the effect of a synthetic calcifying solution on the etched enamel. Part 1. Sixty human anterior teeth (10 complete sets) that were previously stored in 70% ethanol were subdivided into groups I and II. The teeth in group I were divided into five sets of six teeth mounted in a plaster block in the anterior arch form. Each set was stripped with one of the following mechanical abrasive methods: garnet disks, tungsten carbide and fine diamonds burs, coarse and fine diamond burs, diamond wheel and 3M strips, diamond-coated metal and 3M strips. The teeth in group II were similarly treated, except each set was subjected to a further microabrasive chemical stripping with 37% phosphoric acid used in conjunction with 3M strips. The teeth were then prepared for scanning electron microscopy, viewed, and photographed under magnification. Part 2. Ten human central incisor teeth were etched and used to study the effect of remineralization solutions at various time intervals. The results showed that teeth stripped by routine mechanical abrasive methods exhibited deep furrows and roughness. The teeth that received mechanical and chemical abrasive treatments showed a flattened, etched surface free of furrows. These etched surfaces showed marked crystal growth at 5 and 10 hours after remineralization suggesting the possibility of repair of the chemically altered enamel surface.


Effects of in vitro amiodarone exposure on alveolar macrophage inflammatory mediator production. Administration of amiodarone, although often lifesaving, is associated with pulmonary side effects. Patients with amiodarone pulmonary toxicity can present with either a chronic disorder that suggests pulmonary fibrosis or a more acute process. Mechanisms of acute pulmonary injury resulting from amiodarone are unclear. Previous studies have demonstrated that the drug is preferentially concentrated in alveolar macrophages. In the present study, the authors examined whether in vitro exposure to amiodarone resulted in alteration of rat alveolar macrophage superoxide, leukotriene B4, or fibronectin release. In addition, the authors assessed whether macrophages were ultrastructurally altered by in vitro amiodarone exposure. Twenty four hour exposure to therapeutic tissue concentrations of amiodarone resulted in enhancement of phorbol myristate acetate-stimulated macrophage superoxide release. In addition, 48 hours exposure to amiodarone caused a dose-dependent inhibition of spontaneous fibronectin release by macrophages. Macrophages exposed to 48 hours of 10 micrograms/ml amiodarone were ultrastructurally abnormal, containing lamellar inclusions and demonstrating a large degree of vacuolization. The authors concluded that alveolar macrophages are very sensitive to therapeutic tissue concentrations of amiodarone. Alteration of macrophage mediator release by amiodarone may be one mechanism for lung damage induced by the drug.


Melanotic neuroectodermal tumor of infancy. A case report of paratesticular primary with lymph node involvement. A 17-month-old boy had a melanotic neuroectodermal tumor of infancy in the left paratesticular region affecting the retroperitoneal lymph nodes. Immunohistochemical and ultrastructural study showed phenotypical diversity of the proliferating cells within a spectrum of neuroectodermal differentiation. Urinary catecholamine levels were initially elevated but returned to normal values after complete eradication of the tumor. The patient received chemotherapy and is now well, without evidence of disease 28 months after surgery.


Direct assay method for guanosine 5'-monophosphate reductase activity. A sensitive and simple micromethod for the accurate measurement of GMP reductase (EC 1.6.6.8) activity in crude extracts is described. The reaction product of [8-14C]IMP was separated from the substrate [8-14C]GMP by descending chromatography on Whatman DE81 ion-exchange paper. This separation method provides an analysis of the possible interfering reactions, such as the metabolic conversion of the substrate GMP to GDP, GTP, and/or guanosine, and guanine and the loss of the product IMP to inosine, hypoxanthine, and other metabolites. Low blank values (70-90 cpm) were obtained consistently with this assay because the IMP spot moves faster than the GMP spot. The major advantages of this method are direct measurement of GMP reductase activity in crude extracts, high sensitivity (with a limit of detection of < 10 pmol of IMP production), high reproducibility (< +/- 5%), and capability to measure activity in small samples (9 micrograms protein).


The effect of enamel preparation on the tensile bond strength of orthodontic composite resin. The bonding of orthodontic brackets to enamel surface using bis-GMA composite resin is usually accomplished by first cleaning the tooth surface then etching with phosphoric acid. This study compared the tensile bond strength of composite resin applied to a tooth surface which had been cleansed with an air-powder polisher to that of the same resin applied to a surface cleansed using a rubber cup and pumice. A wire loop apparatus was attached to bonded orthodontic brackets and pulled in tension in order to test the adherence of the bracket to the tooth. Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) were used to evaluate the tooth surface to determine whether sodium bicarbonate material remained after the cleaning operation. All data was analyzed by the one way analysis of variants, the Student-Newman-Keuls test and Duncan's multiple comparison test. No statistical differences were found between the tensile strength of the bonds on the teeth cleansed with the air-powder polisher and those cleansed with a rubber cup and pumice. However, a double exposure of the tooth to phosphoric acid may lower the tensile bond strength by a significant amount.


Non-radioisotopic methods for DNA probes. We have recently developed a new colorimetric method, DNA enzyme immunoassay (DEIA), for detecting specific hybrids of complementary nucleic acids. This technology is based on an antibody that selectively recognizes double, but not single-stranded DNA. The molecule does not react with a specific probe immobilized on microwells through an avidin-biotin bridge, nor with non-specific amplified sequences, since they are removed by washing. The antibody reveals the hybridization event, independently of the DNA sequences and, for this reason, the method is broadly applicable and extremely versatile. Although we chose a format based on the immobilization of the probe through an avidin-biotin interaction, DEIA assay can also be applied to other analytical schemes that do not require any modification of the probe. Most importantly, the test has an ELISA format and is rapid and convenient for processing large numbers of samples. This technology has been applied, in our laboratory, to different areas, including virology, genetic and basic immunology. The DEIA assay has been successfully used to detect the presence of hepatitis B (HBV), hepatitis C (HCV) and hepatitis delta virus (HDV) sequences in serum of patients, to discriminate different HLA alleles, to identify mutations in the Cystic Fibrosis gene, and to investigate the role of the T cell receptor in some immunological diseases. The results obtained in all our experiments demonstrated that the advantages offered by the assay do not penalize its analytical performance as compared to conventional Southern blot.


Comparison of the immunoreactive plasma corticotropin and cortisol responses to two synthetic corticotropin preparations (tetracosactrin and cosyntropin) in healthy cats. Plasma cortisol and immunoreactive (IR)-ACTH responses to 125 micrograms of tetracosactrin and cosyntropin--the formulation of synthetic ACTH available in Europe and the United States, respectively--were compared in 10 clinically normal cats. After administration of tetracosactrin or cosyntropin, mean plasma cortisol concentration reached a peak and plateaued between 60 and 120 minutes, then gradually decreased to values not significantly different from baseline concentration by 5 hours. Mean plasma IR-ACTH concentration reached a maximal value at 15 minutes after administration of tetracosactrin or cosyntropin and was still higher than baseline concentration at 6 hours. Difference between mean plasma cortisol and IR-ACTH concentrations for the tetracosactrin or cosyntropin trials was not significant at any of the sample collection times. Individual cats had some variation in the time of peak cortisol response after administration of either ACTH preparation. About half the cats had peak cortisol concentration at 60 to 90 minutes, whereas the remainder had the peak response at 2 to 4 hours. In general, however, peak cortisol concentration in the cats with delayed response was not much higher than the cortisol concentration at 60 to 90 minutes. Overall, these results indicate that tetracosactrin or cosyntropin induce a comparable, if not identical, pattern of adrenocortical responses when administered to healthy cats.


