Comparison of the N-linked oligosaccharide structures of the two major human myelin glycoproteins MAG and P0: assessment and relative occurrence of oligosaccharide structures by serial lectin affinity chromatography of 14C-glycopeptides.

The N-linked oligosaccharide structures of human myelin-associated glycoprotein (MAG) and P0 have been characterized by serial lectin affinity chromatography (SLAC) of 14C-glycopeptides. 14C-Glycopeptides were prepared from purified MAG derivative and P0 by extensive proteolytic digestion and N-14C-acetylation. Assuming that all the 14C-glycopeptides were radiolabelled to the same specific radioactivity, the relative occurrence of the oligosaccharide structures was correlated to the amount of incorporated radioactivity. Sixteen and 15 fractions were generated by SLAC of MAG and P0 14C-glycopeptides, respectively. Despite this tremendous structural heterogeneity, the oligosaccharide "fingerprints" of MAG and P0 obtained by SLAC displayed similarities: (a) of the three types of N-linked oligosaccharides, the complex type accounted for 80.4% and 94.9% of MAG and P0 radioactivity, respectively; (b) biantennary complex oligosaccharides were the major structures present on MAG and P0; (c) approximately 60% of MAG and P0 oligosaccharides possessed a bisecting N-acetylglucosamine residue; and (d) large amounts of oligosaccharides with an alpha(1-6)fucose residue were found in both MAG and P0 and, noticeably, approximately 25% of the tri- and/or tetraantennary and approximately 90% of the bisected biantennary oligosaccharides of both glycoproteins contained alpha(1-6)fucose residues in the core. This study demonstrates that MAG and P0, both belonging to the immunoglobulin superfamily, display structural similarities in their N-linked oligosaccharide contents.


Comparison of the N-linked oligosaccharide structures of the two major human myelin glycoproteins MAG and P0: assessment of the structures bearing the epitope for HNK-1 and human monoclonal immunoglobulin M found in demyelinating neuropathy.

The epitope for HNK-1 and patient's monoclonal autoantibodies in demyelinating polyneuropathy associated with immunoglobulin M gammopathy is borne by different types of N-linked oligosaccharide structures in human P0 and myelin-associated glycoprotein (MAG). Fourteen glycopeptide fractions bearing different oligosaccharide structures were obtained from either MAG or P0 glycopeptides by serial lectin affinity chromatography on concanavalin A-Sepharose, Phaseolus vulgaris erythrophytohemagglutinin-agarose, Pisum sativum agglutinin-agarose, and Phaseolus vulgaris leucophytohemagglutinin-agarose. As shown by dot-TLC plate immunostaining, the same MAG and P0 glycopeptide fractions were recognized by HNK-1 and patient's immunoglobulin M, confirming that these antibodies display similar specificities. The antigenic carbohydrate was present in glycopeptide fractions that either interact with Pisum sativum agglutinin-agarose or were bound by Aleuria aurantia agglutinin-digoxigenin, indicating that these structures contained alpha(1-6)fucose residues. This study demonstrates that the L2/HNK-1 epitope is borne mainly or even exclusively by N-linked oligosaccharide structures alpha(1-6)fucosylated in the core.


c-fos expression as a model for studying the action of hexachlorocyclohexane isomers in the CNS.

The induction of protooncogene c-fos in the CNS after administration of several convulsants has been studied. The organochlorine insecticide gamma-hexachlorocyclohexane (gamma-HCH, lindane) has been shown to induce c-fos expression in different brain areas. Pentylenetetrazole and picrotoxin, a known gamma-aminobutyric acid-receptor antagonist, have also been considered. The administration of two nonconvulsant isomers of gamma-HCH, alpha-HCH, and delta-HCH, before the mentioned toxicants, affects the protooncogene expression in different ways. The differential pattern of expression displayed by c-fos after these treatments suggests the presence of diverse mechanisms of action for the compounds studied.


In vivo neurochemical evaluation of striatal serotonergic hyperinnervation in rats depleted of dopamine at infancy.

Destruction of nigrostriatal dopamine (DA) neurons with 6-hydroxydopamine (6-OHDA) early in development results in hyperinnervation of striatum by the serotonergic afferents deriving from the dorsal raphe nucleus. We have used in vivo microdialysis to investigate the degree to which serotonergic neurotransmission in striatum is altered by this increase in the density of serotonin (5-HT) terminals. The effects of several manipulations known to influence 5-HT function on extracellular 5-HT and 5-hydroxyindoleacetic acid in striatum were compared in adult rats treated neonatally with 6-OHDA and in intact adult rats. Basal levels of 5-HT in extracellular fluid (ECF) of striatum were similar in neonatally DA-depleted rats and in intact rats. Perfusion with the 5-HT reuptake blocker, fluoxetine (100 microM), increased 5-HT in striatal ECF of neonatally DA-depleted rats to levels that were threefold greater than those achieved in intact rats. Likewise, K(+)-depolarization of the 5-HT terminals (100 mM in perfusate) or systemic administration of the 5-HT releaser, (+/-)-fenfluramine (10 mg/kg i.p.), increased the concentration of 5-HT in striatal ECF of neonatally DA-depleted rats to levels approximately threefold greater than those observed in striatum of intact rats. These findings indicate that the 5-HT hyperinnervation of striatum that takes place in rats depleted of DA at infancy is associated with an increased capacity for neurotransmitter release in this system. Concomitant increased in high-affinity 5-HT uptake may prevent the occurrence of any measurable changes in the resting concentration of 5-HT in striatal ECF.


Tau 2: a probe for a Ser conformation in the amino terminus of tau.

We have determined the epitope for Tau 2, a monoclonal antibody that intensely stained tangles, plaque neurites, and curly fibers in the tissue section, and strongly labeled bovine tau, but only very weakly labeled human tau on the blot. The epitope has been localized to Ala95 through Ala108 of bovine tau. Ser101 is critical for Tau 2 reactivities; the replacement of Ser by Pro, which is found in rat, mouse, and human tau, brings about very weak Tau 2 reactivities. The strong Tau 2 staining of tangles and its effective absorption with a synthetic Ser peptide (Ala95 through Ala108) suggest that the tau in paired helical filaments takes a Ser conformation, rather than a Pro conformation, in its amino-terminal portion.


