Workshop on DNA repair.

A workshop on DNA repair with emphasis on eukaryotic systems was held, under the auspices of the EC Concerted Action on DNA Repair and Cancer, at Noordwijkerhout (The Netherlands) 14-19 April 1991. The local organization of the meeting was done under the auspices of the Medical Genetic Centre South-West, The Netherlands (MGC), c/o Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden (The Netherlands). Local organizers were: D. Bootsma (chairman), W. Ferro, J.H.J. Hoeijmakers, A.R. Lehmann, P.H.M. Lohman, L. Mullenders, and A.A. van Zeeland (secretarial assistance: Mrs. C. Escher-van Heerden and Mrs. R. Bontre). Over 190 scientists participated, and the format of the meeting followed that of the 1987 workshop on the 'Molecular Aspects of DNA Repair' (Friedberg et al., 1987). Plenary review talks in the mornings were followed, in the afternoon, by poster viewing in three or four parallel sessions. Groups of 15-20 posters were discussed in detail, and later on, in plenary sessions, chairpersons of the poster discussions reviewed the afternoons' posters. The principal themes of the meeting were the isolation and characterisation of repair genes and proteins, repair in specific sequences, consequences of defective DNA repair, and new methods for detecting DNA damage and repair. Remarkable progress has been made recently in all of these areas, and many exciting new results were presented. It is impossible to summarize all contributions to this (intensive) one-week meeting. Therefore, and for the sake of coherence, presentations that did not fit easily into any of the general themes of the meetings have not been included.


Differences in nucleotide and base DNA excision repair observed during mitogenic stimulation of bovine lymphocytes.

The bromodeoxyuridine density-shift technique was used to examine nucleotide and base DNA excision repair in quiescent and lectin stimulated bovine lymphocytes damaged with either ultraviolet light or dimethyl sulfate (DMS). Compared to a number of human cell lines, quiescent lymphocytes were less proficient in the repair of both types of damage. Repair replication was enhanced upon mitogenic stimulation, but both the amount and time course of the increase in repair depended upon the damaging agent used. A 2-3-fold increase in UV light induced repair replication occurred early during stimulation and subsided only gradually as stimulation proceeded. However, the profile of DMS induced repair increased 7-fold and then decreased, in parallel with measurements of lectin-stimulated DNA replication. Estimates of average repair patch sizes showed that quiescent lymphocytes produced smaller patches of 7 nucleotides in response to DMS damage while UV light irradiation resulted in repair patches of 20 nucleotides. During stimulation, patch sizes appeared to increase to maximum values of 45 and 33 nucleotides in response to UV light and DMS, respectively, one day prior to the peak of DNA replication. These increases in patch size were followed by a gradual decrease towards unstimulated levels. However, the appearance of a DNA species of intermediate density in the gradient profiles made the interpretation of repair patch sizes in stimulated cells difficult. These results are discussed as evidence not only for differences in the mechanisms of nucleotide and base excision repair but also for changes in repair as the cell progresses through the cell cycle.


UV endonuclease-mediated enhancement of UV survival in Micrococcus luteus: evidence revealed by deficiency in the Uvr homolog.

Unlike its phage T4 counterpart (also known as endonuclease V), Micrococcus luteus UV endonuclease (pyrimidine dimer DNA glycosylase/apurinic-apyrimidinic endonuclease) has suffered from lack of genetic evidence to implicate it in the promotion of UV survival of the cell, i.e., mutants with its deficiency are no more UV-sensitive than the wild type. On the assumption that the contribution of UV endonuclease is obscured by the presence of a homolog of Escherichia coli UvrABC endonuclease, which has recently been identified in this bacterium, survival studies were carried out in its absence. With 254-nm UV irradiation, which generates not only pyrimidine dimers but also 6-4 photoproducts as lethal lesions, a double mutant defective in both UV endonuclease and the Uvr homolog was shown to be more sensitive than a single mutant defective only in the latter, with a dose reduction factor of approximately 2 at the survival level of 37%. Furthermore, molecular photosensitization, which produces only pyrimidine dimers, revealed an even greater difference in sensitivity, the dose reduction factor being about 3.4. These results indicate that the contribution to cell survival of UV endonuclease, an enzyme specific for pyrimidine dimers, is manifest if the backup by the Uvr homolog is absent.


Biochemical heterogeneity in xeroderma pigmentosum complementation group E.

Cells from two patients with xeroderma pigmentosum complementation group E (XP-E) have been shown to lack an activity which binds specifically to UV-irradiated DNA (Chu and Chang, 1988). We investigated the occurrence of this binding activity in cell strains from nine additional, unrelated XP-E patients and found that all but one of these strains contained normal levels of the binding protein. Furthermore, the binding activity from these XP-E strains was indistinguishable from that of normal controls in thermal stability, behavior on ion-exchange chromatography, and electrophoretic mobility of protein-DNA complexes, indicating that there were no gross structural alterations in the protein. The association of XP-E with a deficiency in DNA-damage binding protein in cells from 3 of 12 XP-E patients (compared to 0 of 20 non-XP-E controls) is statistically significant (p less than 0.05), but there is no obvious correlation between the biochemical defect and the clinical or cellular characteristics of individual patients. Implications of these findings for the role of the binding protein in XP-E are discussed.


Defective DNA endonuclease activities in Fanconi's anemia cells, complementation groups A and B.

Cells from patients with the inherited disorder, Fanconi's anemia (FA), were analyzed for endonucleases which recognize DNA interstrand cross-links and monoadducts produced by psoralen plus UVA irradiation. Two chromatin-associated DNA endonuclease activities, defective in their ability to incise DNA-containing adducts produced by psoralen plus UVA light, have been identified and isolated in nuclei of FA cells. In FA complementation group A (FA-A) cells, one endonuclease activity, pI 4.6, which recognizes psoralen intercalation and interstrand cross-links, has 25% of the activity of the normal human endonuclease, pI 4.6, on 8-methoxypsoralen (8-MOP) plus UVA-damaged DNA. In FA complementation group B (FA-B) cells, a second endonuclease activity, pI 7.6, which recognizes psoralen monoadducts, has 50% and 55% of the activity, respectively, of the corresponding normal endonuclease on 8-MOP or angelicin plus UVA-damaged DNA. Kinetic analysis reveals that both the FA-A endonuclease activity, pI 4.6, and the FA-B endonuclease activity, pI 7.6, have decreased affinity for psoralen plus UVA-damaged DNA. Both the normal and FA endonucleases showed approximately a 2.5-fold increase in activity on psoralen plus UVA-damaged reconstituted nucleosomal DNA compared to damaged non-nucleosomal DNA, indicating that interaction of these FA endonucleases with nucleosomal DNA is not impaired. These deficiencies in two nuclear DNA endonuclease activities from FA-A and FA-B cells correlate with decreased levels of unscheduled DNA synthesis (UDS), in response to 8-MOP or angelicin plus UVA irradiation, in these cells in culture.


(6-4) photoproducts and not cyclobutane pyrimidine dimers are the main UV-induced mutagenic lesions in Chinese hamster cells.

A partial revertant (RH1-26) of the UV-sensitive Chinese hamster V79 cell mutant V-H1 (complementation group 2) was isolated and characterized. It was used to analyze the mutagenic potency of the 2 major UV-induced lesions, cyclobutane pyrimidine dimers and (6-4) photoproducts. Both V-H1 and RH1-26 did not repair pyrimidine dimers measured in the genome overall as well as in the active hprt gene. Repair of (6-4) photoproducts from the genome overall was slower in V-H1 than in wild-type V79 cells, but was restored to normal in RH1-26. Although V-H1 cells have a 7-fold enhanced mutagenicity, RH1-26 cells, despite the absence of pyrimidine dimer repair, have a slightly lower level of UV-induced mutagenesis than observed in wild-type V79 cells. The molecular nature of hprt mutations and the DNA-strand specificity were similar in V79 and RH1-26 cells but different from that of V-H1 cells. Since in RH1-26 as well as in V79 cells most hprt mutations were induced by lesions in the non-transcribed DNA strand, in contrast to the transcribed DNA strand in V-H1, the observed mutation-strand bias suggests that normally (6-4) photoproducts are preferentially repaired in the transcribed DNA strand. The dramatic influence of the impaired (6-4) photoproduct repair in V-H1 on UV-induced mutability and the molecular nature of hprt mutations indicate that the (6-4) photoproduct is the main UV-induced mutagenic lesion.


Identification of cellular factors that recognize UV-damaged DNA in Drosophila melanogaster.

Using a gel electrophoresis DNA band-shift assay, we have identified 2 DNA-binding protein complexes in wild-type Drosophila embryonic cells which have high affinity for UV-irradiated, double-stranded DNA. Screening of Drosophila mutants deficient in DNA repair led to the identification of 5 mutants which lacked either one of the 2 protein complexes. Four excision repair-deficient mutants (mus-201, phr, mus-308 and mus-205) lacked one protein complex (Factor 2). The other protein complex (Factor 1) was not detectable in the post-replication repair-deficient mutant mus-104. These findings might suggest the possible involvement of these gene products in lesion recognition and repair of UV-induced photoproducts in DNA.


Genetic toxicology of propylene oxide and trichloropropylene oxide--a review.

Aliphatic epoxides are a group of compounds extensively used in industry and as laboratory reagents, and can be produced as metabolic intermediates. An important aliphatic epoxide is propylene oxide, extensively used in the production of propylene glycol and polyols used in the manufacture of polyurethane polymers. The widespread human exposure to propylene oxide is of great health concern. In this review an attempt has been made to evaluate and update the genotoxic effects of propylene oxide and trichloropropylene oxide based on the available literature.


Statistical methods for short-term tests in genetic toxicology: the first fifteen years.

Short-term tests (STTs) for detecting and assessing genotoxic or mutagenic effects have catalyzed the development of biostatistical methods for more than one decade. Most notably, the Ames Salmonella/microsome assay created statistical methodology with a range of applications going beyond genotoxicity. Early approaches with parametric statistical methods appeared to be insufficient and have been replaced by non-parametric ones requiring less restrictive distributional assumptions. There have also been successful attempts to use biomathematical models for establishing dose-response relationships. Overdispersion has been recognized as a major problem for the evaluation of mutagenic count data and methods to cope with it have became available. A theory of generalized linear modelling is emerging to combine dose-response modeling with much less restrictive distributional assumptions, while allowing the inclusion of concomitant factors arising from the experimental conditions. The methodological survey below reviews the present state of this development and is intended to promote further research into biostatistical issues and methods of analysis. Appropriate methods for the design and analysis of STTs are discussed. The progress for the Ames assay was only partially transmitted to the analysis of the large number of other short-term assays. Several such assays are reviewed with respect to their present state of statistical evaluation.