Sequelae of cytomegalovirus pulmonary infections in lung allograft recipients. Indirect effects of cytomegalovirus (CMV) infections in lung transplant recipients (LTX) have not previously been described in detail. We compared spirometric results, development of chronic rejection, rates of respiratory superinfections, and mortality as long as 2 yr after transplantation, between 62 LTX who never developed CMV (CMV-) and 56 LTX with a history of CMV pulmonary infections (CMV+). Initial spirometric parameters were near identical for both groups, but determinations > or = 6 months after transplantation showed that expiratory flows of the CMV+ were significantly reduced. Actuarial prevalences of chronic allograft rejection (CR) at 2 yr were highest among CMV+ with biopsy-proved pneumonitis (74%) compared with 22% among CMV- (p < 0.038). Bacterial or fungal pneumonias developed in 58.9% of the CMV+, whereas the rate among CMV- was 38.7% (p < 0.05). Only 36% of LTX with CMV pneumonitis lived 2 yr compared with 70% survival for CMV- (p < 0.016). Ganciclovir treatment of CMV infections decreased rates of respiratory superinfections and improved survival of patients, but it did not appear to affect subsequent development of CR. We conclude that CMV pulmonary infections among LTX result in serious late sequelae and that current treatment is ineffectual for prevention of viral-associated CR in these patients.


In vitro activity of the new quinolone lomefloxacin against Mycobacterium tuberculosis. Minimal inhibitory concentrations (MIC) of ciprofloxacin, ofloxacin and lomefloxacin were determined for 90 Mycobacterium tuberculosis strains isolated from both AIDS and other patients. Eleven (12.2%) of these strains showed in vitro resistance to one or more first-line antituberculosis drugs. Susceptibility tests were done in 7H12 broth by the radiometric method. The MIC range for ciprofloxacin was 0.125 to 4.0 micrograms/ml; for ofloxacin, 0.25 to 4.0; and for lomefloxacin 0.5 to 4.0 micrograms/ml. On the basis of our data, we believe that the following MIC, when determined in 7H12 broth radiometrically, should be used as break points to classify the strain as susceptible: ciprofloxacin and ofloxacin, 1 microgram/ml or less; lomefloxacin, 2 micrograms/ml or less. Lomefloxacin on a once-daily basis deserves further evaluation as a potential supplementary drug for the treatment of tuberculosis.


Aerosolized lipopolysaccharide increases pulmonary clearance of 99mTc-DTPA in rabbits. Bacterial endotoxins alter the permeability of endothelium, but little is known of their effect on epithelium. We exposed specific pathogen-free rabbits to aerosolized Pseudomonas aeruginosa LPS or saline and performed serial measurements of RL, Cdyn, BP, WBC count and differential, and platelet counts. Pulmonary 99mTc-DTPA half-life was measured 4, 6, or 8 h after exposure. The animals were sacrificed and BAL performed. Background and PMA-stimulated superoxide production was measured from individual AM using electrooptical determination of reduction of NBT. Lung tissue was processed for light microscopy and ratio of wet to dry weight. 99mTc-DTPA half-life was significantly shorter in LPS-exposed animals at 6 h (p < 0.05) and 8 h (p < 0.001). There were no differences in Cdyn, RL, BP, WBC, differential, platelet, or BAL cell count at any time between groups. No histologic changes or differences in lung wet to dry weight ratios were found. PMA-stimulated AM superoxide production was significantly increased (p < 0.01) in LPS-exposed animals. This effect was time dependent and could be duplicated in AM from control animals following a 4-h incubation with LPS, lavage fluid from LPS-exposed animals, or recombinant murine TNF. These results demonstrate that aerosolized Pseudomonas LPS increases pulmonary epithelial permeability and primes AM superoxide production.


Effect of indomethacin on leukotriene4-induced histamine hyperresponsiveness in asthmatic subjects. The effect of indomethacin on the capacity of LTE4 to enhance airway histamine responsiveness was evaluated in eight mild asthmatic subjects. Subjects attended the laboratory on three separate pairs of study days when inhalation challenges with methacholine or LTE4 were performed and the airway responses to histamine were measured 4 and 7 h later. An open pair of study days was followed by a pair of study days during ingestion of either placebo or indomethacin capsules. The dose of agonist that produced a 35% fall in specific airways conductance (PD35 SGaw) was obtained by linear interpolation from the logarithmic dose-response curve. Indomethacin treatment did not affect baseline SGaw or methacholine airway responsiveness. However, indomethacin significantly inhibited LTE4-induced histamine hyperresponsiveness. Maximum enhancement of histamine responsiveness by LTE4 on the open and placebo study days was 4.1 +/- 0.9- (mean +/- SEM) and 5.7 +/- 1.2-fold, respectively (p = 0.36). Maximal enhancement on the indomethacin day was 1.68 +/- 0.46, and this was significantly decreased compared with that on the placebo day (p = 0.02). This suggests that LTE4-induced enhanced responsiveness to histamine is mediated in part by cyclooxygenase pathway-derived products.


Urinary leukotriene E4 after lysine-aspirin inhalation in asthmatic subjects. The FEV1 and urinary leukotriene E4 (LTE4) concentrations were determined in six aspirin-sensitive and six non-aspirin-sensitive asthmatic subjects before and after inhalation challenge with lysine-aspirin or placebo solution. Lysine-aspirin produced a mean fall in FEV1 of 26.7 +/- 4.9% (mean +/- SEM) in subjects with aspirin sensitivity and of 8.5 +/- 6.5% (mean +/- SEM) in non-aspirin-sensitive asthmatic subjects. The mean baseline urinary LTE4 concentration of 83 pg/mg creatinine (geometric mean [GM], range 15 to 326 pg/mg creatinine) in aspirin-sensitive subjects was significantly higher than the 33.8 pg/mg creatinine (GM, range 10 to 111 pg/mg creatinine) in non-aspirin-sensitive subjects (p = 0.02). In aspirin-sensitive subjects, inhalation challenge with lysine-aspirin produced a significant increase in urinary LTE4 concentration to 240 pg/mg creatinine (GM, range 60 to 1,113 pg/mg creatine), which was not observed after placebo challenge. There was no significant change in urinary LTE4 concentration after inhalation challenge with either lysine-aspirin or placebo solution in non-aspirin-sensitive asthmatic subjects. Thus, sulfidopeptide leukotrienes are released after inhalation of lysine-aspirin in aspirin-sensitive asthmatic patients.


Gastrointestinal viscerosynthesis with biofragmentable anastomosis ring. The biofragmentable anastomosis ring (BAR) has been in clinical use since 1987. It yields a standardized intestinal viscerosynthesis leaving no foreign body in the tissues. Several multicentre and single studies have shown its safety and efficacy in elective colonic surgery. Our studies have shown it to be safe and reliable also in small bowel surgery and bilio-digestive anastomosis. Our long-term results in colonic surgery are very promising. A review of anastomosis technique and current literature is given.


Ryanodine receptor autoantibodies in myasthenia gravis patients with a thymoma. Sera from patients with myasthenia gravis were examined by Western blot for the presence of antibodies to proteins of the sarcoplasmic reticulum from rabbit skeletal muscle. Fourteen of 30 patients with myasthenia gravis and a thymoma had IgG autoantibodies to the calcium release channel of the sarcoplasmic reticulum (the ryanodine receptor), which plays a crucial role in the mechanism of excitation-contraction coupling in striated muscle. Ryanodine receptor autoantibodies were not detected in any of the 45 sera from patients with myasthenia gravis without a thymoma. Ryanodine receptor autoantibodies may have pathogenetic relevance in thymoma-associated myasthenia gravis.


Risk assessment of dermal exposure of greenhouse workers to pesticides after re-entry. On 18 farms for rose culture in greenhouses in The Netherlands, dermal exposure of hands and forearms to abamectin (avermectin B1), dodemorph (4-cyclododecyl-2,6-dimethylmorpholinium acetate) and bupirimate (5-butyl-2-(ethylamino)-6-methyl-4-pyrimidinyl dimethylsulphate) was measured during crop activities. Dermal exposure during cutting (75 workers) amounted to 13 micrograms/h, 1.8 mg/h, and 2.2 mg/h for abamectin, dodemorph and bupirimate, respectively. Dermal exposure to abamectin and dodemorph during sorting (21 workers) and bundling (30 workers) was comparable with that during cutting. From the dependence of dermal exposure on the amount of dislodgeable foliar residue (DFR) a transfer factor was estimated to be 1,200, 4,550, and 2,400 cm2/h for abamectin, dodemorph and bupirimate, respectively. For sorting and bundling these factors were of the same order of magnitude. The results suggested that work rate was also a determinant of dermal exposure. The within-farm variance of dermal exposure during cutting appeared to account for approximately 30% of the unexplained part of the variation remaining after regression on DFR and application technique. The final unexplained part in the variation of dermal exposure during cutting was amongst others due to the variation between the different farms in which the measurements were performed. A health risk evaluation of the observed levels of dermal exposure after re-entry of greenhouses led to the conclusion that a health hazard may exist, especially after application of high rates of relatively toxic pesticides which easily penetrate the skin.