Detection of multisulphated N-linked glycans in the L2/HNK-1 carbohydrate epitope expressing neural adhesion molecule P0.

P0, the most abundant glycoprotein of PNS myelin, is a homophilic and heterophilic adhesion molecule. P0 is known to contain a glycoform population that expresses the L2/HNK-1 carbohydrate epitope found on other neural adhesion molecules, and to be functionally implicated centrally in neural cell adhesion and neurite outgrowth. This carbohydrate epitope has been characterized previously from glycolipid structures and contains a sulphated glucuronic acid residue. However, the L2/HNK-1 carbohydrate epitope has not been characterized in glycoproteins. Because P0 possesses only one glycosylation sequon, the number of P0 glycoforms is equal to the heterogeneity of the glycan species. Here we report that the carbohydrate analysis of L2/HNK-1-reactive P0 showed the presence of anionic structures containing sialic acid and sulphate in various combinations. At least one sulphate residue was present in 80% of the monosaccharide sequences, and 20% contained three sulphates. High-resolution P4 gel chromatography of the desialylated and desulphated oligosaccharides showed substantial heterogeneity of monosaccharide sequences. Sequential exoglycosidase digestions indicated that the majority of the structures were of the hybrid class, although the sulphated structures were found to be endoglycosidase H-resistant.


Immunocytochemical localization of the L1 and N-CAM cell adhesion molecules and their shared carbohydrate epitope L2/HNK-1 in the developing and differentiated gustatory papillae of the mouse tongue.

The localization of the cell adhesion molecules L1 and N-CAM, and their shared carbohydrate epitope L2/HNK-1, was investigated at the light and electron microscopic levels in developing and adult fungiform and circumvallate gustatory papillae of the mouse tongue. At embryonic day 13, the earliest stage investigated, the tongue epithelium was still undifferentiated and was not yet innervated by sensory fibres. At this stage none of the three molecules was detectable within the tongue epithelium. At embryonic day 15 the primordia of the gustatory papilla became unequivocally discernible when the papillary epithelium was already innervated by few sensory axons. At this stage N-CAM was the first molecule expressed on epithelial cells and was confined to those parts of the papillary epithelium destined to become the chemosensory cells of the taste buds. The sensory axons were N-CAM-, L1- and L2/HNK-1-positive when fasciculating or contacting their accompanying Schwann cells or the cells of the papillary epithelium. Contacts between Schwann cells were also prominently labelled by antibodies to the three antigens. The mesenchymal tissue underlying the prospective sensory epithelium expressed N-CAM at all embryonic stages, but ceased to be N-CAM positive within the first six postnatal days. From embryonic day 16 onward a weak L1 immunoreactivity was detectable within the basal and intermediate layers of the lingual epithelium and remained present in adulthood. Cytodifferentiation of epithelial cells into spindle-shaped sensory cells and organization into taste buds began at postnatal day two. Simultaneously, L1 and L2/HNK-1 immunoreactivity increased on taste bud cells and N-CAM disappeared from the non-sensory extragemmal parts of the papillary epithelium. At approximately postnatal day six, taste bud formation was complete and the pattern of cell adhesion molecule expression was comparable to that found in the adult in that L1 was strongly expressed on the apposing surfaces of all cells, whereas N-CAM was confined to cell contacts between a subpopulation of intragemmal cells. The L2/HNK-1 epitope was visible on the surfaces of taste bud cells, on intragemmal axons, and in a small portion of extracellular matrix directly underlying the taste buds, but was no longer expressed on those parts of the sensory fibres embedded in the subepithelial mesenchyme. The L2/HNK-1 epitope may thus be regarded as a cell surface marker for the cellular elements of mature taste buds. The highly sialylated form of N-CAM was not detectable at any stage investigated.(ABSTRACT TRUNCATED AT 400 WORDS)


Cysteine proteinases from Schistosoma haematobium adult worms.

To identify and characterize cysteine proteinases from Schistosoma haematobium, lyophilized adult worms were homogenized, and enzymes were isolated and purified. From extracts prepared in acidic buffer, 3 putative cysteine proteinases were identified either directly or indirectly. The first proteinase (ShCP1) was identified by labeling with a radioiodinated inhibitor, Z-Tyr-Ala-CHN2, as a 35-kDa protein. However, it could not be detected by silver staining, amino acid sequencing, or by a monoclonal antibody specific for a similar molecule from Schistosoma mansoni. A second cysteine proteinase, ShCP2, was purified by gel filtration and dialysis. This 32-kDa molecule was thiol-dependent and was labeled with Z-Tyr-Ala-CHN2. The amino terminal amino acid sequence of ShCP2 showed remarkable similarity (up to 77%) to that of S. mansoni cathepsin B (SmCP2) as well as to mammalian cysteine proteinases. Both ShCP1 and ShCP2 reacted with polyclonal antibodies against S. mansoni, suggesting the existence of shared antigenic epitopes. A third activity, ShCP3, was identified as possibly a distinct proteinase based on its similarities to a 28-kDa cysteine proteinase from S. mansoni. This preliminary investigation demonstrates that the overall profile of cysteine proteinases in S. haematobium is very similar to that of S. mansoni.


Nonspecific oral immunity in individuals with HIV infection.

Lactoferrin, lysozyme, interferon, and neopterin levels were determined in parotid saliva from 44 individuals with different clinical stages of human immunodeficiency virus (HIV) infection and 19 HIV-seronegative controls. The secretory output of individual components was calculated according to the fluid flow rate. No parotid interferon activity was found in any of the HIV-infected subjects or controls, and no significant differences in parotid lysozyme or neopterin outputs were observed. The lactoferrin output was significantly decreased in HIV-seropositive subjects in parallel with their markedly reduced parotid secretory IgA output. This combined deficiency of parotid lactoferrin and secretory IgA may well contribute to the frequent oral infections seen in subjects with HIV infection.


Cyclosporine and FK506 inhibition of murine mast cell cytokine production.