DNA adduct formation by 12 chemicals with populations potentially suitable for molecular epidemiological studies.

DNA adduct formation, route of absorption, metabolism and chemistry of 12 hazardous chemicals are reviewed. Methods for adduct detection are also reviewed and approaches to sensitivity and specificity are identified. The selection of these 12 chemicals from the Environmental Protection Agency list of genotoxic chemicals was based on the availability of information and on the availability of populations potentially suitable for molecular epidemiological study. The 12 chemicals include ethylene oxide, styrene, vinyl chloride, epichlorohydrin, propylene oxide, 4,4'-methylenebis-2-chloroaniline, benzidine, benzidine dyes (Direct Blue 6, Direct Black 38 and Direct Brown 95), acrylonitrile and benzyl chloride. While some of these chemicals (styrene and benzyl chloride, possibly Direct Blue 6) give rise to unique DNA adducts, others do not. Potentially confounding factors include mixed exposures in the work place, as well the formation of common DNA adducts. Additional research needs are identified.


CD5+ B lymphocytes and other lymphocyte subsets in primary Sjögren's syndrome.

CD5+ B cells and other lymphocyte subsets were analyzed by flow cytometry in patients with primary Sjögren's syndrome (pSS), in healthy subjects (HS) and in patients with various control diseases. When compared with HS, patients with pSS were found to have similar levels of CD5+ B cells and decreased levels of CD8+ T cells (P = 0.0003). When compared with patients with various other diseases, however, the number of CD5+ B cells in pSS was more than twice as high (P = 0.0002), whereas no difference was found between numbers of CD8+ T cells. When the number of CD5+ B cells was expressed as a percentage of total B cells, the results obtained were similar to those with absolute numbers. Determination of lymphocyte subsets may be used as a diagnostic aid for Sjögren's syndrome in selected patients with suspected immunological diseases of unknown type.


Two brothers with a variant of hereditary sensory neuropathy.

We report two brothers with the clinical symptoms and neuropathological findings of hereditary sensory and autonomic neuropathy (HSAN) type IV but with normal sweating function and absence of recurrent fever. We propose that our patients may have a lower degree of expression of the genetic defect underlying HSAN type IV or that they represent a separate genetic entity.


Monoamine metabolites, iron induced seizures, and the anticonvulsant effect of tannins.

Intracortical injections of iron ions have been shown to induce recurrent seizures and epileptic discharges in the EEG. (-)-Epigallocatechin (EGC) and (-)-epigallocatechin-3-O-gallate (EGCG), isolated from green tea leaves, have been reported to prevent or diminish the occurrence of epileptic discharges induced by iron ions, and to inhibit catechol-O-methyltransferase. Iron ions significantly increased DOPAC and HVA levels in the intrastriatal perfusate 140 and 180 minutes, respectively, after injection. EGC and EGCG inhibited the increases induced by iron ions. Furthermore, EGCG decreased the HVA level in the perfusate 200 minutes after injection whether or not iron ions were injected. Iron ions had no effect on the 5-HIAA level, and EGC and EGCG raised it. These results suggest that formation of an epileptic focus induced by iron ions might be accompanied by activation of dopaminergic neurons, and that EGC and EGCG inhibit that hyperactivity.


Acquisition of signs from American sign language in hearing individuals following left hemisphere damage and aphasia.

Three severely aphasic hearing patients with no prior knowledge of sign language were able to acquire competency in aspects of American Sign Language (ASL) lexicon and finger spelling, in contrast to a near complete inability to speak the English counterparts of these visuo-gestural signs. Two patients with damage in left postero-lateral temporal and inferior parietal cortices mastered production and comprehension of single signs and short meaningful sign sequences, but the one patient with damage to virtually all left temporal cortices was less accurate in single sign processing and was unable to produce sequences of signs at all. These findings suggest that conceptual knowledge is represented independently of the auditory-vocal records for the corresponding lexical entries, and that left anterior temporal cortices outside of traditional "language areas" are part of the neural network which supports the linkage between conceptual knowledge and linguistic signs, especially as they are used in the sequenced activations required for production or comprehension of meaningful sentences.


Overproduction of voltage-dependent Na+ channels in the developing brain of genetically seizure-susceptible E1 mice.

We used E1 mice, a ddY mouse-derived, autosomal mutant strain and a model of hereditary sensory-precipitated epilepsy, to test the hypothesis that epileptic susceptibility may be associated with the activity of voltage-dependent ion channels. We examined the saxitoxin binding capacity of the receptor site 1 of the Na+ channel alpha-subunit, the expression activity of the Na+ channel mRNA, the veratridine-induced 22Na+ influx in the brain synaptosomes, and the regional distribution of Na+ channels in the brain. Compared with control ddY mice, in E1 mice which have not experienced seizures, the number of Na+ channels in the brain synaptosomes increased by approximately 20% starting at the fourth postnatal week through the adult stage as determined by [3H]saxitoxin binding assay. Northern blot hybridization analysis showed excess expression of Na+ channel mRNA (by 30-40%) coincidentally with Na+ channel increases. Regional analysis using the saxitoxin binding assay demonstrated approximately 1.3-fold denser distribution of Na+ channels in the cortex and cerebellum but not the hippocampus and midbrain including thalamus of E1 mice compared to ddY mice. Scatchard plot analysis for saxitoxin binding in the cortex of E1 mouse brains revealed higher maximum binding capacity (Bmax) values (ddY, 4.43 +/- 0.28 pmol/mg protein; E1, 5.43 +/- 0.25 pmol/mg protein) without a change in Kd (ddY, 1.05 +/- 0.03 nM; E1, 1.03 +/- 0.01 nM). Lastly, veratridine-evoked 22Na+ influx, sensitive to tetrodotoxin, was increased approximately 45% in the cortical synaptosomes in six-week-old E1 mice.(ABSTRACT TRUNCATED AT 250 WORDS)


Neurons of origin of zinc-containing pathways and the distribution of zinc-containing boutons in the hippocampal region of the rat.

Recent methods allow the study of neurons that contain zinc in synaptic vesicles of their boutons (Timm-stainable boutons) by the intravital precipitation (local or throughout the CNS) of the vesicular zinc with selenium compounds and its subsequent retrograde transport to the parent neurons, where the precipitate can be silver enhanced. The present study is a description of the distribution of zinc-containing neurons, their possible connections and their terminal fields within the hippocampal region of the rat. Problems inherent to the methods are addressed. Finally, based on the results and a review of literature, the possible function of zinc in the hippocampal region is considered. Neurons which contain silver-enhanced precipitates were observed in layers II, V and VI of the lateral entorhinal area and in layers V and VI of the medial entorhinal area. In the parasubiculum, labeled cells were seen in layer II/III of the parasubiculum a and in layer V. Labeled cells in the presubiculum were concentrated in layers III and V, in the hippocampal pyramidal cell layer and the dentate granule cell layer, but neurons containing precipitates were largely absent from the subiculum. Zinc-containing axonal boutons defined subpopulations within principal hippocampal neuron populations. Within layer II of the lateral entorhinal cortex and the pyramidal cell layer for regio inferior deeply situated neurons were labeled, whereas superficially placed pyramidal cells were labeled in regio superior. The neuropil staining described in the present study corresponded to that found in earlier studies. However, glial and vascular staining or unspecific background were largely absent, and the neuropil staining could unequivocally be identified light microscopically. Methodological problems are most prominently reflected in unstained mossy fibers in some animals. Based on series from animals treated with decreasing doses of sodium selenite and increased survival times, this problem can be related to small amounts of circulating reactive selenium and a competition of zinc compartments (vesicles) for the selenium. Staining will fail where the competition prevents individual compartments from reaching a threshold amount of zinc precipitate for silver amplification. A guide to evaluate histological material is provided. The distribution of zinc-containing boutons and their cells of origin indicate that zinc-containing and zinc-negative projections are not organized as parallel pathways. The mossy fibers provide an example of a pure zinc-containing pathway. Projections from regio superior to the dorsal presubiculum are likely to be zinc-negative while projections from the same area to the subiculum are zinc-containing.(ABSTRACT TRUNCATED AT 400 WORDS)


Intracellular responses of olfactory bulb granule cells to stimulating the horizontal diagonal band nucleus.

The effects of centrifugal afferents on membrane potentials of identified granule cell layer using evoked field potential profiles, and trans-synaptic activation via antidromic stimulation of output cell axon collaterals. Intracellular recordings maintained for 4-30 min showed complex spontaneous spike discharges and allowed characterization of the cell's input resistance, and on some occasions its morphology following intracellular injection of Lucifer Yellow. Stimulation in the nucleus of the horizontal limb of the diagonal band, but not surrounding regions, produced hyperpolarizing responses in 13 of 27 cells in the granule cell layer; four of these were morphologically identified as granule cells of two types, in five the responses had reversal potentials more negative than the resting potential, and six were identified as granule cells by monosynaptic activation from output axon collaterals. A different set of three cells in the granule cell layer responded with depolarization. The results are consistent with the inhibition of tonic activity of granule cells by the nucleus of the horizontal limb of the diagonal band, leading to disinhibition of mitral and tufted cells via dendrodendritic synapses of granule cells on mitral/tufted cell secondary dendrites.


Intravenous injection of Evans Blue labels magnocellular neuroendocrine cells of the rat supraoptic nucleus in situ and after dissociation.

Previous work has demonstrated that intravenous injection of neuronal tracers, e.g. horseradish peroxidase or Fast Blue, can retrogradely label neurons in brain areas that project outside the blood-brain barrier, e.g. magnocellular neuroendocrine neurons of the hypothalamus. Here we have shown that 24 h after intravenous injection of the fluorescent retrograde tracer Evans Blue, the same population of magnocellular neuroendocrine neurons is labeled in the paraventricular, supraoptic and accessory magnocellular nuclei. Parvicellular neuroendocrine cells in the paraventricular nuclei are also labeled. Most Evans Blue-labeled magnocellular neuroendocrine cells in the supraoptic nucleus could be stained immunocytochemically for neurophysins, suggesting that these neurons continue to produce their peptide hormones after taking up the fluorescent dye. Ultrastructural observation of supraoptic cells retrogradely labeled with Evans Blue shows that 95% of the neurons appeared healthy. There was no ultrastructural evidence of degeneration, hyperstimulation, or interruption of the axoplasmic flow. Labeling the neuroendocrine cells with Evans Blue did not alter the size of magnocellular cells, the animal's fluid balance or ingestive behavior. Following enzymatic/mechanical dissociation of the supraoptic nucleus from animals that had been injected with Evans Blue 24 h previously, phase-bright neurons that often contained fluorescent material were observed, thus identifying these neurons as neuroendocrine. Recording from identified neuroendocrine cells showed that these neurons generated spontaneous or current-evoked overshooting action potentials with an afterhyperpolarization and had negative resting membrane potentials. Action potential broadening, a feature of magnocellular neurons, was observed during bursts of action potentials elicited by depolarizing current injection. Taken together, this work would suggest that Evans Blue is non-toxic at the doses used and that it provides a method to identify single neuroendocrine cells in primary cell cultures made from adult hypothalamus for voltage-clamp recordings.