Expression of steroid hormone receptors in benign hepatic tumors. An immunocytochemical study. Many hepatic adenomas have been demonstrated to have a clear relationship with oral contraceptive use, and it is presumed that there may be hormone receptors within the cytoplasm or nucleus of adenoma cells that mediate tumor growth in response to hormonal stimulation. Only a small number of examples of benign hepatic tumors have been analyzed for the presence of estrogen and progesterone receptors, and there has been a lack of consensus with regard to the findings. All previous studies have determined receptor levels by biochemical methods. In a retrospective study, we employed specific monoclonal antibodies against estrogen and progesterone receptors in 10 benign paraffin-embedded hepatic lesions: five examples of hepatic adenoma and five examples of focal nodular hyperplasia. All patients were female, except for one male with adenoma and one male with focal nodular hyperplasia. No patient had received tamoxifen citrate or any other form of hormonal therapy for their hepatic lesion. Positive controls included benign and malignant breast tissue. No positive staining was seen in hepatic adenoma, focal nodular hyperplasia, or normal adjacent liver parenchyma. Intense positive staining was seen in all positive control tissues. This negative result with the use of specific monoclonal antibodies in an established immunohistochemical method for analysis of estrogen and progesterone receptors does not exclude the presence of these receptors in benign hepatic lesions, but does suggest that, if present, they occur in much smaller amounts than in benign and malignant breast tissue. The presence of hormone receptors in benign hepatic tumors deserves further study.


Pathogenesis of cytomegalovirus-associated pneumonitis in ICR mice: possible involvement of superoxide radicals. We have studied the pathogenesis of murine cytomegalovirus (MCMV) pneumonitis in immunocompetent ICR mice and in mice treated with cyclophosphamide (CP). Intranasal infection of immunocompetent mice with MCMV resulted in transient and self-limited pulmonary lesions. When mice were given 200 mg/kg of CP one day before virus infection, transient splenic atrophy and subsequent splenic hypertrophy were induced, and the lesions in the lung were markedly augmented in their number and size although there was no significant enhancement of the virus growth. The augmentation coincided with the period of splenic hypertrophy. A marked increase in the number of pulmonary lesions was also induced in mice given 100 mg/kg of CP every 4 days following the initial dose of 200 mg/kg. In these mice, however, continuous splenic atrophy and augmented replication of MCMV in the lung were observed. When the activity of xanthine oxidase (XO) in lung tissue homogenates was measured, the activity was found to significantly increase after intranasal infection with MCMV irrespective of CP administration and there was a good correlation between the elevation of XO activity and the degree of pathological changes in the lung. In addition, we found that the administration of allopurinol, a specific inhibitor of XO and superoxide dismutase, a superoxide radical scavenger, reduced the number of the pulmonary lesions. These results suggest that superoxide radicals are involved in the pathogenesis of MCMV-associated pneumonitis in ICR mice.


Antigen B of the vaccine strains of Marek's disease virus and herpesvirus of turkeys presents heat-labile group and serotype specific epitopes. Antigen B of Marek's disease virus (MDV) vaccine strains CVI988 and SB1 (serotypes 1 and 2) and herpesvirus of turkeys (HVT) (serotype 3) is formed of oligomeric molecules that are detergent-stable and heat-labile. Immunoblots of native membranal extracts of HVT- and MDV-infected chick embryo fibroblasts (CEF) probed with avian monoserotypic antisera, murine monoclonal antibodies (mAb) to the three serotypes and mAb to antigen B showed two distinct patterns of high molecular weight oligomeric antigens. Serotypes 1 and 3 vaccine viruses formed one set and serotype 2, the other. Avian monotypic sera to serotypes 1 and 3 viruses detected two high molecular weight bands of 230 and > or = 300 kDa in MDV-1 and HVT-infected CEF but only a weak diffuse zone ranging from 130 to 230 kDa in extracts of SB1-infected CEF. No 300 kDa band was discernible in the SB1 extract when blotted with avian monotypic 1 and 3 antisera. MAbs to MDV serotypes 1 and 3 and to antigen B also detected the 230 and > or = 300 kDa antigens, while the mAb to SB1 detected a 50 kDa antigen in the SB1-infected extract only. Furthermore, the antigen B mAb did not reveal high mol. wt. oligomers in SB1-infected CEF extracts. Antigen B oligomers were rapidly destroyed by heating at 95 degrees C and the rate of denaturation of the 230 and > or = 300 kDa oligomers differed for each of the three vaccine viruses. We propose that antigen B of MDV1 and HVT has a complex conformation created by juxtaposition of dimers (230-250 kDa) and trimers (> or = 300 kDa), and is inserted in the infected cell membrane so that conformational, discontinuous epitopes are formed in addition to continuous epitopes. It appears that HVT protects chickens against oncogenic strains of MDV1 by virtue of the cross reactivity of the conformational determinants located on these oligomers. Serotype 2 vaccine shares some of its antigenic determinants with serotypes 1 and 3, while its unique immunogenic features form the basis of the protective synergism achieved when serotypes 2 and 3 vaccines are combined together.


Beneficial effect of insulin on the decreased gastrointestinal beta-adrenergic responses in streptozotocin-diabetic rats: a contributing role of protein biosynthesis. The beneficial effect of insulin on the decreased gastrointestinal beta-adrenergic responses in experimentally diabetic rats has been reported. The effects of streptozotocin-induced diabetes and insulin on the isolated rat duodenum, precontracted with bethanechol, were examined in the absence and in the presence of protein synthesis inhibitors in isometric conditions. Streptozotocin-induced diabetes caused a decrease of the beta-adrenergic responses of the rat duodenum. In vitro insulin treatment corrected the decreased beta-adrenergic responses of rat duodenum due to streptozotocin-induced diabetes. In the presence of protein synthesis inhibitors, actinomycin D or cycloheximide, the beneficial effect of insulin on the decreased intestinal beta-adrenergic responses was significantly inhibited. These findings suggest that the beneficial effect of insulin in the rat duodenum might be related to a new receptor synthesis.


Biological roles of the major capsid proteins and relationships between the two existing serotypes of infectious bursal disease virus. Neutralizing monoclonal antibodies (n-MAbs) were produced against infectious bursal disease virus (IBDV) of serotypes 1 and 2. The n-MAbs recognizing the major antigenic proteins VP2 and VP3, were characterized using different strains of IBDV representing the existing two serotypes and a variant subtype of serotype 1. The biological properties of these viral antigens as defined by the MAbs in vitro, were studied utilizing post-adsorption virus neutralization tests and fluorescence-activated cell sorter analysis. The MAbs directed against the immunodominant epitopes on VP2 were capable of enhanced virus neutralization but did not inhibit the virus attachment to susceptible cells. These MAbs were able to neutralize the virus by interfering with an event subsequent to virus adsorption, possibly inhibiting virus penetration or uncoating. On the contrary, a MAb that immunoprecipitated the other capsid protein VP3 was able to prevent virus attachment although it possessed lower neutralization titers. Cross-immunoprecipitations of various virus strains by these MAbs and antisera revealed interrelationships between the two serotypes of IBDV.