The ability of cyclosporine (CSA) and FK506 to inhibit cytokine production by factor-dependent murine mast cell lines was investigated. The mast cell clone, MC/9, and two mast cell lines, MCIII and MCVI, were stimulated to produce cytokines with phorbol myristate acetate plus the calcium ionophore A23187. The production of cytokines by stimulated mast cells cultured in the presence or absence of drug was monitored by bioassay of culture supernatants for induction of proliferation by factor-dependent cell lines and inhibition of these responses by neutralizing monoclonal antibodies. Both CSA and FK506 inhibited mast cell cytokine production at concentrations comparable to those observed with T cells. However, the degree of inhibition of cytokine production varied among the mast cell lines as well as between different cytokines produced by a given mast cell line. For example, CSA completely inhibited interleukin-2 (IL-2), IL-3, IL-4 and granulocyte-macrophage colony stimulating factor secretion by all three lines, with the exception that IL-2/IL-4 production by MCIII was partially resistant to inhibition by CSA. Similarly, FK506 completely inhibited cytokine production by MC/9, partially inhibited cytokine production by MCIII and had differential effects on IL-3/granulocyte-macrophage colony-stimulating factor and IL-2/IL-4 production by MCVI. Consistent with their ability to selectively inhibit cytokine gene transcription in T cells, neither CSA nor FK506 inhibited factor-dependent proliferation by these mast cell lines. In view of the putative role of cytokines in inflammation and late phase asthmatic reactions, these observations may be of particular significance in development of methods of pharmacologic intervention.


Relief of experimental spasticity and anxiolytic/anticonvulsant actions of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline.

Spasticity is characterized by pathological overactivity in spinal stretch reflex circuits and may be associated with disturbances in excitatory amino acid-mediated transmission in the cord. A genetically determined syndrome of spasticity in the rat permits the quantitative evaluation of the antispastic effects of drugs by recording activity in the electromyogram (EMG) from a hind limb extensor muscle. In genetically spastic rats, systemic administration of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F) quinoxaline (NBQX), normalized pathologically increased EMG activity, whereas the AMPA agonist, alpha-amino-3-hydroxy-5-tertbutyl-4-isoxazolepropionate (ATPA), exacerbated the EMG measures of spasticity. The reflex mechanisms in the spinal cord can be studied in mice using EMG recordings from the tibial muscle (Hoffmann reflex) or from the plantar foot muscle (flexor reflex) after electrical stimulation of the tibial nerve. Systemic and i.t. administration of NBQX blocked Hoffmann reflexes in mice, leaving flexor reflexes unchanged. ATPA enhanced Hoffmann, and had no effect on flexor reflexes. The effects of NBQX on spinal reflexes were seen in doses which do not affect locomotor activity, but show anxiolytic and some antiepileptic activity in rodents. These data suggest that the design of novel muscle relaxant drugs acting at the AMPA subtype of glutamate receptors may be feasible.


Botulinum C2 toxin and steroid production in adrenal Y-1 cells: the role of microfilaments in the toxin-induced increase in steroid release.

Exposure of adrenal Y-1 cells to C2 toxin results in an increase in steroid release that is accompanied by a rounding of the cell. The actions of C2 toxin mimic those of adrenocorticotropin and cholera toxin except that there is no increase in intracellular cyclic AMP content. In the present study we provide evidence that C2 toxin increases steroid output from Y-1 cells through an alteration in the microfilament network of the cell. C2 toxin significantly increased steroid output after 3 hr of exposure. This effect was accompanied by a significant increase in the transport of [3H]cholesterol to the mitochondrial fraction, independent of cholesterol uptake by the cell. The toxin was unable to increase steroid output from cells prerounded in suspension culture. The protease inhibitors benzamidine and phenylmethylsulfonyl fluoride did not attenuate the ability of C2 toxin to alter the morphology of Y-1 cells. A 3-hr exposure to C2 toxin resulted in the ADP-ribosylation of 50 to 60% of the total actin pool. Fluorescein isothiocyanate-labeled phalloidin visualization of the cytoskeleton of toxin-treated cells confirmed that the toxin caused a decrease in the stress fiber network. C2 toxin treatment of a protein kinase A mutant Y-1 cell (Kin 8) resulted in morphological changes and an increase in steroid output that was not different from that observed for wild type Y-1 cells. The data suggest that C2 toxin increases steroid output from adrenal Y-1 cells by a cyclic AMP-independent mechanism that involves the microfilament network of the cell.


Tin-protoporphyrin: a potent inhibitor of hemoprotein-dependent steroidogenesis in rat adrenals and testes.

The present investigation provides evidence of the ability of Sn-protoporphyrin to cause striking alterations in adrenal and testicular cytochrome P-450-dependent steroidogenesis and defines the potential of this metalloporphyrin to serve as a cellular toxin. Sn-protoporphyrin is currently used on an experimental basis for treatment of hyperbilirubinemias in humans, including newborn infants. Specifically, in the adrenals of rats treated with a moderate regimen of Sn-protoporphyrin (two doses of 50 mumol/kg, s.c.), marked decreases of 60 to 70% in the microsomal 21 alpha-hydroxylase and the mitochondrial 11 beta-hydroxylase activities were observed after 7 days. Concomitant with these decreases was a significant depression in the adrenal mitochondrial cytochrome P-450 content and a notable reduction (approximately 30%) in serum corticosterone levels. Similarly, in the testes, significant decreases in the microsomal and mitochondrial cytochrome P-450 contents and the microsomal 17 alpha-hydroxylase activity were observed. Serum testosterone level, however, was not decreased, reflecting an absence of decrease in side chain cleavage activity. Metalloporphyrin caused a striking decrease of 65 to 80% in the microsomal heme oxygenase activity in the testes and the adrenals, as well as significant reductions in NADPH-cytochrome P-450 reductase activity of the organs. The decrease in heme oxygenase activity, however, as suggested by Western immunoblotting, apparently resulted, to a large extent, from the loss of enzyme protein integrity rather than a competitive inhibition of activity. At the transcript level, Northern blot analysis using a full length rat testis cDNA probe for heme oxygenase-2 mRNA indicated that Sn-protoporphyrin treatment did not decrease the amount of message for either of the heme oxygenase-2 transcripts (1.3 and 1.9 Kb).(ABSTRACT TRUNCATED AT 250 WORDS)


Cranial ontogeny in the direct-developing frog, Eleutherodactylus coqui (Anura: Leptodactylidae), analyzed using whole-mount immunohistochemistry.