Identification of spinal electromotoneurons in the ray Raja clavata (Rajidae).

Retrograde transport of horseradish peroxidase injected ipsilaterally in several parts of the electric organ and light microscopy were employed to study motoneurons and electromotoneurons of the spinal cord in the weakly electric ray Raja clavata (Rajidae). The horseradish peroxidase-labelled electromotoneurons form a chain of cells located ventrolaterally in the gray matter of the spinal cord segments adjacent to the electric organ. The diameters of cells in the electromotor nucleus vary from 70 to 110 microns. When comparing other motoneurons of the spinal cord with electromotoneurons, the latter reveal no segmentation, whereas motoneurons yield accumulations which are close to metametric ones and are essentially smaller in size (averaging 33 microns in diameter).


Serotonin-, substance P- and glutamate/aspartate-like immunoreactivities in medullo-spinal pathways of rat and primate.

Serotonergic neurons of the medulla oblongata have been proposed to play a role in the control of sensory, motor and autonomic cells in the spinal cord. Many of these raphe neurons have been shown to contain the undecapeptide substance P as well as the tripeptide thyrotropin-releasing hormone, but evidence for the presence of an excitatory amino acid in these pathways has not yet been documented. In colchicine-treated rats, we have used a combination of retrograde tracing and tri-color immunohistofluorescence techniques to study co-localization of serotonin- and substance P- with glutamate- or aspartate-like immunoreactivities in medullary neurons and the possible spinal projections of these cells. In addition, the distributions of serotonin-, substance P- and glutamate-immunoreactive terminal fields in the dorsal, ventral and lateral horns of the spinal cord were examined with tri-color immunofluorescence in the rat and the primate Macaca fasciculata. In colchicine-treated rats, glutamate- and aspartate-like immunoreactivity was found in practically all serotonin- and substance P-immunoreactive neurons of the B1, B2 and B3 cell groups. Some of these neurons also contained wheat-germ agglutinin conjugated to inactivated horseradish peroxidase and colloidal gold particles retrogradely transported from the spinal cord. In the spinal cords of non-colchicine-treated monkeys and rats, striking co-localization of serotonin, substance P- and glutamate-like immunoreactivities was seen in large boutons, surrounding the dendrites and cell bodies of large alpha motor neurons in the ventral horn. These observations suggest the existence of spinally projecting serotonin/substance P neurons containing excitatory amino acids such as glutamate or aspartate.(ABSTRACT TRUNCATED AT 250 WORDS)


Recovery from unilateral 6-hydroxydopamine lesion of substantia nigra promoted by the neurotachykinin substance P 1-11.

Previous work has indicated that the neurotachykinin substance P may have nootropic and neurotrophic effects in vivo and in vitro raising the possibility that this neuropeptide may promote functional recovery from brain damage. This hypothesis was tested using the unilateral 6-hydroxydopamine lesion of the nigrostriatal dopamine system, as there is close anatomical and functional interaction between dopamine and substance P in this system. Rats were unilaterally injected with 6-hydroxydopamine into the substantia nigra, and, starting with the day after the lesion, were treated daily with peripheral injections of substance P (50 micrograms/kg, i.p.). The analysis of open-field behavior showed that, compared with vehicle-treated control lesions, substance P prevented the lesion-induced ipsiversive asymmetry in turning behavior and accelerated recovery from the ipsilateral asymmetry in thigmotactic scanning. The facilitatory effects of substance P were dependent on the degree of the lesion, as they were observed in animals with subtotal neostriatal dopamine depletions but not in those with near-total depletions. These results are discussed, firstly, with regard to the possible mechanisms of substance P on dopaminergic and non-dopaminergic systems, and secondly, with respect to their possible relevance in the study of neurodegenerative diseases.


Distribution of neuronal cannabinoid receptor in the adult rat brain: a comparative receptor binding radioautography and in situ hybridization histochemistry.

The neuronal distribution of cannabinoid receptor in the adult rat brain is reported, combining receptor binding radioautography using the synthetic psychoactive cannabinoid ligand CP55,940 with in situ hybridization histochemistry using oligonucleotide probes complementary to rat cannabinoid receptor cDNA. In the cerebral cortex, especially in the frontal and cingulate cortex, dense binding was found in layers I and VI together with slight mRNA levels in a majority of both pyramidal and non-pyramidal-shaped neurons and of high mRNA levels in a moderate number of non-pyramidal-shaped neurons especially in layers II-III and V-VI. In the hippocampal dentate gyrus, very dense staining was found in the molecular layer together with high mRNA levels in a moderate number of hilar neurons close to the granular layer. In Ammon's horn, especially in the CA3 sector, very dense binding was found in the dendritic layers together with slight mRNA levels in the majority of the pyramidal cells and high mRNA levels in a moderate number of interneurons. In the basal ganglia, binding was very dense in the lateral putamen, substantia nigra pars reticulata, globus pallidus and entopeduncular nucleus, moderate in the medial putamen and caudate; and slight in the accumbens, together with slight to moderate mRNA levels in the striatal medium-sized neurons. Together with slight binding, slight to moderate mRNA levels were found in the majority of the neurons in the subthalamic nucleus. No binding and mRNA were found in the substantia nigra pars compacta and ventral tegmental area. Slight to moderate binding was found together with slight to moderate mRNA levels in the majority of neurons in the anterior olfactory nucleus; septum, especially medial septum and diagonal band of Broca; amygdala, especially basolateral amygdala; lateral habenula; ventromedial hypothalamic nucleus; lateral interpeduncular nucleus; central gray, dorsal cochlear nucleus; parabrachial nucleus; dorsal pontine tegmentum; pontine nuclei; commissural part of the nucleus tractus solitarius; inferior olive and dorsal horn of the spinal cord. In the cerebellum, very dense binding was found in the molecular layer together with slight mRNA levels in the majority of the granule cells and moderate mRNA levels in the basket and stellate cells. In conclusion, this study provides, for the first time, indirect assessment of the neurons containing cannabinoid receptor in the entire adult rat brain and will serve as a basis for future direct morphological confirmation using receptor immunohistochemistry and for functional studies.


In situ hybridization: mRNA levels of secretogranin II, neuropeptides and carboxypeptidase H in brains of salt-loaded and Brattleboro rats.

In situ hybridization was used to study the mRNA levels for vasopressin, galanin, secretogranin II and carboxypeptidase H in salt-loaded and Brattleboro rats. These animals represent an in vivo model for the chronic stimulation of the hypothalamo-neurohypophyseal neurons. As shown by immunelectron microscopy secretogranin II is co-stored with vasopressin in these neurons. In salt-loaded rats the levels of mRNA for vasopressin, galanin and secretogranin II are increased in the paraventricular and supraoptic nuclei. Analogous changes were observed for Brattleboro rats with the exception of the vasopressin message which was decreased in these animals. The secretogranin II message was also increased in neurons which do not contain the vasopressin mRNA, i.e. in magnocellular neurons of the lateral hypothalamus and in the subfornical organ. Carboxypeptidase H message was also found in the paraventricular and supraoptic nuclei and in the subfornical organ; however, in both models the changes in mRNA in these nuclei were much lower than those observed for the secretory peptides or non-existent. We conclude that chronic stimulation of vasopressin neurons leads to a concomitant up-regulation of the biosynthesis of neuropeptides and secretogranin II. We suggest that the secretogranin II message might be a useful general marker for identifying chronically stimulated neurons.


Corticotropin-releasing factor innervation of the locus coeruleus region: distribution of fibers and sources of input.

Electrophysiologic studies support the hypothesis that corticotropin-releasing factor, the neurohormone that initiates adrenocorticotropin release during stress, also serves as a neurotransmitter in the pontine noradrenergic nucleus, the locus coeruleus. To elucidate the circuitry underlying proposed corticotropin-releasing factor neurotransmission in the locus coeruleus, the present study utilized immunohistochemical techniques to characterize corticotropin-releasing factor innervation of rat locus coeruleus and pericoerulear regions. Corticotropin-releasing factor-like immunoreactive fibers were identified in the locus coeruleus of colchicine- and non-colchicine-treated rats. However, corticotropin-releasing factor innervation of pericoerulear regions rostral and lateral to the locus coeruleus was more dense than that of the locus coeruleus proper. Double-labeling studies utilizing antisera directed against corticotropin-releasing factor and tyrosine hydroxylase indicated that corticotropin-releasing factor-like immunoreactive fibers overlap with tyrosine hydroxylase-like immunoreactive processes of locus coeruleus neurons, particularly in rostral medial and lateral regions. A group of corticotropin-releasing factor-like immunoreactive neurons was localized just lateral to the locus coeruleus and numerous corticotropin-releasing factor-like immunoreactive neurons were visualized just ventral to the rostral pole of the locus coeruleus in a region corresponding to Barrington's nucleus. None of these corticotropin-releasing factor-like immunoreactive neurons were tyrosine hydroxylase-positive. To determine the source of corticotropin-releasing factor-like immunoreactive fibers in the locus coeruleus, injections of the retrograde tracer [wheat germ agglutinin conjugated to inactivated (apo) horseradish peroxidase coupled to gold particles] were made into the locus coeruleus and sections were processed for corticotropin-releasing factor-like immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)


Ultrastructural localization of hyaluronan in myelin sheaths of the rat central and rat and human peripheral nervous systems using hyaluronan-binding protein-gold and link protein-gold.

Neural tissue of central (rat spinal cord) and peripheral origin (rat sciatic nerve, nerve fascicles of rat skin and iris and of human conjunctiva) was processed by osmium tetroxide/microwave fixation and embedded in epoxy resin. Hyaluronan-binding proteins and link proteins coupled to 15-20-nm gold particles were used as markers in a one-step post-embedding procedure for identifying hyaluronan (hyaluronic acid) at the ultrastructural level. All myelin sheaths in both rat and human material were found to be intensely labelled. The specificity of the hyaluronan-binding probes was demonstrated by the total loss of labelling following treatment of sections with hyaluronidase or by preincubating either the probes with hyaluronan oligosaccharides or the sections with unlabelled hyaluronan-binding protein. The identified hyaluronan appears to be located extracellularly, but is precise role here remains to be elucidated.