Feline immunodeficiency virus: quantification in peripheral blood mononuclear cells and isolation from plasma of infected cats. The titer of feline immunodeficiency virus in peripheral blood mononuclear cells (PBMC) and the presence of infectious virus in plasma was investigated over 20 week period in 8 experimentally infected cats, 3 uninfected cats and 2 naturally infected cats by end point dilution cultures using a feline T-lymphoblastoid cell line (MYA-1). FIV was isolated from PBMC of all infected cats, but not from the uninfected cats. FIV was also isolated consistently from 100 microliters plasma from most of the experimentally infected cats, but not from the 2 naturally infected cats. The virus titer in PBMCs in both experimentally and naturally infected cats was comparatively high, ranging from 10 TCID/10(6) PBMC to 14,286 TCID/10(6) PBMC. The titers in PBMC of individual cats remained unchanged or varied only slightly over the 20 week period. In contrast, the titers varied substantially between cats, with significantly higher titers in the youngest litter (4 cats) than in the oldest litter (3 cats). This suggests that there is an age-related factor influencing the level of PBMC virus titers in experimental infection with FIV. A similar age-related susceptibility has been shown with feline leukemia virus. More importantly, the sustained titers in the experimentally infected cats bear close resemblance to infection of children with human immunodeficiency virus. These data reinforce suggestions that age and immune maturity have a fundamental influence on PBMC and plasma titers in lentivirus infections.


Factors influencing the interaction of herpes simplex virus glycoprotein C with the third component of complement. The factors influencing the interaction of herpes simplex virus (HSV) glycoprotein C (gC) with the third component of complement (C3) were investigated in this study. The ability of gC of HSV type 1 (gC-1) to bind to the C3b fragment of C3 was found to be influenced by cell specific processing of gC-1 in a different manner, binding being remarkably enhanced in some cell lines following removal of sialic acid residues. Testing several intertypic recombinants of HSV we found that only strains expressing gC-1 exhibited binding to C3b, even though their genome consisted mainly of HSV-2 sequences in some recombinants. Expression of type-2 glycoproteins gB, gD, gE, gG, gH, and gI did not alter the ability of gC-1 to bind to C3b. Rosetting of HSV-1 infected Vero cells with C3b-coated red blood cells (EAC) was found to be temperature dependent and could be inhibited with purified C3b and anti-C3 antibodies. Polyanions like heparin or dextran sulfate were also inhibitory in a dose dependent manner, whereas C3d, neomycin and other aminoglycoside antibiotics failed to block. As the tested polyanions are also known to inhibit the infectivity of HSV, it could be speculated, that the complement binding function and the heparin-binding/attachment function of gC might be related.


Stimulation of thymidine kinase activity in baculovirus infected cells is not due to a virus-coded enzyme. A polyhedrin-positive recombinant autographa californica nuclear polyhedrosis virus (AcNPV) carrying a herpes simplex virus thymidine kinase gene under the control of the SynXIV promoter, a fusion of synthetic and linker-modified polyhedrin promoters, has been constructed. When this recombinant baculovirus was used to infect a variant of Spodoptera frugiperda cells deficient in thymidine kinase (TK-), a high level of TK activity was detected. These results, in conjunction with the demonstration that AcNPV could replicate in TK- S. frugiperda cells and no TK activity was found throughout infection, imply that the wild type virus-stimulated TK activity observed in S. frugiperda (TK+) cells is not contributed by a virus-coded enzyme.


Immunohistochemical localization of the rabbit haemorrhagic disease viral antigen. Immunohistochemical investigations were carried out to determine organ and cellular localization of the rabbit haemorrhagic disease viral antigen (RHDVA). It was found in certain parenchymal liver cells near the interlobular septs and in some macrophages and pseudoeosinophils of all studied organs and blood. Whereas in morphologically preserved hepatocytes and macrophages the RHDVA accumulated in the nuclei, in cells with disintegrated nuclei it was distributed throughout the cytoplasm.


Susceptibility of mouse embryo to murine cytomegalovirus infection in early and mid-gestation stages. The susceptibility of mouse embryonic cells to murine cytomegalovirus (MCMV) infection was studied by injecting the virus in the early and mid-gestation stages. For the early stage, blastocysts from BDF1 mice were injected with MCMV or minimal essential medium (MEM) by micromanipulator and returned to the uteri of pseudopregnant ICR mice. On day 11 of gestation, the embryos were examined immunohistochemically, using antibody specific to the early antigen of MCMV, and the placentae were examined by plaque assay. No infection was detected by either method. Furthermore, no infection was detected in MCMV-infected blastocysts that were cultured and examined for infection by immunofluorescence. For mid-gestation embryos, the conceptus was injected with MCMV on day 8.5 of gestation and was subjected to immunohistochemical analysis from day 10.5 to 12.5 of gestation. Viral antigen-positive cells were first observed in the placentae, then antigen-positive cells appeared among the blood cells, endothelial and mesodermal cells of the embryos. On day 12.5 of gestation, clusters of viral antigen-positive cells were sometimes observed in the hearts and livers. Although the incidence was lower, viral antigen-positive cells were also observed in the neuroectoderm and the eyes. These results suggest that MCMV does not infect early embryos and that infection first occurs in the placenta of postimplantation embryos, whence it extends through the blood cells to the endothelial and mesodermal cells of different embryonic regions, eventually extending to the neuroectoderm.


Identification of a protein encoded in the EB-viral open reading frame BMRF2. Using monospecific rabbit sera against a peptide derived from a potential antigenic region of the Epstein-Barr viral amino acid sequence encoded in the open reading frame BMRF2 we could identify a protein-complex of 53/55 kDa in chemically induced B95-8, P3HR1 and Raji cell lines. This protein could be shown to be membrane-associated, as predicted by previous computer analysis of the secondary structure and hydrophilicity pattern, and may be a member of EBV-induced membrane proteins in lytically infected cells.


Identification and nucleotide sequence of a gene in feline herpesvirus type 1 homologous to the herpes simplex virus gene encoding the glycoprotein B. The nucleotide sequence of the glycoprotein B (gB) homologous gene of feline herpesvirus type 1 (FHV-1) was determined. The gene was found to be located within a 9.6 kbp SalI fragment by Southern-blot hybridization with a probe derived from the herpes simplex virus type 1 (HSV-1) gB DNA sequence. Furthermore, the predominant portion of the coding sequences was mapped to a 1.9 kbp Hin cII-EcoRI and its flanking 2.7 kbp Eco RI-Eco RI subfragments in the 9.6 kbp SalI fragment. The entire nucleotide sequence revealed that the FHV-1 gB homologous gene is capable of encoding a polypeptide of 948 amino acids. The predicted precursor polypeptide derived from this open reading frame could have a calculated M(r) of 106 kDa in unglycosylated form and contains ten potential N-linked glycosylation sites and a probable internal proteolytic cleavage site. By Northern-blot analysis using portions of the open reading frame as a probe, 3.9 and 3.3 kb RNA transcripts were identified in FHV-1 infected cells. The alignment of the amino acid sequence of the FHV-1 gB homologue with those of 14 other herpesviruses revealed that 10 cysteine residues were completely conserved. Meanwhile, when evolutionary trees were generated among these herpesvirus gB counterparts, the FHV-1 gB homologous nucleotide sequence seems to be closely related to equine herpesvirus type 4 and its amino acid sequence to pseudorabies virus.


Temperature sensitivity of herpes simplex virus type 1 is a tissue-dependent phenomenon. The temperature sensitivity of herpes simplex virus type 1 (HSV-1) was assessed in primary cultures of mouse central nervous system (MNS) cells and mouse embryo cells (MEC). Infectious yields were determined and the ultrastructural morphogenesis of HSV-1 particles was compared following incubation at 37 or 40.5 degrees C. Yields of infectious virus were significantly reduced for both types of cell cultures following incubation at 40.5 degrees C. However, the effect of supraoptimal temperature (40.5 degrees C) on HSV-1 replication in MEC was significantly greater than the effect of supraoptimal temperature on virus replication in MNS cells. With respect to viral morphogenesis, no significant differences were found in either the quantity or the appearance (empty versus electron opaque core) of intranuclear particles present per infected nucleus, regardless of cell type or incubation temperature. However, complete virus particles (enveloped capsids with dense cores) were never observed in MEC at 40.5 degrees C, either intracytoplasmically or extracellularly. In contrast, complete virus particles were observed in MNS cell cultures at 40.5 degrees C, albeit in reduced numbers. At the permissive temperature (37 degrees C), complete intracytoplasmic and/or extracellular virus particles were associated with every infected cell in the MNS cell or MEC cultures. Thus an interactional effect on HSV-1 replication was found between cell culture type and incubation temperature.