Direct development in amphibians is an evolutionarily derived life-history mode that involves the loss of the free-living, aquatic larval stage. We examined embryos of the direct-developing anuran Eleutherodactylus coqui (Leptodactylidae) to evaluate how the biphasic pattern of cranial ontogeny of metamorphosing species has been modified in the evolution of direct development in this lineage. We employed whole-mount immunohistochemistry using a monoclonal antibody against the extracellular matrix component Type II collagen, which allows visualization of the morphology of cartilages earlier and more effectively than traditional histological procedures; these latter procedures were also used where appropriate. This represents the first time that initial chondrogenic stages of cranial development of any vertebrate have been depicted in whole-mounts. Many cranial cartilages typical of larval anurans, e.g., suprarostrals, cornua trabeculae, never form in Eleutherodactylus coqui. Consequently, many regions of the skull assume an adult, or postmetamorphic, morphology from the inception of their development. Other components, e.g., the lower jaw, jaw suspensorium, and the hyobranchial skeleton, initially assume a mid-metamorphic configuration, which is subsequently remodeled before hatching. Thirteen of the adult complement of 17 bones form in the embryo, beginning with two bones of the jaw and jaw suspensorium, the angulosplenial and squamosal. Precocious ossification of these and other jaw elements is an evolutionarily derived feature not found in metamorphosing anurans, but shared with some direct-developing caecilians. Thus, in Eleutherodactylus cranial development involves both recapitulation and repatterning of the ancestral metamorphic ontogeny.(ABSTRACT TRUNCATED AT 250 WORDS)


Necrosis and inhibition of growth of human lung tumor by anti-alpha-human chorionic gonadotropin antibody.

Human lung tumor cells (ChaGo) established in culture from a bronchogenic squamous cell carcinoma synthesize and secrete large amounts of human chorionic gonadotropin (HCG), predominantly the alpha subunit of the glycoprotein hormone. ChaGo cells lose their transformation phenotypes following treatment with anti-alpha-HCG antibody or following inhibition of intracellular synthesis of alpha-HCG by the anti-sense RNA technique. We report that tumors induced by ChaGo cells in female athymic mice undergo necrotic degeneration following local or intraperitoneal administration of alpha-HCG-specific-antibody. The alpha-HCG antibody did not affect the growth of tumor induced by alpha-HCG nonproducing human tumor cells. Histopathological examinations of the anti-alpha-HCG antibody-treated tumor tissues showed active necrosis. When the antibody treatment was discontinued, the tumorigenesis process resumed. When anti-alpha-HCG antibody was administered simultaneously with ChaGo cells, a concentration-dependent inhibition of tumor growth in athymic mice was completely prevented at higher concentrations of the specific antibody.


The dominant linear neutralizing antibody-binding site of glycoprotein gp86 of human cytomegalovirus is strain specific.

Bacterial fusion proteins, constructed from overlapping fragments of the open reading frame coding for gp86 of human cytomegalovirus (HCMV) strain AD169, were used to localize antigenic regions recognized by antibodies from human convalescent sera. A major domain for binding of conformation-independent antibodies was localized on fusion protein AP86, containing amino acids 15 to 142 of gp86. Human antibodies, affinity purified on AP86, neutralized infectious virus in tissue culture. In addition, a mouse monoclonal antibody (AP86-SA4), raised against AP86, also neutralized HCMV. AP86-SA4 was reactive with viral gp86 in immunoblot assays and showed a plasma membrane staining on intact HCMV-infected fibroblasts late in infection. After exonuclease III deletions of the viral gene, the binding site of neutralizing human as well as mouse antibodies was localized between amino acid residues 34 and 43. The domain has sequence variation between laboratory strains AD169 and Towne, and binding of the antibodies was strain specific. To our knowledge, this is the first characterization of a strain-specific neutralizing epitope on HCMV.


Characterization of the interaction of polyomavirus middle T antigen with type 2A protein phosphatase.

Two cellular proteins of 36 and 63 kDa which bind the small T and middle T antigens of polyomavirus recently have been identified as the catalytic and regulatory subunits of the phosphoserine/threonine-specific type 2A protein phosphatase (PP2A). We report here the presence of phosphoseryl phosphatase activity associated with polyomavirus small T and middle T antigens in immunoprecipitates prepared from virus-infected and transformed cells. Phosphatase activity was also found associated with middle T-antigen mutants, some of which had been defined previously to associate with 36- and 63-kDa cellular proteins. Middle T-antigen-associated phosphatase activity was sensitive to okadaic acid and microcystin-LR, inhibitors of PP2A, and insensitive to inhibitor 1 or 2, orthovanadate, or EDTA. Using antiserum specific for the catalytic subunit of PP2A, we found that unlike the majority of PP2A, middle T-antigen-bound PP2A was membrane associated. However, no gross change in the amount, activity, or localization of PP2A could be attributed to middle T-antigen expression in transformed cells. Anti-PP2A antibodies coprecipitated a 63-kDa protein from normal cells and in addition coprecipitated middle T antigen, 60- and 61-kDa proteins (identified as src family members), and an 81-kDa protein from middle T-antigen-transformed cells. Furthermore, we detected protein kinase activity in PP2A immunoprecipitates and protein phosphatase activity in src immune complexes from extracts of middle T-antigen-transformed, but not normal, cells. These results reinforce the notion that at least a portion of middle T antigen bridges a protein kinase with a protein phosphatase.


Ty3 GAG3 and POL3 genes encode the components of intracellular particles.