Health problems among Army children.

Of 782 Army families with children that were surveyed, over 10% reported one or more member with health problems or handicapping conditions requiring special ongoing treatment. However, less than half of these were enrolled in the Exceptional Family Member Program. Families with illnesses/handicaps that were enrolled in the program reported on the average more health problems than families with illnesses that were not enrolled.


Effect of vitamin E on acetylcholine-induced current in molluscan neurons: role of cytoplasmic free calcium and arachidonic acid.

Role of cytoplasmic concentration of free Ca2+ ([Ca]in) and arachidonic acid in potentiating the effect of vitamin E (DL-alpha-tocopherol) on acetylcholine receptor activity in Helix pomatia neurons was studied using a two-microelectrode intracellular recording, voltage clamp and fluorescent calcium probe fura-2 technique. Elevation of [Ca]in by intracellular injection from a microelectrode or by depolarizing pulses and application of 0.1 microM-0.1 mM vitamin E enhanced the acetylcholine-induced chloride current both in LP11 and RBc4 neurons. Application of 10 microM arachidonic acid to the same neurons decreased this current. The use of fluorescent probe showed that vitamin E did not essentially change [Ca]in, but an increase of [Ca]in intensified both the enhancing effect of vitamin E and the depressing effect of arachidonic acid. The enhancing effect of calcium influx was considerably decreased after vitamin E application. The antioxidant action of vitamin E was probably not involved in the mechanism of its enhancing effect on acetylcholine-induced current, since synthetic antioxidant, ionol, depressed acetylcholine responses. A spectrum analysis has shown the interaction between vitamin E and arachidonic acid in solution. This interaction may be considered as the molecular mechanism responsible for the prevention by vitamin E of steady arachidonic acid production from membrane phospholipids and its down-regulatory effect on acetylcholine receptor activity. Our results support this suggestion, since an inhibitor of phospholipase A2, 4-bromophenacyl bromide, mimicked the enhancing effect of vitamin E.


A Golgi study on the olfactory bulb in the red stingray, Dasyatis akajei.

The intrinsic organization of the olfactory bulb (OB) was studied in the red stingray using the rapid Golgi method. The OB is horse shoe-shaped, surrounding the equator region of the nasal capsule. As seen in the sagittal sections, the OB is round with the long olfactory peduncle extending from the dorsocaudal region and the olfactory fibers in a thick bundle entering from the rostroventral aspect. Although not so distinct, the following areas are distinguished. A rostroventral ovoid area adjacent to the entrance of the olfactory fibers consists exclusively of the olfactory fibers running in various directions. Dorsocaudal to the olfactory fiber area is a wide crescent region containing thin bundles of olfactory fibers, olfactory glomeruli, mitral cells and a few disseminated granule cells. A narrow crescent area made up of scattered granule cells is located dorsocaudally to the above wide crescent area. The outermost region consists of a fiber layer encapsulating the dorsal to caudal aspect of the OB. Thus, while the major constituents of the vertebrate OB are recognized, the lamination is very obscure.


Dendritic arbolization of large pyramidal neurons in the motor cortex of normal and reeler mutant mouse.

Reeler, an autosomal recessive mutation in mice, is characterized by abnormal positioning of the neurons in the cerebral cortex. We performed a descriptive analysis on the arborization of dendritic processes of large pyramidal neurons in the motor cortex (hindlimb area) of normal and reeler mice, as seen in the Golgi preparations. In the normal mouse, somata of large pyramidal neurons were located in the layer V, and their apical dendrites ascend vertically to the pial surfaces. Their basal dendrites proceed horizontally or inferiorly. In the reeler mouse, typical large pyramidal neurons with a normal (upright) apical dendrite and a variety of atypical large pyramidal neurons with a disoriented apical dendrite were radially scattered within the motor cortex. Typical large pyramidal neurons occupied the lower half of the motor cortex, whereas atypical large pyramidal neurons were predominantly observed in the upper half of the motor cortex. Atypical large pyramidal neurons were further divided into inverted, tumbled, V-shaped, bipolar and superficial polymorphic cells, as previously reported (Terashima et al., J. Comp. Neurol. 218:314-326, 1983). Superficial polymorphic cells localized in the layer of polymorphic cells and the layer of the large pyramidal cells were characterized by the extremely poor dendritic arborizations and the smooth surface of the dendrites, which suggests development of dendrites of these neurons was deranged by the reeler genetic locus.


Analyses of salivary components in leukemia patients receiving chemotherapy.

We analyzed several salivary components in stimulated whole saliva from patients with acute leukemia who were undergoing chemotherapy. Saliva samples were collected at the time of diagnosis and longitudinally during the treatment period. Data analyses showed that patients with leukemia had significantly higher peroxidase and amylase activity and elevated concentrations of total protein at the time of diagnosis. After induction chemotherapy these parameters returned to normal values and remained constant during the observation period. At the time of diagnosis no significant differences in thiocyanate (SCN-) concentrations were found in saliva samples from control subjects and patients with leukemia. Treatment with cytotoxic agents resulted in granulocytopenia and a concomitant decrease in the SCN- concentration in saliva. The function of the salivary peroxidase system is impaired by the decrease in SCN- concentration, which may be a contributing factor to some of the oral complications that occur in patients undergoing chemotherapy.


Effect of hypoxemia on fetal hemoglobin synthesis during late gestation.

This study was carried out to investigate the effect of fetal hypoxemia, as a result of acute intermittent maternal hypoxia, on the switchover from fetal to adult type Hb as well as Hb oxygen affinity and 2,3-diphosphoglycerate levels in the near-term fetus. These experiments were carried out using 10 fetal lambs with gestational ages ranging from 132 to 140 d. After the installation of appropriate fetal catheters, five of the ewes were exposed to an air mixture containing 10% O2 for 90 min/d for 4 consecutive d. Blood samples were withdrawn before the beginning of the hypoxic experiments and between the 5th and 6th d after the first episode of hypoxia. These samples were for the determination of 2,3-diphosphoglycerate concentration, arterial O2 pressure at which Hb is 50% saturated, and Hb type synthesis. Blood gases were monitored during each hypoxic episode. During the hypoxia, fetal arterial O2 pressure decreased from 2.43 +/- 0.36 kPa (18.2 +/- 2.7 mm Hg) to 1.57 +/- 0.17 kPa (11.8 +/- 1.3 mm Hg). These values returned to their initial levels after cessation of the maternal hypoxia. Five control animals of the same gestational age were also followed. During the interval of the study, a decrease of fetal Hb synthesis was noted (71.7 +/- 12.1 to 57.4 +/- 10.2%, p less than 0.001) in the control group. However, the level of fetal Hb synthesis did not significantly change in the hypoxic group (85.1 +/- 11.1 versus 80.6 +/- 18.9%).(ABSTRACT TRUNCATED AT 250 WORDS)


Nursing care of patients with esophageal cancer.

Esophageal cancer is an uncommon but aggressive disease with the potential for early metastasis. The majority of patients will present with metastatic disease, and treatment is usually palliative. This affects the physical and psychosocial needs of the patient and family. The nurse plays a crucial role in the care of these individuals and, therefore, needs a current and in-depth understanding of the disease, its treatment, and its nursing management.


Relative incidence and mortality of congenital heart defects diagnosed by angiohemodynamic methods: a 17-year study.

Over a 17-year period (January 1971 to January 1988), 2322 children, aged 0-14 years, were diagnosed as having congenital heart disease (CHD) by cardiac catheterization and angiography. Excluding those with highly complex or undiagnosed defects, there were 2156 children with CHD, 72.4% of whom were treated surgically, with a total surgical mortality rate of 24.1%. After a mean follow-up of 9 years the overall mortality of the cohort was 29.9%, 29.1% occurring in the first month of life, 39.6% between 1 month and 1 year, and 31.2% between 1 and 14 years. The incidence, mortality, and age at death of each cardiac defect are presented and compared with the results of other studies. The overall mortality for congenital heart defects in eastern Spain remains elevated, whereas there has been a significant decrease in neonatal mortality and a trend towards a lower mortality in the last years of the study.


Balloon atrial septostomy under transesophageal echocardiographic guidance.

Balloon atrial septostomy is an established method of palliation for several forms of congenital heart disease. Previously performed under fluoroscopic x-ray control, recent reports have demonstrated the utility of transthoracic echocardiographic monitoring. We report the first application of uniplane transesophageal echocardiography (TEE) (6.7-mm probe) as an alternative imaging modality for control of balloon atrial septostomy on neonates in the intensive care unit.


Dyskeratosis congenita associated with elevated fetal hemoglobin, X-linked ocular albinism, and juvenile-onset diabetes mellitus.

An 11-year-old boy had dyskeratosis congenita, elevated fetal hemoglobin level, X-linked ocular albinism, and juvenile-onset diabetes mellitus. A review of the international literature revealed that elevated fetal hemoglobin has been noted in 15 reported cases of dyskeratosis congenita. It is a previously unrecognized, commonly associated finding in dyskeratosis congenita that may provide insight into the location and function of the gene for dyskeratosis congenita.


Eruptive vellus hair cysts: case report and review of the literature.

A 6-year-old Caucasian girl had dozens of asymptomatic, flesh-colored, 2- to 5-mm eruptive vellus hair cysts. These papules on the buttocks, thighs, and groin increased in number for three months. Histologic examination revealed poorly defined, keratin-filled cysts in the upper middermis, containing numerous transversely or obliquely cut portions of vellus hair. The histopathologic differential diagnosis with other epithelial cysts containing hair shafts is debated, and new clinical differential diagnoses are proposed. Review of the literature suggests that eruptive vellus hair cyst is not a rare disorder, but its frequency is probably underestimated due to paucity of symptoms. Nevertheless, the clinical relevance of some of the differential diagnoses should convince clinicians to obtain histologic confirmation.


Kinetic study of the helix to coil dark reaction of poly(spiropyran-L-glutamate).