Naturally occurring-neutralizing monoclonal antibody escape variants define the epidemiology of infectious bursal disease viruses in the United States. A panel of two non-neutralizing and six neutralizing monoclonal antibodies (Mabs) were used in antigen-capture enzyme immunoassays (AC-ELISA) to examine the antigenicity of 1301 wild type infectious bursal disease viruses (IBDV) isolated from different poultry flocks throughout the United States over a three year period. Analysis of these isolates with protective, neutralizing Mabs directed against the VP2 structural protein of IBDV showed that four antigenically distinct groups of serotype 1 IBDV could be separated on the basis of the presence or absence of one or more Mab defined, conformation-dependent, multivalent neutralization site. AC-ELISA reactivity patterns of the Mabs with isolates demonstrated that IBDV field populations were relatively antigenically homogeneous per premise isolation. Geographically, various antigenic species were more or less prevalent, or nearly absent. Competition analysis with neutralizing Mabs coupled with AC-ELISA results suggested that neutralization epitopes for IBDV are distinct, spatially arranged, yet closely linked. Of 5 Mab defined neutralization epitopes, shown to be related to protection from virulent challenge by Classic IBDV strains isolated prior to 1985, only two of the epitopes remain unaltered on the most recent emergent variant field strain of IBDV isolated.


Investigation, follow-up and recurrence after resection of colorectal cancer. A study was undertaken to determine the patterns of management in the 2 years following resection of colorectal cancer by Victorian surgeons. Patients were identified by the Victorian Cancer Register as having colorectal cancer diagnosed between July 1 and December 31 1987. The surgeon of each of the 947 eligible patients who underwent surgery was sent a questionnaire seeking information about the pre-operative investigation, type of surgery and subsequent line of referral. Only 16% of colonic cancers and 39% of rectal cancers were biopsied pre-operatively and colonoscopy was undertaken in one-half and one-third respectively. Of the 737 responses, 555 patients were considered to have had curative surgery, and details of their follow-up during the four 6-month periods following surgery was analysed; this includes the effect of tumour stage and surgeon activity on the use and frequency of each test. Most patients had a clinical examination in each of the 6 month periods, but almost half did not have a colonoscopy and two-thirds did not have the serum CEA level measured at all. Only one in eight had a chest X-ray and fewer had the liver scanned during this 2 year period. Eighty-two patients (20% of those satisfactorily followed) suffered a recurrence during this period. Twenty-six were asymptomatic at the time of recurrence and were diagnosed by a routine test and of these, eight were diagnosed by tests used infrequently. Sixteen (20%) were considered surgically curable.


Attenuation by the benzodiazepine receptor antagonist, ZK 93 426, of the deficit in spatial navigation induced by nucleus basalis lesions. The effects of the benzodiazepine receptor antagonist, beta-carboline ZK 93,426 treatment were studied both in NB-lesioned (ibotenic acid) and in unoperated Kuo-Wistar rats in a water maze task. The ZK 93,426 administered in the doses of 1 and 5 mg/kg, 30 min prior to the testing in a water maze apparatus, attenuated the NB lesion-induced spatial navigation deficit, although it had no effect on the performance of unoperated rats. The results suggest functional interactions between GABAergic system and ibotenic acid-induced lesion of the basal forebrain in rats.


Relationship between vomiting and taste aversion learning in the ferret: studies with ionizing radiation, lithium chloride, and amphetamine. The relationship between emesis and taste aversion learning was studied in ferrets (Mustela putorius furo) following exposure to ionizing radiation (50-200 cGy) or injection of lithium chloride (1.5-3.0 mEq/kg, ip). When 10% sucrose or 0.1% saccharin was used as the conditioned stimulus, neither unconditioned stimulus produced a taste aversion, even when vomiting was produced by the stimulus (Experiments 1 and 2). When a canned cat food was used as the conditioned stimulus, lithium chloride, but not ionizing radiation, produced a taste aversion (Experiment 3). Lithium chloride was effective in producing a conditioned taste aversion when administration of the toxin was delayed by up to 90 min following the ingestion of the canned cat food, indicating that the ferrets are capable of showing long-delay learning (Experiment 4). Experiment 5 examined the capacity of amphetamine, which is a qualitatively different stimulus than lithium chloride or ionizing radiation, to produce taste aversion learning in rats and cats as well as in ferrets. Injection of amphetamine (3 mg/kg, ip) produced a taste aversion in rats and cats but not in ferrets which required a higher dose (> 5 mg/kg). The results of these experiments are interpreted as indicating that, at least for the ferret, there is no necessary relationship between toxin-induced illness and the acquisition of a CTA and that gastrointestinal distress is not a sufficient condition for CTA learning.


Changes of mitochondrial respiratory functions and superoxide dismutase activity during liver regeneration. The general objective of this study was to examine the relationship between mitochondrial respiratory function and liver regeneration in the rat. The role that free radicals may play in the process was also evaluated. It was found that the respiratory control and ADP/O ratios were concomitantly decreased to the lowest level at 6 hr after hepatectomy and gradually recovered thereafter. Both ratios were significantly increased at 48 hr and quickly reached plateau levels. Assays of mitochondrial respiratory functions revealed that the activities of Complex I+III, Complex II+III and Complex IV all decreased drastically at 6 hr after hepatectomy and then gradually returned to the original level during 18-24 hr after hepatectomy. Interestingly, the activities of all these enzyme complexes continuously increased and were elevated significantly above the normal levels (145-200%). In contrast, the liver mitochondrial electron transport activities of sham-operated rats returned only to the original level after recovering from the operation-induced decline at 6 hr post-hepatectomy. We measured the superoxide dismutase (SOD) activity of liver mitochondria of the hepatectomized and sham-operated rats. The results showed that the Mn-SOD activity started to increase after hepatectomy, reached a maximum (900% of control) at 6 hr, and then returned to normal levels at 24 hr after operation. The Cu, Zn-SOD activity was increased 9-fold in hepatectomized rats and about 3-fold in sham-operated rats as compared with control rats. The maximum activity of Mn-SOD was found to be about 4 times higher than that of Cu, Zn-SOD after hepatectomy. The amount of lipoperoxides in the liver mitochondria was found to be increased to 140% in hepatectomized rats and to 120% in sham-operated rats as compared with that of the control rats. Taken together these results suggest that the changes in mitochondrial respiratory functions in the early phase of hepatectomy are due to tissue damage caused by the transient elevation of free radicals. These free radicals are then quickly disposed of by the ever-increasing activities of the Mn-SOD and Cu, Zn-SOD in the liver mitochondria, thereby protecting the liver from further damage and gearing the organ to the regeneration process.


Serum catalase as the protective agent against inactivation of alpha 1-proteinase inhibitor by hydrogen peroxide; comparison between normal and rheumatoid sera. Human serum contains catalase activity, which can protect alpha 1-proteinase inhibitor from inactivation by H2O2. The primary source of serum catalase is probably erythrocytic. The enzyme activity correlates with haemoglobin concentration in sera from control subjects but not in sera from patients with rheumatoid arthritis. Catalase is inactivated by oxidants, such as H2O2 and hypochlorous acid and it is suggested that the decrease in catalase/haemoglobin ratio observed in rheumatoid serum is due to oxidant stress associated with inflammation.


Fluidity alterations induced by chemical modification of erythrocyte membrane proteins. Using EPR technique we have examined the effect of chemical agents on pig erythrocyte membranes. Treatment of the erythrocyte membranes with SH-oxidizing and denaturing or specific for amino groups reagents affects both the membrane proteins and lipids. These results suggest that modified proteins may perturb the interactions of the membrane components and lead to alterations of the membrane organization in the polar region.