Ty3 is a Saccharomyces cerevisiae retrotransposon that integrates near the transcription initiation sites of polymerase III-transcribed genes. It is distinct from the copialike Ty1 and Ty2 retrotransposons of S. cerevisiae in both the sequences of encoded proteins and gene order. It is a member of the gypsylike family of retrotransposons which resemble animal retroviruses. This study was undertaken to investigate the nucleocapsid particle of a transpositionally active gypsylike retrotransposon. Characterization of extracts from cells in which Ty3 expression was induced showed the presence of Ty3 nucleoprotein complexes, or viruslike particles, that migrated on linear sucrose gradients with a size of 156S. These particles are composed of Ty3 RNA, full-length, linear DNA, and proteins. In this study, antibodies raised against peptides predicted from the Ty3 sequence were used to identify Ty3-encoded proteins. These include the capsid (26 kDa), nucleocapsid (9 kDa), and reverse transcriptase (55 kDa) proteins. Ty3 integrase proteins of 61 and 58 kDa were identified previously (L. J. Hansen and S. B. Sandmeyer, J. Virol. 64:2599-2607, 1990). Reverse transcriptase activity associated with the particles was measured by using exogenous and endogenous primer-templates. Immunofluorescence studies of cells overexpressing Ty3 revealed cytoplasmic clusters of immunoreactive proteins. Transmission electron microscopy showed that Ty3 viruslike particles are about 50 nm in diameter. Thus, despite the unusual position specificity of Ty3 upstream of tRNA-coding regions, aspects of the Ty3 life cycle are fundamentally similar to those of retroviruses.


High-frequency intracellular transposition of a defective mammalian provirus detected by an in situ colorimetric assay.

We devised an indicator gene for retrotransposition, nlsLacZRT, which contains the Escherichia coli lacZ gene fused to a nuclear location signal (nlsLacZ), engineered in such a way that the gene is expressed only if the structure in which it has been inserted transposes itself through an RNA intermediate. A cloned murine leukemia retrovirus with an ecotropic host range (Moloney murine leukemia virus), rendered defective by a large deletion encompassing the three viral gag, pol, and env open reading frames, was marked with this indicator gene and introduced by transfection into heterologous feline cells. No beta-galactosidase activity could be detected among the clonal cell population, unless the defective provirus was complemented in trans by the gag-pol gene products. Under these conditions, cell variants which disclosed an easily detectable nuclear blue coloration upon in situ 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining were observed. Fluorescence-activated cell sorting of the beta-galactosidase-positive cells, followed by Southern blot analysis, demonstrated an unambiguous correlation between nlsLacZRT activation and retrotransposition of the marked provirus. Transposition occurs at a high frequency (up to 10(-4) events per cell per generation), which is dependent on the level of expression of the gag-pol gene and is concomitant with the release of noninfectious retroviruslike particles which are the hallmarks, but not the intermediates, of the intracellular transposition process.


The baculovirus-integrated retrotransposon TED encodes gag and pol proteins that assemble into viruslike particles with reverse transcriptase.

TED is a lepidopteran retrotransposon found inserted within the DNA genome of the Autographa californica nuclear polyhedrosis virus mutant, FP-D. To examine the proteins and functions encoded by this representative of the gypsy family of retrotransposons, the gag- and pol-like open reading frames (ORFs 1 and 2) were expressed in homologous lepidopteran cells by using recombinant baculovirus vectors. Expression of ORF 1 resulted in synthesis of an abundant TED-specific protein (Pr55gag) that assembled into viruslike particles with a diameter of 55 to 60 nm. Expression of ORF 2, requiring a -1 translational frameshift, resulted in synthesis of a protease that mediated cleavage of Pr55gag to generate p37, the major protein component of the resulting particles. Expression of ORF 2 also produced reverse transcriptase that associated with these particles. Both protease and reverse transcriptase activities mapped to domains within ORF 2 that contain sequence similarities with the corresponding functional domains of the pol gene of the vertebrate retroviruses. These results indicated that TED ORFs 1 and 2 functionally resemble the retrovirus gag and pol genes and demonstrated for the first time that an invertebrate member of the gypsy family of elements encodes active forms of the structural and enzymatic functions necessary for transposition via an RNA intermediate. TED integration within the baculovirus genome thus represents one of the first examples of transposon-mediated transfer of host-derived genes to an eukaryotic virus.


Analysis of T- and B-cell epitopes of capsid protein of rubella virus by using synthetic peptides.

A nested set of 11 overlapping synthetic peptides covering the entire sequence of rubella virus capsid protein was synthesized, purified, and tested against human rubella virus-specific T-cell lines and rubella virus-seropositive sera. T-cell lines derived from four donors responded strongly to four synthetic peptides containing residues 96 to 123, 119 to 152, 205 to 233, and 255 to 280. Only one peptide (residues 255 to 280) was recognized by all four T-cell lines. Two human immunodominant linear B-cell epitopes were mapped to residues 1 to 30 and 96 to 123 by using peptide-specific enzyme-linked immunosorbent assay. All 11 synthetic peptides were highly immunogenic and induced strong antibody responses in rabbits against the respective immunized peptides. Seven of the 11 rabbit antipeptide antisera (anti-1-30, -74-100, -96-123, -119-152, -205-233, -231-257, and -255-280) specifically recognized the capsid protein on immunoblots. Identification of these T- and B-cell epitopes represents the first step toward rational design of synthetic vaccines against rubella.


A nuclear single-stranded-DNA binding factor interacts with the long terminal repeats of the 1731 Drosophila retrotransposon.

Using gel mobility assays, we have detected two proteins that bind in the U3 region of the 1731 retrotransposon long terminal repeats (between positions -110 and -73) in nuclear extracts from Drosophila melanogaster cultured cells. The first one binds double-stranded DNA, whereas the other binds the mRNA-like strand in a sequence-specific manner. We report here the characterization of the latter protein, named NssBF for nuclear single-stranded-DNA binding factor. Gel filtration shows an apparent molecular mass of 95 kDa for NssBF. The points of contact between NssBF and its single-stranded DNA target were determined. This protein binds neither the complementary strand nor the corresponding RNA sequence. A possible role of NssBF in transcription is discussed.


Transformation-defective mutants of polyomavirus middle T antigen associate with phosphatidylinositol 3-kinase (PI 3-kinase) but are unable to maintain wild-type levels of PI 3-kinase products in intact cells.