An investigation of kinetics of the helix to coil dark reaction of light adapted poly(spiropyran-L-glutamic acid) (PSLG) dissolved in hexafluoroisopropanol was performed. The reaction was associated with the spiropyran (SP) to merocyanine (MC) ring opening. The ring opening reaction monitored with UV/VIS spectroscopy showed first order kinetics. Chromophore and polypeptide backbone circular dichroism data fit to an expression consistent with a single intermediate series mechanism. By FTIR, we monitored the polypeptide alpha-helix amide I, the MC chromophore--C = C--stretch and the protonated unmodified carboxylate C = O stretch bands. During the first step of the series mechanism, changes in the hydrogen bonding of the unmodified carboxylate groups occurred, suggesting breakup of polypeptide aggregates. The second step of the proposed series mechanism was dominated by the helix to coil transition and the ring opening of SP to MC. The CD spectrum of MC in the dark adapted PSLG was red shifted and had a narrower bandwidth than the UV/VIS spectrum. The kinetic and spectroscopic data suggested that a fraction (population I) of the MC chromophores experienced optical activity induced by the chiral polypeptide environment, while the remainder of the MC chromophores (population II) were solvated and enantiomeric.


[Use of octreotide in the treatment of digestive endocrine tumors. A French multicenter study].

Morbidity and mortality in endocrine gastro-enteropancreatic (GEP) tumours are mainly related to the clinical consequences of tumoral peptide hypersecretion. Surgical resection at an early stage is the only curative treatment. However, most tumours are detected only when the hypersecretory state reflects the presence of metastases; surgery and chemotherapy then give only palliative results counterbalanced by serious side-effects. Somatostatin inhibits most endocrine secretions of the GEP tract and thus can alleviate invalidating symptoms. Its use is limited by its short half-life (2 min), the necessity of i.v. infusion and the possibility of a rebound phenomenon. Octreotide, a synthetic somatostatin analogue with a long duration of action, is administered subcutaneously and allows ambulatory treatment. In our series of 78 patients we observed about 80 percent of excellent or good clinical results, enabling the patients to resume normal life. Only minor and transient side-effects were noted. The overall tolerance of the drug was considered excellent or good. Prolonged administration of octreotide is a safe and effective symptomatic treatment in patients without any restriction of anti-tumoral procedures. Furthermore, it prevents the severe carcinoid crises that occur during surgery or embolization in patients with carcinoid syndromes.


[The US-guided positioning of intraprostatic metal spirals. Preliminary experience].

The authors report their preliminary experience with the insertion of intraprostatic spirals in the treatment of chronic urinary retention due to prostatic hypertrophy in 8 inoperable patients. The value is stressed of transrectal US guidance during spiral insertion together with the advantages of US over other positioning techniques (i.e., better visualization of prostatic urethra and urethral sphincter, real time visualization of spiral progression and correct positioning). All patients were successfully treated without complications; spiral positioning was easier in the patients with a Foley's catheter (22 CH). US-guided positioning of intraprostatic spirals appears an easy and safe treatment of chronic urinary retention in inoperable patients.


Role of sodium cromoglycate on analgesia, locomotor activity and opiate withdrawal in mice.

The role of sodium cromoglycate (CRO) on analgesia, locomotor activity and morphine withdrawal in mice was studied in morphine-dependent and drug-naive mice. CRO (0.5, 1, 5, 10, 30, 50 and 100 mg/kg, SC) induces analgesia (hot plate), an effect blocked by previous administration of the opiate antagonist naloxone (1 mg/kg). Furthermore, CRO (30 mg/kg) potentiates morphine analgesia. In morphine-tolerant mice, moderate doses of CRO (0.5, 1, 5, 10 and 30 mg/kg) do not induce analgesia, which suggested the development of cross tolerance between CRO and morphine, whereas coadministration of CRO and morphine in morphine tolerant animals restored the sensitivity to morphine. Administration of CRO (10 and 30 mg/kg) induces an increase in spontaneous locomotor activity, and previous administration of naloxone (1 mg/kg) blocks this effect, whereas CRO (10 mg/kg) blocks morphine (10 mg/kg) and amphetamine (3 mg/kg)-induced hyperactivity. CRO (10, 50 and 100 mg/kg) induces a significant and dose-dependent reduction in the number of jumps ("jumping up") during naloxone (1 mg/kg)-induced withdrawal in morphine-dependent mice. Finally, CRO (100 mg/kg) reduces the "wet dog shake" phenomenom during naloxone-induced withdrawal in morphine-dependent mice. These results suggest a possible stabilizing effect of CRO on the membranes of neurones that mediate analgesia, locomotor activity and opiate abstinence. Changes and inhibition of DA, NA and 5-HT release may also explain these effects.


In vivo and in vitro interaction of trichloroethylene with macromolecules from various organs of rat and mouse.

Trichloroethylene was covalently bound in vivo to DNA, RNA and proteins of rat and mouse organs 22 hr after ip injection. The covalent binding index values of rat and mouse liver DNA classify trichloroethylene as a weak initiator. Labeling of RNA and proteins from various organs of both species was higher than that of DNA. In vitro, trichloroethylene was bioactivated by microsomal fractions dependent on cytochrome P450, mainly from liver of both species, to intermediate(s) capable of binding to exogenous DNA. No particular species-specific difference was evident except for mouse lung microsomes which were more efficient than rat lung microsomes. GSH-transferases capable of bioactivating P450-dependent were present in mouse lung microsomes and in liver microsomes of both species. These data, along those previously reported, provide sufficient evidence for a weak ability of TCY to interact covalently with DNA.


Analysis of premature ventricular counts in long term ECG following myocardial infarction.

Frequent and recurrent ventricular premature beats (VPB's) are considered to be associated with a higher risk of sudden cardiac death, particularly for survivors of myocardial infarction (MI). The distribution of VPB's has a large probability mass at zero, and a very heavy right hand tail. In this research, we fitted a model to VPB for patients for which VPB was present. The model was fitted on the basis of a relatively large data set on MI survivors in Israel. The model was fitted by a method which is based on the generalized linear model. This method, which was introduced by Zeger and Liang, is designed for longitudinal data and uses the quasi-likelihood concept. No specific assumptions are required on the shape of the distribution of the dependent variable. The results indicate that the model fits the data quite well but underestimates the very extreme high values. This research demonstrates the applicability of generalized linear models for longitudinal non-Gaussian data. Such data often arise in medical studies. The study also points out the distributional properties of VPB counts. In particular, it shows their associations with simple clinical and epidemiological variables, and with certain time periods during the day.


[Acute liver failure: current hepatological-surgical therapy strategies].

Patients with acute liver failure, characterized by deep jaundice, hepatic encephalopathy within eight weeks from the start of symptoms, are subjected to intensive care treatment. New drugs (Interferons, Prostaglandins) have been applied to these patients, with variable results. Mortality of fulminant hepatic failure is high in patients who don't respond to intensive therapy. These patients are characterized by progressive hepatic encephalopathy, decrease of clotting factors, especially of factor 5 and constantly increased serum bilirubin. Results of a multivariate analysis disclosed a poor prognosis in the individual patient more then 40 years old or under 11 years, with a prothrombin time below 10% of control, serum bilirubin over 300 mmol/l and with a late onset of hepatic encephalopathy after long standing jaundice.


[Diagnosis and therapy of hepatorenal syndrome].

Hepatorenal syndrome (HRS) is defined as severe vasoconstriction of renal cortical vessels with critical impairment of glomerular filtration rate, initiated by deteriorating liver function. In addition, systemic vasodilation, predominantly of splanchnic vessels is present. Although the urinary status is basically normal, during the course of HRS kidneys are loosing their ability to concentrate and at an urinary production rate of less than 100 ml/day the urinary osmolarity and Na(+)-concentration drops significantly. Because HRS can not be improved by conventional medical treatment regimens, its prophylaxis is of major importance. Besides improvement of liver function and avoidance of drastic volume depletion (bleeding, paracentesis, high dose diuretic therapy) and sepsis, the therapeutic goal is to reverse systemic vasodilation and to achieve a concomitant relaxation of renal arteries. Expansion of plasma volume and application of renal vessel dilating prostaglandins is one strategy. Alternatively, therapy with ornipressin was shown to be successful, which improves renal perfusion and normalizes the hyperdynamic state of systemic circulation.


[Early diagnosis of hepatocellular carcinoma].

In every patient, in particular males of all ages presenting with chronically progressive diseases or cirrhosis of the liver, ultrasonography and an AFP test should be performed at intervals of six months. If hepatocellular carcinoma of the liver (HCC) is suspected (i.e. by increase of AFP or a positive result in ultrasonography), diagnosis should be confirmed by further investigations such as fine-needle biopsy guided by sonography, angiography and CT-scan. Adequate therapeutical measures such as resection of the tumor, chemotherapy, injection of alcohol or liver transplantation can thus be initiated in time. Besides efforts for early diagnosis of carcinoma of the liver, preventive measures (vaccination for hepatitis B, restrictive use of blood transfusion, reduction of alcoholism, thorough therapy of hemochromatosis, etc.) may contribute to the reduction of chronic diseases of the liver and of associated HCC.


Real time analysis of antibody-antigen reaction kinetics.

Surface plasmon resonance, i.e. detection of changes in refractive index on a surface, was used in a biosensor to evaluate the dissociation/association rate and affinity constants of human monoclonal IgG and IgM antibodies and Fab fragments. The results showed that an observed difference in affinity constants between intact and fragmented IgG anti-tetanus antibody was related to approximately 10-fold differences in dissociation rate constants, since the association rate constants were in the same range, i.e. 2-3 x 10(5) (M-1 s-1). Affinity constants, as determined by conventional solid phase enzyme immunoassay, were substantially higher than the constants produced by the biosensor. Human monoclonal IgM anti-Tn alpha antibodies showed, furthermore, one order of magnitude higher association rate constants, as compared with the IgG antibodies, but since the dissociation rate constants were more than ten times higher, the resulting affinity constants of the anti-carbohydrate IgM antibodies were still somewhat lower than those of the IgG antibodies.


Spontaneous IgM autoantibody production in vitro by B lymphocytes of normal human neonates.

Human neonate B lymphocytes display unique phenotypic and functional characteristics: in addition to CD1c antigens, CD5+ and CD5- subsets both express activation markers such as CD23 and Bac-1. They proliferate strongly in the presence of various lymphokines (rIL-2, rIL-4, low molecular weight BCGF), but differentiate poorly in the presence of the same lymphokines, pokeweed mitogen and Epstein-Barr virus. It has also been reported that human neonate B lymphocytes produce polyreactive autoantibodies after in vitro activation by Staphylococcus aureus Cowan I and transformation by Epstein-Barr virus. We now show that, in the absence of in vitro stimulation, human neonate B lymphocytes produce polyreactive antibodies of the IgM isotype against several autoantigens. The B lymphocytes involved expressed membrane IgD, IgM, CD23 and CD11b molecules; CD5 expression was variable. This phenotype was consistently found on a minority of B lymphocytes and is similar to that of polyreactive autoantibody-producing B cells in mice. We also found that autoantibody production in vitro could occur in the absence of any T helper effect. The function of these autoantibodies is not clearly established, but their occurrence in a large proportion of human neonates strongly suggests that they play an important role in the development of the immune system.