Dependence of spectral and structural properties of resting cytochrome oxidase on temperature. The temperature effect on isolated resting oxidized oxidase was investigated by optical and EPR spectroscopy. The reversible decrease of the optical alpha-band absorption is linear with temperature increase from 5 to 30 degrees C. The temperature increase also causes an irreversible change of the suspension turbidity and the Arrhenius plot of mobility of lipid-soluble spin labels is nonlinear. A significant enhancement of turbidity is observed on heating from 30 to 50 degrees C, with an apparent transition temperature at 43 degrees C. The change of turbidity and the nonlinear behaviour of spin label mobility are being ascribed to denaturation of non-heam-containing subunits followed by an aggregation of the enzyme.


Responsiveness to gonadotropin releasing hormone of human term trophoblast cells in vitro: induction by estradiol. Human trophoblast cells isolated from term placentae by enzymatic treatment and maintained in culture, spontaneously secrete hCG after 72hrs but fail to increase their gonadotropin secretion in response to exogenous GnRH. Treatment with estradiol for 24 hrs prior to challenge with GnRH induces the responsiveness to GnRH stimulation. This action of estradiol on trophoblast cells in independent of their stage of differentiation. The competitive displacement analysis of [125I] GnRH binding to estradiol-primed trophoblast cells indicated a single class of low affinity sites with Ka of 1.6 x 10(6) M-1, similar to that observed previously in placental membranes. To our knowledge this is the first demonstration of induction of GnRH receptors by estradiol in human trophoblast cells.


Immortalization of human aortic smooth muscle cells with origin-minus simian virus 40 DNA. We established two cell lines of human smooth muscle cells (SMC) by transfection of cells from the aortic intima and aortic media with origin-minus simian virus 40 (ori-minus SV40) DNA. Ori-minus SV40 DNA very efficiently immortalized human smooth muscle cells in culture. Proteins that these cell lines produced included type I, III, IV, and V collagens, fibronectin, and human matrix metalloproteinases (MMP)-1 (tissue collagenase), -2 ("type IV collagenase"), and -3 (stromelysin). The protein production in these cell lines generally mimicked that of normal SMC, but the immortalization stimulated the cell line of medial SMC to produce excessive MMP-2 and to secrete MMP-9 (92-kDa gelatinase). However, since these cell lines did not show a fully malignant phenotype, we concluded that, in addition to the degradation of extracellular matrix macromolecules, including basement membrane components by MMP-2, -3, and/or -9, some additional factors must be involved for the malignancy of fully transformed cells and that these immortalized human aortic SMC, which share many characteristics with normal SMC, will prove useful to study the role(s) of metalloproteinases in atherosclerosis.


Binding characteristics and thermal behaviour of cytochrome-C oxidase, inserted into phospholipid-coated, magnetic nanoparticles. A strategy for the immobilization of cytochrome-c oxidase, used as a representative membrane-bound enzyme, into so-called magnetoliposomes has been developed. The latter structures consist of a phospholipid bilayer which covers nanometer-sized Fe3O4 colloids. Incorporation of the enzyme into the phospholipid envelope is facilitated by a short sonication step. Upon adsorption, the reaction characteristics of the lipid-depleted enzyme are drastically changed. With double-layered phosphatidylcholine (PC) magnetoliposomes the activity increases by a factor of approximately 5. After a first magnetic fractionation step, approximately 67% of the activity remains with the magnetoliposome retentate. Subsequent magnetophoresis cycles show that the adsorbed enzyme is firmly fixed into the phospholipid coat. Upon immobilization, the thermal behavior is also profoundly affected. The heating inactivation curves show two sigmoidal transition zones. Irrespective of the PC type used, a first inflection point is located near 39 degrees C, whereas a second one, which is located at higher temperatures, clearly depends on the acyl chain length (56 degrees C with dimyristoyl-PC and 60 degrees C for dioleoyl-PC and Ovothin-200). An identical behavior is observed with classical proteoliposomes with an equal phospholipid composition. By contrast, monolayer-coated dimyristoyl-PC magnetic structures are inferior with respect to both their reactivation potency and their ability to strongly affix cytochrome-c oxidase and to improve the thermal stability of the enzyme.


A procedure for large-scale plasmid isolation without using ultracentrifugation. An expedient procedure for large-scale plasmid isolation from Escherichia coli strains without using ultracentrifugation or special setups or reagents is described. The protocol, which utilizes a modified alkaline extraction procedure as well as differential precipitations by isopropanol and lithium chloride, is simple and rapid and yet produces plasmid DNA with a yield of about 2 mg/liter culture. The isolated plasmids consisted of mostly monomeric and dimeric covalently closed circular DNA. The plasmids could be digested by various restriction endonucleases and were compatible with gene cloning, transfection-gene expression, and viral production.


Molecular analysis of pseudorabies viral vaccines and their rapid differentiation from wild-type isolates using DNA-amplified glycoprotein I and thymidine kinase gene segment polymorphisms. Several conventional and genetically recombinant modified-live viral (MLV) vaccines are used to control pseudorabies virus infections (Aujeszky's disease, PRV) in swine. Differentiating vaccinal PRV (V-PRV) from wild PRV (WT-PRV) is important for herd health, regulatory and forensic purposes, and for studies of PRV latency and epidemiology. All PRV vaccines used currently contain glycoprotein I (gI) and/or thymidine kinase (TK) gene deletions, whereas WT-PRV typically contain intact gI and TK genes. Utilizing these differences we developed an effective but simple differential polymerase chain reaction (PCR) approach based upon the amplification of gI and TK gene polymorphisms. The primary immunoreactive epitope-encoding region of the gI gene and nearly the entire TK gene were amplified and analyzed using nested PCR procedures. TK and gI PCR products were cleaved with Sal I and Sac I, and Nco I restriction enzymes respectively. PCR product and restriction fragment length polymorphisms enabled most V-PRV to be clearly distinguished from each other, and all of them, as a group, clearly differentiated from typical WT-PRV. Mixtures of V-PRV and WT-PRV could be identified as such. The uncommon but occasional occurrence of atypical WT-PRV containing altered gI and/or TK genes indicates the need for interpretive caution, particularly if aberrant gene segment polymorphisms are observed. This rapid and precise molecular approach will facilitate regulatory monitoring, epidemiological investigations, diagnostic differentiation, purity testing and latency/recrudescence studies with the class of biologicals and offers a model for similar analyses of other MLV biologicals as well.


The Second International Standard for anti-poliovirus sera types 1, 2 and 3. The Second International Standard for anti-poliovirus sera types 1, 2 and 3 was established by the WHO Expert Committee on Biological Standardization in 1991 on the basis of an extensive collaborative study. Nine laboratories from eight countries participated and all used neutralizing antibody assays. The standard is a human serum pool which contains antibodies to all three poliovirus types and replaces the three previously established monovalent standards which were all hyperimmune monkey sera. The standard was assigned an activity of 25 IU of anti-poliovirus serum (type 1) human: 50 IU of anti-poliovirus serum (type 2) human; and 5 IU of anti-poliovirus serum (type 3) human. The study also showed significant interlaboratory differences in relative potency are observed when human sera are compared to hyperimmune monkey sera. It was therefore recommended that National laboratory references are established from human sera.


Colorectum cell-derived growth factor (CRDGF) is homologous to amphiregulin, a member of the epidermal growth factor family. We have previously shown that an autocrine factor (CRDGF) of molecular weight 25,000 is produced by the HT29 human colon cancer cell line. Although CRDGF was shown to inhibit the binding of epidermal growth factor (EGF) to its receptor, several lines of evidence suggested that it was distinct from EGF or transforming growth factor-alpha (TGF-alpha). In order to check the possibility that CRDGF represents a new member of the EGF family, a four-step purification protocol involving acid gel filtration, cation-exchange high-performance liquid chromatography (HPLC), C18 reversed-phase HPLC and gel permeation HPLC was used to purify this protein to homogeneity. The purified material exhibited a 22 kDa molecular mass on SDS-PAGE. Partial N-terminal amino acid sequence of CRDGF showed identity to amphiregulin (AR), an EGF-related protein. Western blotting experiments using AR-specific antiserum confirmed that CRDGF and AR are identical proteins. In addition, we showed that AR, like EGF or TGF-alpha stimulated the phosphorylation of the epidermal growth factor receptor (EGF-R) on tyrosine residues. This indicates that the AR intracellular signalling pathway involves the activation of EGF-R kinase.