Middle T antigen (MT) of polyomavirus causes transformation by associating with a number of cellular proteins. The association with and activation of two such proteins, phosphatidylinositol 3-kinase (PI 3-kinase) and pp60c-src, appears to be necessary for transformation by MT. The tyrosine kinase activity of MT-associated pp60c-src is significantly increased when assayed in vitro, and levels of phosphotyrosine-containing proteins are elevated in vivo. Similarly, levels of the PI 3-kinase products phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and phosphatiylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] are constitutively elevated in MT-transformed cells. However, the formation of a complete MT/cellular protein complex and the activation of tyrosine kinase are not sufficient to cause transformation, since the transformation-defective mutants 248m and dl1015 associate with all wild-type MT-associated proteins, including PI 3-kinase and pp60c-src, and neither mutant appears to be defective in MT-associated tyrosine kinase activity. Studies presented here compared (i) the amount of PI 3-kinase activity associated with the MT complex and (ii) levels of [3H]inositol incorporation into PI 3-kinase products in cells expressing mutant or wild-type MT. The results show that dl1015 is defective in both assays, whereas 248m is defective only for incorporation of [3H]inositol into PI(3,4,5)P2 and PI(3,4)P3. These findings identify a biochemical defect in the 248m mutant and corroborate previous results correlating transformation and elevated levels of PI 3-kinase products in vivo. In addition, they indicate that PI 3-kinase product levels are affected by factors other than simply the amount of PI 3-kinase activity associated with the MT complex.


Resistance to influenza virus infection of Mx transgenic mice expressing Mx protein under the control of two constitutive promoters.

Transgenic mice constitutively expressing in the brain the influenza virus resistance protein Mx1 controlled by the HMG (3-hydroxy-3-methylglutaryl coenzyme A reductase) promoter showed specific resistance against the neurotropic influenza A virus strain NWS. Control mice of the A2G strain express Mx1 protein in all organs, but only after induction by interferon type I upon or without viral infection. The extent of specific resistance in transgenic mice of the best-expressing line reached about two-thirds that of controls, most likely because of considerably less total-body Mx protein activity in the transgenic mice. Thus, the theoretical advantage in these mice of the continuous presence of Mx protein with early inhibitory potential to viral replication was apparently offset by restricted organ expression. Strong evidence that the Mx1 protein on its own is a specific anti-influenza A virus agent and that its efficiency in the experimental setting is independent of interferon actions could be derived from the treatment of experimental and control mice with anti-interferon antibodies at the time of virus tests. Whereas in A2G mice, Mx1 mRNA and Mx1 protein synthesis were abolished and viral resistance was markedly reduced or abolished, resistance in the transgenic mice persisted to almost the same degree. Transgenic mice generated with a mouse albumin/Mx1 cDNA construct showed liver-specific expression. However, in two expressing transgenic lines, Mx1 protein synthesis was suppressed after a few months. The mechanism of suppression could not be elucidated, but increasing methylation of the transgene's coding region was not the cause. It is possible that continuous Mx1 protein expression in the liver is less well tolerated than that in the brain. Whether this partial suppression and, with the HMG promoter, restricted organ expression are the organism's responses to interference of Mx1 with normal cellular activities such as nucleocytoplasmic transport of RNA and proteins cannot be determined until the molecular mechanisms of antiviral activity of Mx1 protein are understood.


Incompletely reverse-transcribed human immunodeficiency virus type 1 genomes in quiescent cells can function as intermediates in the retroviral life cycle.

Using a quantitative polymerase chain reaction (PCR) method, we have previously shown that a molecularly cloned isolate of human immunodeficiency virus type 1 (HIV-1) can efficiently enter quiescent primary lymphocytes; however, the reverse transcription process is not completed in these cells. In this study, we further characterized the reverse transcription of HIV-1 in quiescent cells, and our results indicate that while initiation of reverse transcription occurs simultaneously in both activated and quiescent lymphocytes, it not only ends prematurely but also proceeds more slowly in quiescent cells. We also performed experiments to address the role of partial reverse transcripts as intermediates in the viral life cycle. We used azidothymidine either before or after infection with HIV-1 to prevent formation of and further DNA synthesis by partial reverse transcripts, respectively. Decreases in virus production from these cells following mitogenic stimulation indicated that partial reverse transcripts can contribute significantly to virus rescue from infected quiescent cells stimulated subsequent to infection. Furthermore, we established that mitogenic stimulation of infected quiescent cells induces reinitiation of DNA synthesis from partial reverse transcripts. However, the virus rescue is inefficient relative to the initial multiplicity of infection, and this is explained by inefficient completion of DNA synthesis from the partial reverse transcript. Thus, the arrest of reverse transcription in quiescent cells may play an important role in HIV-1 pathogenesis by contributing to the inefficient infection of potential target cells in the peripheral blood of HIV-1-infected individuals.


Cloning of noncultivatable human rotavirus by single primer amplification.

A novel, sequence-independent strategy has been developed for the amplification of full-length cDNA copies of the genes of double-stranded RNA (dsRNA) viruses. Using human (Bristol) group C rotavirus as an example, a single amino-linked modified oligonucleotide (primer 1) was ligated to either end of each dsRNA genome segment by using T4 RNA ligase. Following reverse transcription, annealing, and repair of cDNA strands, amplification of the viral dsRNA genome was accomplished by polymerase chain reaction using a single complementary oligonucleotide (primer 2). Northern (RNA) hybridization of cDNA to virus dsRNA indicated that it was possible to generate cDNA representing the complete genome from very small clinical samples. This technique was used to determine the complete nucleotide sequence (728 bp) and coding assignment of gene 10, which revealed an open reading frame of 212 amino acids with limited homology to NS26 from human group A rotavirus. In contrast to previous tailing methods, the addition of one defined primer allowed unequivocal identification of terminal nucleotides and should be generally applicable to viruses with segmented dsRNA genomes and especially for analysis of clinical samples, for which very limited quantities of biological material are available.


Estrogen receptor in carcinoma in situ of the cervix.