Expression and regulation of adhesion molecules ICAM-1 (CD54) and LFA-3 (CD58) in human intestinal epithelial cell lines.

T cells and other leucocytes regularly occur within and subjacent to the gut epithelium. Recent data suggest that intestinal epithelial cells may exert accessory immunological functions. Although interactions between leucocytes and accessory cells usually require expression of adhesion molecules, intestinal epithelium has generally been considered negative for intercellular adhesion molecule-1 (ICAM-1) by immunohistochemical techniques. We therefore studied the expression of ICAM-1 and lymphocyte function-associated antigen-3 (LFA-3) by two colonic epithelial cell lines and found that both adhesion molecules were constitutively present at low levels. ICAM-1 protein expression could be enhanced within 4 h by cytokines, particularly after co-incubation with interferon-gamma (IFN) and tumour necrosis factor-alpha (TNF), interleukin-1 beta (IL-1), or IL-6. IFN also resulted in accumulation of ICAM-1 mRNA. Conversely, the LFA-3 expression was virtually unaffected by cytokine stimulation. These data imply that intestinal epithelial cells under certain conditions may bear adhesion molecules required for cooperation with juxtaposed leucocytes in situ, and that the expression of some of these molecules is modulated by cytokines from activated mucosal leucocytes.


Resistance to collagen-induced arthritis in Biozzi mice is not associated with a defect in autoantibodies to accessible epitopes on cartilage collagen.

In order to understand why most strains of Biozzi mice are unexpectedly either arthritis-resistant, or susceptible, we investigate the autoantibody reactivity against matrix-embedded collagen molecules. We show a close correlation between these tissular and the free collagen antibodies, irrespective of arthritis susceptibility. These results are against an antibody response restricted to hidden non-pathogenic epitopes in the resistant mice.


Cells of cerebrospinal fluid of multiple sclerosis patients secrete antibodies to myelin basic protein in vitro.

To characterize the role of B lymphocytes in the pathogenesis of Multiple Sclerosis (MS), we have isolated mononuclear cells from cerebrospinal fluid (CSF) and stimulated them with a polyclonal B-cell mitogen (pokeweed mitogen). This study has been done with MS patients selected for the occurrence of an acute attack in the course of the disease and with patients hospitalized for other neurological diseases. Five of the 11 MS patients had B lymphocytes producing in vitro antibodies (Abs) directed against purified human myelin basic protein (hMBP), as revealed by Western blot analysis. None of the 20 patients with other neurological diseases showed such a reactivity. The produced Abs recognized only 1 or 2 hMBP peptides without dominance for a certain peptide. This result emphasizes the presence of B cells producing Abs against MBP in CSF of MS patients and shows the interest of studying mononuclear cells of CSF as a good marker of the pathogenesis.


Soluble interleukin-2 receptors in children with juvenile chronic arthritis.

Concentrations of interleukin-2 receptors were studied in sera of 81 patients with pauciarticular-, polyarticular-, and systemic-onset juvenile chronic arthritis using a double antibody "sandwich" enzyme-linked immunosorbent technique. Serum concentrations were significantly increased in all subgroups when compared with the healthy controls. Higher concentrations were found in systemic onset disease than in pauci- or polyarticular onset disease. Serum levels were found to be increased in both inactive and active disease, showing maximal values in patients with systemic features. A correlation to C-reactive protein, alpha 1-acid glycoprotein, and erythrocyte sedimentation was observed. Increased sIL-2R values suggest disease activity despite the absence of clinical symptoms.


Dissimilar biosynthesis of interleukin-6 by different areas of synovial membrane of patients with rheumatoid arthritis and osteoarthritis.

In this study the IL-6 production was studied by synovial cells isolated from patients with either rheumatoid arthritis (RA) or osteoarthritis (OA). The kinetics of spontaneous IL-6 production differs in both groups. Furthermore, the induction of IL-6 by bacterial lipopolysaccharide (LPS) in synovial cell cultures of RA is much more rapid than in those of OA patients. On the other hand, more PGE2 was detected in culture supernatants from synovial adherent cells of OA than in those of RA patients. We also compared the IL-6 production and the amount of IL-6 mRNA in fragments derived from the areas of synovial tissue showing macroscopic signs of intensive inflammation (area A), with those from relatively intact (area B) synovial sites. In synovial fragments, but not in isolated adherent cells at area A the in vitro IL-6 production starts earlier in RA than in OA. In the area A, significantly more CD14+, CD43+ and HLA-DR+ cells were detected than in the other compartment less involved in local inflammatory events.


[Therapy of arrhythmias in clinical practice].

The primary care physician should be cautious and restrictive in the treatment of cardiac arrhythmias and adhere to the well established modalities only. For supraventricular arrhythmias digitalis, verapamil, quinidine and betablocking agents are the drugs of choice. In atrial fibrillation oral anticoagulants and in some cases aspirin seem to be beneficial for the prevention of stroke in selected subgroups of patients. Criteria for their use are outlined in Tables 1 and 2. Ventricular arrhythmias in healthy persons generally should not be treated. In patients with coronary artery disease and severely compromised left ventricular function amiodarone is very effective for the treatment of complex ventricular arrhythmias. If other antiarrhythmics are to be used, the advice of a cardiologist is strongly recommended, as proarrhythmic effects are particularly common in severe cardiac conditions. Severely symptomatic patients with recurrent ventricular tachycardia and syncope should be offered invasive evaluation in order to determine effective medical or surgical treatment.


Therapeutic landscapes: medical issues in light of the new cultural geography.

Employing an expanded meaning of the concept of landscape taken from the 'new' cultural geography, this paper explores why certain places or situations are perceived to be therapeutic. Themes from both traditional and recent work in cultural geography are illustrated with examples from the literature of the social science of health care. The themes include man-environment relationships; humanist concepts such as sense of place and symbolic landscapes; structuralist concepts such as hegemony and territoriality; and blends of humanist concerns, structuralist concerns, and time geography. The intention of this broad overview is to bring some particularly useful concepts developed in cultural geography to the attention of social scientists interested in matters of health and to stimulate research along new lines.


Child developmental delay and socio-economic disadvantage in Australia: a longitudinal study.

Socio-economic inequalities in adult and child health in Australia have been an issue of national concern. While a large body of data has discussed adult health, there have been relatively few Australian reports of socio-economic inequalities in child health. This occurs in a context where there have been increases in the proportion of Australian children living in poverty and where there has been an increased interest in child developmental delay as an indicator of child health status. This paper reports the result of a longitudinal study of pregnancy outcomes and one indicator of child health, namely child developmental delay. Three indicators of socio-economic status (chronic socio-economic disadvantage, mother's education, family income) were used to predict child developmental delays observed some 5 1/2 years after the study commenced. Mothers who had the lowest socio-economic status (using any of the indicators) had substantially higher rates of children manifesting developmental delays.


'Culture' in culture-bound syndromes: the case of anorexia nervosa.

Anorexia nervosa is presently considered a Western culture-bound syndrome. A cultural focus on dieting and ideals of thinness for women are assumed to be implicated in the disorder. While research indicates that the majority of non-anorectic women in the United States are preoccupied with body weight and dieting, it is not clear what 'thinness' means to anorectics themselves or that norms about dieting are always involved in subjective experiences of anorexia. Meaning-centered studies of anorectics--especially those in non-clinical settings--are needed to clarify the cultural contexts of the disorder. Case studies of two anorectic women from Minneapolis-Saint Paul, Minnesota, show that for some anorectics self-starvation is encoded in religious idioms and symbols about the body, food, and self. A review of the literature illustrates a long-standing relation between self-starvation and religious ideals in Western culture and points to an association between contemporary anorexia nervosa and asceticism. The case studies presented here demonstrate that this asceticism may be subjectively expressed through religious concepts about the body and food and suggest that future research formally investigate the religious practices and beliefs of anorectics seen clinically. The author explores the implications of these findings for definitions of 'normality' and 'abnormality,' key issues in ethnopsychiatry. These findings also suggest that future cross-cultural research might examine asceticism about the body and food in religions other than Judeo-Christian, cultural groups with rituals of fasting and vomiting, and the presence of fundamentalist churches and missionaries in those non-Western cultures for which there are recent reports of eating disorders. Anorexia nervosa's designation as a syndrome limited to Western cultures or to those cultures influenced by them may reflect unexamined assumptions on the part of researchers that dieting and secular ideals of slimness are primarily involved in the disorder.


Human platelets deficient in dense granules contain normal amounts of pp60c-src.

We have determined that pp60c-src, a protein tyrosine kinase abundant in normal platelets, is present at comparable levels in platelets that are deficient in dense granules (Hermansky-Pudlak syndrome). Relative quantitation of pp60c-src was performed by immunoblot analysis after protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our data suggest that human platelet dense granules, unlike chromaffin cell secretory granules, are not the major intracellular site of localization of pp60c-src.


The effect of FK506 treatment on pancreaticoduodenal allotransplantation in the primate.

The effect of FK506 monotherapy on pancreaticoduodenal allotransplantation was studied in a primate model. The recipients were made diabetic by total pancreatectomy, thus glycemic control depended on the graft. In control animals hyperglycemia occurred after 14 +/- 3 days and the animals died after 36 +/- 15 days. For FK506-treated animals, the time until development of hyperglycemia was 60 +/- 12 days (P less than 0.05). The animals were then sacrificed by day 90 (P less than 0.05). All three control animals lost their graft function because of severe rejection, as verified by postmortem examination. Only one of the five treated animals showed evidence of a moderate-to-severe rejection at 90 days, one animal having a mild rejection as judged by the histological findings. The drug was clinically well tolerated in all except one animal that became apathetic and refused to eat. Hyperglycemia and an elevated serum creatinine level, which were probably related to the FK506 treatment, occurred in one animal each. Both of these side-effects were reversed by reducing the dose. Thus the use of FK506 permits successful single pancreatic transplantation in primates.


Prediction by postrevascularization biopsies of cadaveric kidney allografts of rejection, graft loss, and preservation nephropathy.