Simian T-cell leukemia virus type I infection in captive baboons. Human T-cell leukemia virus type (HTLV-I) is a type C retrovirus that has been linked to both adult T-cell leukemia and neurological disorders in humans. Baboons and other Old World non-human primates harbor a related virus termed simian T-cell leukemia virus type 1 (STLV-I), which may also be associated with neoplastic disease. To explore the utility of the baboon as a model for HTLV-I infection and disease, 329 baboons from a colony of 3200 at the Southwest Foundation for Biomedical Research (SFBR) were analyzed for the presence of antibodies against STLV-I. An overall seroprevalence rate of > 40% was found, with higher rates in females versus males. Furthermore, seroprevalence rates increased dramatically with age, reaching greater than 80% in animals over the age of 16. Molecular and antigenic analysis of proviral DNA isolated from both tumor tissue and a cell line isolated from a baboon with non-Hodgkin's lymphoma (NHL) indicates that STLV-I in this colony is closely related to HTLV-I. Furthermore, monoclonally integrated provirus isolated from lymphoma tissue was detected, strongly implicating STLV-I in the etiology of this malignancy. DNA primer pairs homologous to HTLV-I sequences amplified both HTLV-I and STLV-I, but not HTLV-II, providing further evidence for a close genetic relationship between baboon-derived STLV-I and HTLV-I. The detailed study of a large population of naturally infected baboons may therefore shed some light into the complex processes required for the induction of disease associated with HTLV-I infection in humans.


Selectivity of antagonist and partial agonist activity of celiprolol in normal subjects. 1. The aims of this study were to assess the relative beta 1/beta 2 selectivity of the antagonist and partial agonist activity (PAA) of celiprolol in man. 2. Eight normal males received single oral doses of celiprolol 200 mg (C200), 400 mg (C400) and 800 mg (C800); atenolol 50 mg (A50), 100 mg (A100) and 200 mg (A200); nadolol 40 mg (N40) and placebo (PL), administered in a single-blind, randomised crossover design. 3. At rest, in the presence of low levels of circulating adrenaline and noradrenergic tone, a low dose of celiprolol (C200) showed evidence of beta 1-PAA by significant increases in systolic blood pressure and resting heart rate. At higher doses (C400, C800), beta 2-PAA became evident by a significant increase in postural finger tremor, whereas C200 had no effect. 4. In the presence of a beta 1-adrenoceptor agonist, as assessed by reduction of exercise tachycardia, increasing doses of celiprolol produced significantly less beta 1-adrenoceptor blockade compared with atenolol. Furthermore, there was no increase in beta 1-adrenoceptor blockade beyond C400. 5. In the presence of a beta 2-adrenoceptor agonist, as assessed by blunting of terbutaline-induced chronotropic, hypokalaemic and finger tremor responses, celiprolol exhibited less beta 2-adrenoceptor blockade than comparable doses of atenolol used in clinical practice. 6. Exercise hyperkalaemia was blunted significantly by C400 and C800 in comparison with all doses of atenolol and nadolol.(ABSTRACT TRUNCATED AT 250 WORDS)


The low-chloride cough response is not inhibited by a single, high dose of aspirin. The effect of a single, high dose of aspirin has been assessed against low chloride cough challenge. The drug does not affect the cough response, suggesting that airway prostaglandin generation is not responsible for the tussive activity of low chloride solution.


Immune aspects of myositis.

Immune aspects of myositis. Myositis describes a heterogeneous group of disorders whose main pathologic feature is chronic inflammation of the affected muscles. The association of myositis with other autoimmune diseases, the response to corticosteroid and immunosuppressive therapy, the frequent occurrence of autoantibodies, and the presence of chronic inflammatory cells in the affected muscles of patients with myositis indicate that the myositis syndromes are autoimmune diseases. This review summarizes recent observations on the role of humoral and cellular mechanisms in myositis. During the past year, the most notable contributions included studies on the relationship among autoantibodies and various clinical and epidemiologic features of patients with myositis; further evidence for T-cell involvement in the pathogenesis of myositis; demonstration of amyloid proteins in muscle fibers of patients with inclusion body myositis; and a controlled trial of plasma exchange and leukapheresis in myositis.


Effects of sodium bicarbonate, vinegar, acetic and citric acids on growth and survival of Yersinia enterocolitica. The effects of sodium bicarbonate, vinegar, acetic and citric acids on growth and survival of Yersinia enterocolitica in a laboratory medium were investigated. Acetic acid was found to be the most effective agent used, which completely inhibited the growth of the test organism at concentration of 0.156% (v/v) in the media incubated at 22 degrees C for 48 h. The antimicrobial activity of acetic acid and vinegar on the survival of the test bacterium in media incubated at 22 degrees C for 60 min was dependent on the inocula levels.


[ACE inhibitors and vagal activity: the effect of captopril and lisinopril on cardiovascular reflexes]. The influence of the ACE-inhibitors captopril and lisinopril on parasympathetic activity in normotensive subjects was evaluated. Three cardiovascular tests which explored chiefly parasympathetic function (deep breathing, lying to standing and Valsalva test) were performed in 10 normotensive volunteers (mean age 26.1 years) in both basal conditions and after four days of treatment with either captopril (25 mg twice a day) or lisinopril (20 mg once a day). Mean blood pressure was not influenced by captopril, whereas it was significantly lowered with lisinopril (from 94.4 +/- 6.8 to 88.7 +/- 5.7 mmHg; p < 0.05). Neither drug interfered with heart rate or with the results of the deep breathing and Valsalva tests. The 30/15 ratio, an index of heart rate variability during the lying to standing test, significantly worsened after assumption of both captopril (from 1.37 +/- 0.18 to 1.21 +/- 0.14; p < 0.05) and lisinopril (from 1.31 +/- 0.17 to 1.20 +/- 0.11; p < 0.05). Although our subjects had a lisinopril-induced drop in blood pressure, their heart rate remained steady. This finding confirms previous studies reporting the lack of reflex tachycardia during ACE-inhibition. The slight effect of ACE-inhibitors on the results of deep breathing and Valsalva tests suggests that such drugs do not directly stimulate vagal activity; the significant decrease of the 30/15 ratio may be due to a functional impairment of the baroreflex mechanism.


Lower urinary tract function after exstrophy closure. Twenty-eight children with bladder exstrophy, prior to surgery for continence, underwent a urodynamic evaluation, cystogram, ultrasound and dimercaptosuccinic acid scan, to define parameters of lower urinary tract function that are a risk for upper tract damage and/or impair development of bladder capacity. The bladders in 7 of 28 demonstrated no storage function [leak pressure)LP) = 0]; but storage parameters could be measured in the other 21. Their LP was 10-35 cm H2O; 17 of 21 revealed involuntary contractions and 7 of 21 had an end-fill pressure greater than 10 cm H2O. Bladder capacity was better in those with a higher LP and those with no involuntary contractions. The 4 patients with bilateral hydronephrosis (3 of whom also had bilateral multiple scars) were among the 6 with LP greater than 30 cm H2O. Of the 7 with an end-fill pressure greater than 10 cm H2O, 5 had upper tract damage. Involuntary contractions may impair development of capacity. A LP of greater than 30 cm H2O is a risk factor for upper tract damage and an end-fill pressure greater than 10 cm H2O may also be a factor.


Distinction of two different classes of small-cell lung cancer cell lines by enzymatically inactive neuron-specific enolase. Neuron specific enolase (NSE) is widely used as a neuro-endocrine marker. However the presence of NSE in many non-neuroendocrine tissues has raised questions on the specificity of NSE. We have investigated NSE immunoreactivity (NSA-ag), gamma-enolase activity and total enolase activity in small cell lung cancer (SCLC) cell lines. During well-controlled exponential growth comparison of NSE-ag content and gamma-enolase activity with the doubling-time (Td) and NSE-ag content with gamma-enolase and total enolase activity led to a clear distinction of two types of cell line: variant cell lines plus part of the classic cell lines (type I) and the remaining classic cell lines (type II). The distinction was based upon both an abrupt 6-fold increase of gamma-enolase activity and an 18-fold increase of NSE-ag, which for the larger part was enzymatically inactive. Within each group the increase of NSE-ag content was significantly correlated with the increase of gamma-enolase activity and both NSE-ag content and gamma-enolase activity increased linearly with Td. It is concluded that gamma-enolase seems to be associated with the regulation of growth rate and that a compound with the gamma-enolase antigen but without enzyme activity can distinguish two different classes of SCLC cell lines. Furthermore the demonstration that NSE-ag can represent the active enzyme as well as an enzymatically inactive compound may explain why a controversy about neuron- or non-specificity of NSE exists.