The lining epithelium of the human cervix uteri is an estrogen dependent tissue containing specific intracellular receptors for this hormone. However, the influence of estrogen on an early neoplastic lesion arising from this epithelium, such as carcinoma in situ of the cervix, has not been determined. We evaluated 24 formalin fixed paraffin embedded tissue specimens of cervical carcinoma in situ for the presence of estrogen receptor by the immunoperoxidase technique. The antigenic sites of estrogen receptor were exposed by DNAse treatment followed by peroxidase-antiperoxidase (PAP) staining with monoclonal antibody against estrogen receptor. Parallel negative controls were run using negative control antibody and rat serum. Quality control for positive staining was performed using breast cancer tissue sections from specimens with known estrogen receptor detected by the radioreceptor method. Strongly positive staining was observed in all specimens in the nuclei of glandular epithelium, stromal cells, and basal and parabasal cells. However, nuclei within carcinoma in situ of the cervix showed no evidence of positive staining. Due to lack of specific intracellular receptor for estrogen, it appears that carcinoma in situ of the cervix will not be under direct influence of estrogen.


Distribution of 111In-bleomycin complex in small cell lung cancer cells by autoradiography.

The distribution of 111In-bleomycin Complex (111In-BLMC) in small cell lung cancer (SCLC) cells was studied by autoradiography. SCLC cells were exposed to 111In-BLMC and 111Indium chloride (111InCl3) for 1 hour, 3 hours, and 4 hours; washed with fresh medium; and spread on slides. The slides were smeared with NTB2 (NTB3) emulsion by wet or dry-mount technique and exposed 3 to 15 days. 111In-BLMC was found to localize in the cell nucleus and nuclear membrane (78.3%); 111InCl3 located mainly in the cytoplasm (52.3%). This distribution of labeled BLM may explain the mechanism of killing SCLC cells by 111In-BLMC.


Hyaluronan synthesis in the adult guinea pig endolymphatic sac.

The endolymphatic sac is believed to play a major role in membranous labyrinth homeostasis by controlling the volume of endolymph, removing debris, and participating in the immune response of the inner ear. The endolymphatic sac is postulated to absorb endolymph and to synthesize and secrete high-molecular-weight and osmotically active glycosaminoglycans (GAGs). The present study examines the ability of in vitro adult guinea pig endolymphatic sac cells to synthesize complex proteins and polysaccharides. The intent is to characterize the nature of these compounds by studying carbon-14 (14C) glucose incorporation in tissue cultured endolymphatic sac specimens using autoradiographic and specific enzymatic digestion techniques. Our results suggest that sac cells can synthesize GAGs and proteins in vitro in proportionately larger amounts than surrounding connective tissue and dura. The principal GAG synthesized by the endolymphatic sac appears to be hyaluronan.


Endorphin-like immunoreactivities in uncultured and cultured human peripheral blood mononuclear cells.

It has been reported that cells of the immune system produce and release considerable amounts of pro-opiomelanocortin (POMC) -derived peptides in response to coculture with a variety of stimulatory agents. The present study investigated whether extracts of human peripheral blood mononuclear cells (PBMC) contain immunoreactivity for beta-endorphin (beta E) and related peptides. Using four endorphin RIA systems with different specificities, extracts of freshly isolated PBMC and PBMC cultured in the presence or absence of mitogens or of corticotropin releasing factor (CRF) and vasopressin (VP), were analyzed. With a radioimmunoassay (RIA) system directed to the midportion of beta E, immunoreactivity (MP beta E-IR) was readily detectable, although the concentration was extremely low (ca. 200 pg/10(7) cells). beta E immunoreactivity (beta E-IR) and alpha-endorphin immunoreactivity (alpha E-IR), as determined in C-terminally directed RIA systems, were present in even lower concentrations. gamma-Endorphin immunoreactivity (gamma E-IR) was hardly detectable. Of subsets enriched in T-cells, B-cells or monocytes, the highest concentration of MP beta E-IR was detected in extracts of monocytes. Coculture of PBMC with the mitogen Concanavalin A (Con A) or Phytohaemagglutinin (PHA) increased the amount of MP beta E-IR in extracts of the cells. No increase in alpha E-IR, however, was detected, whereas beta E-IR was only increased in extracts of cells cultured in the presence of Con A. No increase, in any of the immunoreactivities, was observed in extracts of PBMC cultured with bacterial lipopolysaccharide (LPS) or with the combination of CRF and VP, both stimuli that have been reported to induce POMC peptides in cultured PBMC. The present data show that human PBMC contain endorphin-like immunoreactivity, but in very small amounts. The extremely low concentrations and the ineffectiveness of LPS and the combination of CRF and VP to increase the endorphin-like immunoreactivity raise questions about the reported capacity of PBMC to synthesize POMC-derived peptides.


Effects of the S2-serotonergic receptor antagonist, ketanserin, on cerulein-induced pancreatitis in the rat.

We investigated the effects of ketanserin, a S 2 (5-hydroxytryptamine 2; 5-HT 2)-serotonergic receptor antagonist, on cerulein-induced pancreatitis in the rat. Large pharmacological doses of cerulein induced acute pancreatitis in the rat. Ketanserin reduced the cerulein-induced increase in serum amylase concentration in a dose-dependent manner. Treatment with 10 mg/kg of ketanserin per os markedly improved cerulein-induced pancreatitis and was associated with a significant reduction of the increase in serum amylase concentration. In addition, a very specific serotonin S 2 antagonist, ritanserin which has no antihypertensive effect, also reduced the cerulein-induced increase in the serum amylase concentration. These results suggest that S 2 (5-HT 2) may play a role in pathophysiology of cerulein-induced pancreatitis in the rat.


The copy number of plasmid pLS1 is regulated by two trans-acting plasmid products: the antisense RNA II and the repressor protein, RepA.

The promiscuous plasmid pLS1 encodes two transacting elements that regulate its copy number: protein RepA and antisense RNA II. In vitro transcription showed that RNAs for both repressors are synthesized from two promoters, PAB and PII. From PAB, genes encoding RepA (transcriptional repressor) and RepB (initiator of replication) are cotranscribed, the target of RepA being located within PAB. Mutants in repA or in PAB are still sensitive to RepA. However, cloning of the repA gene in a compatible replicon did not result in incompatibility towards pLS1. From PII, the 50-nucleotide RNA II is synthesized. The main incompatibility determinant towards pLS1 corresponds to the coding sequence for RNA II. The RNA II target could be reduced to 21 nucleotides, including the RepB initiation of translation signals. We propose that plasmids of the pLS1 family (pE194, pADB201, and pLB4) share functional and structural characteristics for the regulation of their copy numbers.