This prospective study of postrevascularization biopsies was undertaken to determine if pathological changes might be correlated with subsequent allograft rejection and loss. Such a relationship, if identified, could be used to predict graft outcome, thus permitting earlier intervention for individuals at an increased risk for rejection or graft loss. Fifty-seven biopsies were obtained, and the number of polymorphonuclear leukocytes marginating in the glomerular loops and peritubular capillaries was documented along with risk factors associated with the recipients' immunological status and with risk factors associated with ischemic preservation injury. The presence of seven PMN leukocytes in the peritubular capillaries is related to the subsequent occurrence of cellular rejection and accurately predicted in 82% of the patients studied whether or not rejection would occur. Mean glomerular PMN leukocyte count was related to cold ischemia time and subsequent graft loss, while an elevated mean glomerular PMN leukocyte count in conjunction with an elevated peritubular PMN leukocyte count was always associated with hyperacute rejection. Focal glomerular thrombosis (less than 50%) and tubular cast formation are manifestations of preservation nephropathy and had no effect on graft outcome. These findings suggest that the peritubular capillaries are a more sensitive target for immune changes and that minor donor/recipient disparities can be detected in the peritubular capillaries while preexisting sensitization to the donor is reflected by concurrent changes in the glomerular and peritubular capillaries.


A prospective, randomized, blind comparison of three biopsy techniques in the management of patients after renal transplantation.

Although conventional histology (CH) of needle core biopsies has been accepted as the gold standard for diagnosis of renal allograft rejection, this assumption has never been tested. Fine-needle aspiration cytology (FNAC) and monoclonal antibody (panleukocyte) staining of needle core biopsies (MABS) have been suggested to be superior to CH. A total of 50 patients received a cadaveric renal transplant followed by immunosuppression with triple therapy. Biopsies were taken routinely at days 7,14,21,28, and 90, with additional biopsies taken between these times if rejection was suspected (total biopsy sessions = 219). Specimens were taken for CH, FNAC, and MABS at each biopsy session, but only the result of one technique (previously randomly allocated) was communicated back to the clinical team, using a standardized grading system. Subsequently the presence or absence of rejection was determined by retrospective analysis of the clinical and biochemical course by 4 clinicians, without reference to the biopsy result. Graft survival was not significantly different in the three groups. The sensitivities for CH, FNAC, and MABS were 75%, 59%, and 77%, respectively, while the specificities were 87%, 96%, and 80%, respectively. Inadequate samples for analysis occurred frequently with the MABS technique--and, to a lesser extent, with CH--and both techniques tended to overdiagnose rejection. FNAC most often gave an answer but did miss clinically important rejection episodes. Needle-core biopsy processed for CH remains the most reliable biopsy technique for the diagnosis of rejection of renal allografts. FNAC is a useful technique for monitoring grafts with stable function or nonfunction. MABS does provide information equivalent to CH, but, in this study, had a high incidence of inadequate samples.


Evidence that changes in expression of major histocompatibility complex antigens may underlie the immunosuppressive effect of heat-treated cells in vivo and in vitro.

The prior transfusion of heat-treated (60 degrees C for 1 hr) allogeneic spleen cells is known to bring about specific prolongation of the survival of subsequent donor strain heart allografts. In this communication we show that some polymorphic and monomorphic class 1 determinants on spleen cells are heat denatured so that they no longer provoke antibody formation in naive allogeneic hosts. By contrast, the heated cells remain able to provoke the formation of anti-class 2 antibodies. When measured in a binding assay, the levels of anti-class 2 antibodies are similar irrespective of whether the immunizing inoculum consists of normal or heated cells. In a cytotoxic assay, the antibodies produced following exposure to normal cells are cytotoxic; this activity is substantially reduced when the cellular immunizing inoculum is heated. Cells heated to 60 degrees C for 1 hr are unable to stimulate in a mixed lymphocyte reaction, but reactivity can be partially restored by the addition of exogenous IL-2 to the culture. From previous evidence it seems unlikely that suppressor cells play a major role in the immunosuppression effected by cells heated to 60 degrees C. The results presented in this communication suggest a possible role for anticlass 2 antibodies and also imply the defective production of a costimulatory signal that normally follows the presentation of allogeneic MHC antigens.


HIV-1 genomic RNA diversification following sexual and parenteral virus transmission.

Human immunodeficiency virus type 1 (HIV-1) genomic RNA variation was studied in seven presumed donor-recipient pairs directly following sexual (6/7) or parenteral (1/7) transmission. The first RNA-positive serum sample of each recipient and the serum sample of the virus transmitter, identified by epidemiological history and taken within a time bracket of three months of the recipient seroconversion, were analyzed by polymerase chain reaction amplification followed by sequencing of eight cDNA clones of 276 bp, including the V3 coding region. The sequence populations of the recipients were without exception homogeneous, while the sequence populations of the transmitters showed varying degrees of heterogeneity. Nucleotide distance between consensus sequences of unrelated individuals from the Amsterdam population (interpatient variation) averaged 11% (range 7-15%). The largest distance between two clonal sequences of one individual (intrapatient variation) was also 11%. Consensus sequences of five recipients differed by only 0-1% from the consensus sequence of the presumed transmitter, including two pairs of which the transmission was either proven or highly probable. This contrasted with a difference of 10-12% in two pairs, casting doubt on the epidemiological relatedness. Antibody reactivity to a panel of V3 peptides with varying degrees of similarity to the V3 sequences obtained did not augment the discriminatory power of sequence analysis. Results of the sequential sequencing of samples of one transmitter suggest that this was due to an anamnestic antibody response of the transmitter to early variants. From the loss of sequence heterogeneity following transmission and the consensus sequence similarities observed within five transmitter-recipient pairs, we conclude that HIV-1 transmission results in the selection of a limited number of genomes carrying on the infection in the new host, but does not generally lead to a shift in the sequence population as defined by the consensus sequence.


Structural determination of gangliosides that bind to influenza A, B, and C viruses by an improved binding assay: strain-specific receptor epitopes in sialo-sugar chains.

An improved binding assay for detection of ganglioside receptors for influenza A, B, and C viruses was developed. In this system, the virions bound to gangliosides that were developed on a silica gel thin-layer plate were detected by mouse monoclonal antibody against viral hemagglutinin and peroxidase-conjugated anti-mouse immunoglobin. No hydrolysis of the gangliosides by viral receptor-destroying enzyme was detected in the present condition. The reactivity of the viruses to gangliosides depended on the amount of developed gangliosides (10 pmols-10 nmols), the molecular species of sialic acid, and their sugar sequences. Human influenza A (PR/8/34), B (Lee/40), and C (Ann Arbor/1/50) viruses bound different receptor epitopes of sialo-sugar chains of gangliosides. The A/PR/8 virus bound most effectively to Neu5Ac-containing lacto-series gangliosides carrying type I and type II sugar chains, followed by ganglio-series and hematoside-series gangliosides. The A/PR/8 virus weakly bound to Neu5Ac alpha 2,6lactotetraosylceramide [IV6(Neu5Ac)Lc4Cer] and Neu5Ac alpha 2,6paragloboside [IV6(Neu5Ac)nLc4Cer] carrying Neu5Ac alpha 2,6Gal sequence, although their Neu5Ac alpha 2,3Gal derivatives were the most potent gangliosides tested. B/Lee/40 bound restrictively to IV6(Neu5Ac)Lc4Cer and IV6(Neu5Ac)nLc4Cer, which carry Neu5Ac alpha 2,6Gal sequence, and type I and type II lacto-series sugar chain, respectively. C/Ann Arbor/1/50 reacted only with 9-O-Ac-Neu5Ac-carrying sugar chains in all the gangliosides tested. This method also allowed the microanalysis of receptor gangliosides of unknown samples. ESK cells, sensitive to the influenza A viruses infection, expressed several kinds of receptor active gangliosides, while those from ESK-R cells, resistant to the virus infection, were undetectable.


Mouse hepatitis virus nucleocapsid protein-specific cytotoxic T lymphocytes are Ld restricted and specific for the carboxy terminus.

Infection of mice with the JHM strain of mouse hepatitis virus (MHV) results in an acute encephalomyelitis associated with primary demyelination of the central nervous system. Efforts at understanding the components of the immune response in the development of chronic MHV-induced demyelination have implicated the antibody response and both the CD4+ and CD8+ T cell responses. In this report, we demonstrate that Balb/c (H-2d) mice immunized with the JHM (JHMV) strain of MHV develop a CD8+ cytotoxic T lymphocyte (CTL) response. One population of these virus-specific CTL recognize the nucleocapsid (N) protein. Recombinant vaccinia viruses expressing either the entire N protein or carboxy-terminal deletions were used to determine the number and location of the epitope(s) recognized. The CTLs were found to recognize a peptide contained within the carboxy-terminal 149 amino acids of the N protein. Analysis of infected cell lines expressing transfected major histocompatibility genes demonstrated that the anti-N protein CTLs were restricted exclusively to the Ld molecule. These data provide the first definition of a MHV-specific CTL response directed to a viral protein and suggest that the anti-N protein CTL response is one potential mechanism used by the host to clear JHMV from the central nervous system.


Flow cytometric analysis of African swine fever virus-induced plasma membrane proteins and their humoral immune response in infected pigs.

African swine fever (ASF) virus-induced plasma membrane proteins may contribute to the protective immune response against the disease since they can be involved in the antibody-mediated lysis of infected cells. In this study we describe the regulation of ASF virus-induced plasma membrane protein expression and its antibody induction in pigs after viral infection by flow cytometric analysis. More than 80% of infected cells contained viral antigens on the surface membranes at 6 hr postinfection (hpi), and the relative amount of viral antigen expression was increased at 12 and 20 hpi. The kinetics of individual viral protein expression on cell surfaces was studied by a collection of monospecific antibodies directed against the six viral plasma membrane proteins p12, p15, p16, p23.5, p30, and p35. Most of these proteins were expressed at 6 hpi, with the exception of p35, which was first detected at 12 hpi. The percentage of cells expressing each antigen at different hpi was also determined. The immune response against virus-induced plasma membrane proteins in pigs infected with an attenuated ASF virus strain was studied. Antibodies against viral epitopes exposed on plasma membranes reached a plateau at 20 days postinfection (dpi). The relative amount of antibodies induced during infection with these specificities was not directly related to the antibody titer of the sera. Sera obtained at 20 and 40 dpi contained antibodies against most of the viral plasma membrane proteins and were most efficient in recognition of viral antigens exposed on the surface of infected cells at early times.


Sulfated polymers inhibit the interaction of human cytomegalovirus with cell surface heparan sulfate.