Expression of the ERBB3 gene product in breast cancer. Abnormalities of the EGF receptor and/or the related ERBB2 receptor occur in a significant proportion of cases of human breast cancer and are important influences in the behaviour of this tumour type. In this report we demonstrate by nucleic acid analysis and immunohistochemistry that the recently recognised third member of this gene family, ERBB3, shows a wide range of expression in breast cancer, and shows stronger immunoreactivity than that observed in normal tissue in 43 out of 195 cases (22%) of primary breast cancer. Overexpression of ERBB3 appears to result from increased levels of gene transcription since in none of the cell lines or primary cancers analysed did we find evidence of gene amplification. High expression of ERBB3 is positively associated with the presence of lymph node metastases, but there was no demonstrable relationship with patient survival in this series.


Altered expression of E-cadherin in gastric cancer tissues and carcinomatous fluid. Expression of E-cadherin in 21 patients with various histological types of gastric carcinomas was studied by immunoperoxidase staining. Intercellular boundaries of almost all cancer cells in well and moderately differentiated adenocarcinomas stained as deeply for E-cadherin as normal gastric mucosa. However, singly infiltrating cells of those histological types were poorly stained. In poorly differentiated adenocarcinomas, cancer cells forming clusters stained lightly and those infiltrating singly stained even less. In signet ring cell carcinomas, hardly any staining was observed. In each histological type, the staining patterns and intensity at different layers of the gastric wall, were essentially the same. Cancer cells from carcinomatous ascites of gastric adenocarcinomas and pancreatic adenocarcinomas, and those from pleural effusion of lung adenocarcinomas were also studied by immunofluorescence staining. Of 11 specimens, ten were negative and only one from a lung adenocarcinomas was positively stained. By phase-contrast microscopic observations, none of these cancer cells including those from the lung adenocarcinomas, formed obvious cell-cell contacts. Cell aggregation assays confirmed the above results. The molecular weight of E-cadherin of cancer cells of lung adenocarcinomas was less than intact E-cadherin as revealed by Western blot analysis. These results suggest that depressed expression and/or impaired function of E-cadherin in cancer cells, facilitates their liberation from primary sites to infiltrate freely into tissue or fluid.


Induction of soluble tumour necrosis factor receptors during treatment with interleukin-2. Interleukin-2 (IL-2) treatment induces other cytokines such as tumour necrosis factor (TNF) TNF may mediate some of the anti-tumour activity of IL-2, but conversely, may contribute to its dose limiting toxicities. Cleaved extracellular domains of the p55 and the p75 TNF receptors (sTNF-R1 and R2) bind to and inhibit the biological activity of TNF in vitro, but may also act as carrier molecules. We have assayed TNF and sTNFR-1 and 2 in the plasma of advanced cancer patients, before and during treatment with IL-2. Plasma levels of TNF in 22 patients were not significantly different from 25 normal controls, but levels of sTNFR-1 and sTNFR-2 were higher (P < 0.001). Levels of TNF and both its soluble receptors were significantly increased in 13 patients receiving IL-2 therapy. Maximum induced levels of sTNFR-1 and sTNFR-2 correlated closely with maximum induced levels of TNF (P < 0.001), but peak levels of sTNFR-1 and two were achieved 24-48 h after peak TNF. Levels of TNF and sTNF-Rs did not correlate with toxicity. Treatment with IL-2 leads not only to induction of TNF but also soluble binding proteins at levels which may modulate its biological activity.


Ubiquitinated proteasome inhibitor is a component of the 26 S proteasome complex. Western blot analysis, using a polyclonal antibody to the 240-kDa endogenous inhibitor of the 20 S proteasome, revealed that the inhibitor is a component of the 26 S complex. Although isolated inhibitor displayed a single 40-kDa band on SDS-PAGE, the antibody detected a 55-kDa component in the 26 S proteasome complex. Ubiquitin polyclonal antibody recognized the same 55-kDa component but did not react with free 40-kDa inhibitor subunit. Addition of purified 40-kDa inhibitor to a ubiquitin ligating system also generated the 55-kDa species. In crude erythrocyte extracts, most of the inhibitor migrated at 55 kDa in the presence of ATP but shifted to 40 kDa in the absence of ATP, consistent with removal of ubiquitin. It is suggested that ubiquitination of the inhibitor may be involved in regulating assembly and/or activity of the 26 S proteasome complex.


Induction of mammalian DNA topoisomerase I mediated DNA cleavage by antitumor indolocarbazole derivatives. DNA topoisomerases have been shown to be important therapeutic targets in cancer chemotherapy. We found that KT6006 and KT6528, synthetic antitumor derivatives of indolocarbazole antibiotic K252a, were potent inducers of a cleavable complex with topoisomerase I. In DNA cleavage assay using purified calf thymus DNA topoisomerase I and supercoiled pBR322 DNA, KT6006 induced topoisomerase I mediated DNA cleavage in a dose-dependent manner at drug concentrations up to 50 microM, while DNA cleavage induced by KT6528 was saturated at 5 microM. The maximal amount of nicked DNA produced by KT6006 was more than 50% of substrate DNA, which was comparable to that of camptothecin. Heat treatment (65 degrees C) of the reaction mixture containing these compounds and topoisomerase I resulted in a substantial reduction in DNA cleavage, suggesting that topoisomerase I mediated DNA cleavage induced by KT6006 and KT6528 is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex". Both KT6006 and KT6528 did not induce topoisomerase II mediated DNA cleavage in vitro. KT6006 and KT6528 were found to induce nearly identical topoisomerase I mediated DNA cleavage patterns, which was distinctly different from that with camptothecin. In contrast to the similarity between KT6006 and KT6528 in their structures and the nature of their cleavable complex with topoisomerase I, these drugs have different properties with respect to their interaction with DNA: KT6006 is a very weak intercalator whereas KT6528 is a strong intercalator with potentials comparable to that of adriamycin. These results indicate that KT6006 and KT6528 represent a new distinct class of mammalian DNA topoisomerase I active antitumor drugs.


Solution conformation of a deoxynucleotide containing tandem G.A mismatched base pairs and 3'-overhanging ends in d(GTGAACTT)2. We have used 31P and 1H NMR spectroscopy and circular dichroism to define the solution conformation of d(GTGAACTT)2 which contains tandem G.A mismatched base pairs and 3'-overhanging TT ends. Measurements of coupling constants and NOE intensities show that the sugar puckers of the nucleotides are predominantly in the south domain (i.e., near C2'-endo) and that the glycosidic torsion angles are anti. The sequential NOE intensities indicate the presence of a right-handed helix. Analysis of the 31P and 1H NMR spectra of the duplex shows that the tandem mismatch forms a block in which there are unusual backbone torsion angles (i.e., in the BII state), within an otherwise B-like structure. The chemical shift of the N1H of the mismatched guanosine and NOEs between the mismatched base pairs and their nearest neighbors are inconsistent with the imino pairing present in single A.G mismatches or in the X-ray structure of a tandem mismatch [Privé, G. G., et al. (1987) Science 238, 498-503] but the data are consistent with the amino pairing found by Li et al. (1991) [Li, Y., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 26-30]. The strong base-base stacking both within the tandem G.A block and between the G.A mismatches and their other nearest neighbors offsets the intrinsic destabilizing effects of the mismatch. Further, the 3'-TT overhangs stack onto the ends of the helix and stabilize the duplex against fraying, which accounts for the observed increase in the melting temperature compared with the flush-ended duplex.