Several sulfated polysaccharides (dextran sulfate, pentosan polysulfate, heparin) and copolymers of acrylic acid with vinylalcohol sulfate have proved to be potent inhibitors of human cytomegalovirus (CMV) infectivity in vitro. Sulfated alpha-cyclodextrins are only weak inhibitors of CMV. A close correlation was found between the 50% inhibitory concentrations of the sulfated polymers for CMV cytopathogenicity, virus-cell binding, and expression of immediate early antigens (IEA) in human embryonic lung (HEL) cells. CMV particles bound specifically to heparin-Sepharose. Sulfated polymers specifically eluted the virus particles from this matrix. Enzymatic digestion of cell surface heparan sulfate, but not of chondroitin sulfate, prevented the cells from being infected with CMV. Moreover, radiolabeled CMV bound efficiently to, and were infective for wild-type Chinese hamster ovary (CHO) cells, whereas virus binding to, and infection of, mutant CHO cell lines that were deficient in either all glycosaminoglycans or heparan sulfate only was significantly impaired. The mechanism of action of the sulfated polymers can be attributed to an inhibitory effect on the binding of CMV particles to the host cells. Presumably, the sulfated polymers interact with the viral envelope site(s) involved in the attachment of the CMV virions to cell surface heparan sulfate.


Increased synthesis of polycistronic mRNA associated with increased polyadenylation by vesicular stomatitis virus.

Electron microscopy suggested that the mRNA produced in vitro by tsG16(I), a temperature-sensitive mutant of vesicular stomatitis virus, contained an increased proportion of polycistronic mRNAs. Using hybrid selection, we found that the poly(A)+ mRNA synthesized in vitro by tsG16(I) contained approximately two to three times more polycistronic mRNA than did poly(A)+ mRNA synthesized in vitro by the parental wild-type (wt) virus. The increase in polycistronic mRNA occurred at all intergenic junctions examined. In vitro, tsG16(I) has an increased polyadenylation phenotype and a temperature-sensitive transcriptase activity that appear to be due to different mutations. Partial revertants of tsG16(I), which have lost the aberrant polyadenylation phenotype but retain the in vitro thermosensitive transcriptase, produced wt amounts of polycistronic mRNA. This suggested that the increased production of polycistronic mRNA by tsG16(I) may be associated with the increased polyadenylation phenotype of this mutant. These data further support the hypothesis that an increase in size of poly(A) tracts is associated with increased production of polycistronic mRNA.


Location of neutralizing epitopes on the hemagglutinin-esterase protein of influenza C virus.

Neutralization-resistant variants of influenza C/Ann Arbor/1/50 virus were selected with monoclonal antibodies against four different antigenic sites on the hemagglutinin-esterase (HE) glycoprotein, and their HE genes were sequenced to identify amino acid residues important for the integrity of each site. Twelve different amino acid substitutions in a total of 18 antigenic variants were all located on the HE1 subunit. Although variants for antigenic site A-2 had a change at position 367, all substitutions in the variants for sites A-1, A-3, and A-4 occurred in the central region of the HE1 spanning amino acid positions 178 to 283. Furthermore, it was found that many of the substitutions in the variants selected with antibodies to sites A-1 and A-3 were clustered within or near one of the three variable regions revealed previously by comparing amino acid sequences of the HEs among various influenza C isolates (Buonagurio, D. A., Nakada, S., Fitch, W. M., and Palese, P., Virology 146, 221-232, 1985). The antigenic variants were also examined for their ability to agglutinate chicken and human erythrocytes in order to obtain information concerning the receptor-binding site on the HE molecule. The results suggested that the amino acid changes at residues 178, 186, 187, 190, 206, 212, and 226 decreased the hemagglutinating activity whereas those at residues 245, 266, and 283 produced an opposite effect.


National Ambulatory Medical Care Survey: 1989 summary.

Based on data collected from a national sample of office-based physicians, statistics are presented on the provision and utilization of ambulatory medical care services in physicians' offices during 1989. Ambulatory medical care services are described in terms of patient characteristics, physician practice characteristics, and visit characteristics. A summary of trends in office-based ambulatory medical care from 1975-89 is included.


[Locoregional chemotherapy in the treatment of liver metastasis of breast carcinoma].

Locoregional chemotherapy enables specific treatment of organ metastases by using a dosage of medication that is maximally effective but only has minimal side effects on other organs. Since 1983, we have applied this procedure in patients whose liver represented the only target-organ of metastases. Between 1983 and 1990, locoregional chemotherapy was performed in 59 women suffering from liver metastases of breast cancer. The average age of our patients was 52 years. 4 patients were treated for solitary metastases that could not be resected, in 5 patients the infiltration of hepatic parenchyma with metastases accounted to 25%, in 32 patients hepatic infiltration ranged between 25% and 75% and in 18 patients it surpassed 75%. In 39% of our patients, a partial remission occurred. The mean period of survival after beginning of treatment was 149 days, whereas the longest survival time lasted 1009 days. We conclude that the locoregional chemotherapy, destined for treatment of liver-metastases of breast cancer, with a mean survival time of only five months could insufficiently satisfy our expectations. Therefore, a decision to administer such palliative therapy should be made on an individual basis. In our opinion, one indication represents solitary metastases that are not resectable, a further indication is pain due to expansion of the liver capsule because of diffuse metastases.


[What therapeutic benefits are there in the early diagnosis of metastases in patients with breast carcinoma?].

Early detection and curative treatment of metastasis of breast cancer is seldom. Local surgery and radiation could be helpful. The survival time was sometimes not a psychological advantage for the patients with early detection of metastasis.


Contribution of cell culture to the study of the physiology of the stria vascularis and Reissner's membrane.

Cell culture is a well-established tool in cell biology which may overcome the limitations of the experimental methods currently used to investigate the physiology of labyrinthine fluids. Using explant and dissociated cell culture technique, sheets of oriented cells derived from the stria vascularis and Reissner's membrane may be obtained, which allows new experimental approaches such as the patch-clamp technique. Other experimental approaches are considered which might improve our understanding of inner ear physiology.


Alterations of spinal dorsal horn substance P following electroacupuncture analgesia--a study of the formalin test with immunohistochemistry and densitometry.

Substance P (SP), released from thin afferent terminals, is believed to be a neurotransmitter for pain transmission in the spinal dorsal horn. It has been demonstrated that in addition to analgesia, morphine increases the accumulation of SP possibly due to the inhibition of its release. The present work investigated the level of spinal SP like immunoreactivity (SPLI) following electroacupuncture analgesia in rats using immunohistochemistry and image analysis. Experiment results revealed that formalin injected into the hind paw elicited marked pain response and accumulation of SP in the spinal dorsal horn. Electroacupuncture of Tsu-San-Li could depress the pain response, however increasing further the SP accumulation. It is thus suggested that pain stimulation itself may activate the endogenous opioid mechanism to inhibit SP release and acupuncture is able to enhance the process. This may be one mechanism of acupuncture analgesia.


Topics in clinical pharmacology: filgrastim, a myeloid colony stimulating factor.

Filgrastim (granulocyte colony stimulating factor) recently became commercially available for the treatment of chemotherapy-induced neutropenia. Studies have shown that filgrastim induces a dose-dependent granulocytosis in humans, thereby shortening the period of neutropenia in patients treated conventionally with submarrow ablative doses of chemotherapy, as well as with marrow ablative therapy given in the bone marrow transplant setting. By reducing the incidence and severity of infections and mucositis in patients treated with chemotherapy, it has a significant economic impact since it shortens the duration of antibiotic administration and hospitalization. Adverse reactions reported are limited to mild to moderate bone pain. Several other potential applications are being investigated for filgrastim, including treatment of patients with myelodysplastic syndrome and congenital neutropenia.


Monosomy 18q12.1----21.1: a recognizable aneuploidy syndrome? Report of a patient and review of the literature.

This report of a patient with an interstitial deletion 18q and review of previously described cases suggest a clinically recognizable syndrome. The phenotype appears to result from a microdeletion of part of 18q12.2 or q12.3, or a deletion of parts of both bands.


New variant in exon 3 of the proteolipid protein (PLP) gene in a family with Pelizaeus-Merzbacher disease.

A C--greater than G transversion has been found in exon 3 of the PLP gene of affected males and their mother in a single sibship with Pelizaeus-merzbacher disease (PMD). The transversion should not result in an amino acid change in the protein but it does result in the loss of a HaeIII restriction endonuclease cleavage site. It is concordant with the disease in this family. One-hundred-ten unrelated X chromosomes are negative for this mutation. No other sequence defect was found in the PLP exons of the affected males. The cause of disease in this family remains unknown, but the association between this rare mutation and PMD is intriguing. The mutation can serve as a marker for following segregation of the PLP gene.


Immunolocalization of a glycosylphosphatidylinositol-specific phospholipase D in mast cells found in normal tissue and neurofibromatosis lesions.

A large number of eukaryotic proteins have been shown to be anchored to the cell membrane by glycosylphosphatidylinositol (GPI). This glycolipid anchor can serve as a substrate for anchor-specific phospholipases that convert the GPI-anchored membrane proteins into soluble forms. Soluble forms of many GPI anchored proteins have been identified in vivo in connective tissue, plasma, and urine. The authors have discovered that mammalian plasma contains a GPI-specific phospholipase D (GPI-PLD). Because it recognizes a portion of the conserved glycan core structure, all GPI-anchored proteins are potential substrates. The authors report the development of a murine monoclonal antibody specific for one form of the human GPI-PLD and the immunohistochemical localization of this enzyme to mast cells.


Post-transplant recurrent hepatitis B viral liver disease. Viral-burden, steatoviral, and fibroviral hepatitis B.

Recurrence of hepatitis is a well-documented complication of hepatitis B liver disease, post-transplantation. It is well established also that the earliest hepatocellular change is the appearance of hepatitis B viral (HBV) markers and that the disease is rapidly progressive. In this article on 17 liver transplants in 16 HBV positive patients with long-term follow-ups (100-1234 days), the distinctive pathologic features of this disease are emphasized: the extreme viral load, the steatosis, and/or fibrosis. An attempt to quantitate the magnitude of the viral burden was made and the result was a staggering figure. In one patient, an estimated 10(18) HBV core particles were present in the liver. One of two patterns of progression were noted. In four patients in addition to the massive nuclear hepatitis B core antigen (HBcAg) and cytoplasmic hepatitis B surface antigen (HBsAg) positivity, superimposed hepatitic changes led to diffuse hepatic fibrosis (fibroviral hepatitis B); and in another six patients, extraordinary hepatocellular viral marker positivity and steatosis were the hallmarks (steatoviral hepatitis B). Steatosis is not usually considered a feature of HBV liver pathology. These results suggest that more than one type of posttransfusion recurrent hepatitis B liver disease exists pathologically.