Hydrolysis of substance P in the rabbit retina: I. Involvement of acetylcholine and acetylcholinesterase. An in vivo study.

The laminar patterns of acetylcholinesterase (AChE) activity and substance P (SP) immunoreactivity within the inner plexiform layer (IPL) of the rabbit retina show striking similarities. Discrete bands of SP-immunoreactivity were seen at 1-7%, 40-48% and 85-95% depth of IPL. AChE activity was present throughout the entire thickness of the IPL with moderately stained bands in each sublamina (3-24% in sublamina a and 62-89% in sublamina b depth IPL). These bands were bordered on both sides by bands of even greater density (in sublamina a 0-3% and 24-34% and in sublamina b 55-62% and 89-100% depth IPL). Cell processes staining for choline acetyltransferase (ChAT) have previously been shown to ramify at 19-24% and 63-79% depth levels. Thus, SP- and ChAT-immunoreactive bands are located in both sublaminae, positioned within regions of moderate AChE activity and flanked by bands with greater AChE activity. This strong morphological correspondence and reported interactions between acetylcholine (ACh), AChE and SP in vitro provide the basis for the present study to determine whether such interactions can be demonstrated in vivo. Retinas infused with ACh showed a 60% average increase in SP-IR as compared with untreated retinas from the same animals. Treatment with diisopropylfluorophosphate (DFP) also resulted in a 56% increase in SP-IR. The ability of ACh to induce increased levels of SP was not inhibited by CoCl2, atropine or mecamylamine, ruling out the possibilities of polysynaptic transmission or involvement of muscarinic or nicotinic receptors. Infusion of ACh did not increase the levels of preprotachykinin-mRNA indicating that the increase in SP-IR is not due to de novo synthesis but rather to inhibition of the enzyme(s) responsible for SP degradation. Whether AChE functions alone or in concert with other enzymes to hydrolyze SP cannot be determined from these experiments but is addressed in a separate study.


Hydrolysis of substance P in the rabbit retina: II. The role of a membrane-associated acetylcholine-sensitive metalloendopeptidase. An in vitro study.

Studies in the rabbit retina have shown that infusion of exogenous acetylcholine (ACh) into the vitreal chamber leads to an increase in the amount of substance P (SP) immunoreactivity (Goebel and Pourcho, submitted). This increase was determined to be independent of new peptide synthesis, suggesting that the elevated level of SP is the result of ACh inhibition of an SP-degrading protease. This phenomenon has now been confirmed in vitro in both tissue slice and retinal homogenate assays. These studies have shown that ACh decreases the rate of SP hydrolysis in a concentration dependent manner. Recovery of SP hydrolytic activity following ACh inhibition was found to be directly proportional to the amount of acetylcholinesterase (AChE) activity in the membrane fraction. Specific protease inhibitors were used to determine the relative contributions of membrane associated retinal enzymes to SP-hydrolysis. In the presence of 1 mM 1,10-phenanthroline or p-chloromercuribenzenesulfonic acid all SP-hydrolytic activity was abolished, indicating that the enzyme(s) responsible for the degradation of the peptide is a metallopeptidase. The ACh sensitive retinal enzyme was found to be concentrated in the membrane fraction where it accounts for approximately 70% of the SP hydrolytic activity. Although the precise identity of this enzyme remains to be determined, the present evidence indicates that it shares many of the characteristics of the enzyme substance P-degrading endopeptidase (Endo et al. 1988, 1989). Enkephalinase activity was also found, contributing to 28% of the hydrolytic activity in the membrane fraction. However, the activity of this enzyme was insensitive to elevated levels of ACh. After initial cleavage of SP by the primary hydrolytic enzymes, further degradation of the fragments appears to be carried out by membrane associated serine protease(s). The activity exhibited by this class of enzymes was inhibited by DFP treatment and was not sensitive to ACh. Although AChE does not make a major contribution to the hydrolysis of SP, it does participate in peptide degradation via its esterase activity which controls the level of ACh, thereby modulating the primary SP-hydrolytic enzyme.


Microvascular decompression and partial sensory rhizotomy in the treatment of trigeminal neuralgia: personal experience with 220 patients.

The results of the treatment of trigeminal neuralgia by neurovascular decompression or partial sensory rhizotomy in a personal series of 220 patients are presented. Microvascular decompression was performed in 178 patients and partial sensory rhizotomy in 42. The mean follow-up was 5.2 years. Immediate pain relief was achieved in 94% of all patients, but the rate dropped to 84% during the follow-up period. The recurrence rate in the microvascular decompression group was 6% and in the PSR 49%. Permanent sequelae occurred in 4 patients (loss of hearing, 1; loss of corneal reflex, 1; lesion of the portio minor, 2), but transitory complications (impaired hearing caused by hematotympanum and diplopia) were more frequent, especially in the beginning of the series. Elderly patients tolerated the procedure very well and the percentage of complications was evenly distributed in all age groups. Three patients died. No patient developed painful dysesthesias or anesthesia dolorosa. There were no differences in the outcome, considering sex and age. The duration of symptoms did not influence the prognosis. Patients with severe compression did better than those with a mild one, and patients with an arterial compression did better than those with a venous one. Trigeminal neuralgia in multiple sclerosis is seldom relieved by microvascular decompression. The experience of the surgeon reduces the number of negative findings considerably.


A flow cytometric study of 137 fresh hydropic placentas: correlation between types of hydatidiform moles and nuclear DNA ploidy.

Hydropic placentas may be classified by histopathology into hydropic abortus, partial hydatidiform mole, and complete hydatidiform mole. We studied 142 hydropic placentas: 39% were complete hydatidiform moles, 35% partial hydatidiform moles, and 26% hydropic abortuses. Villous vesicle size was predictive of histologic diagnosis. We determined DNA ploidy in 137 cases. Seventy-three percent of hydropic abortuses were diploid and 11% were triploid. Ninety percent of partial moles were triploid or near-triploid; one partial mole was haploid and one diploid. Of the complete moles, 50% were diploid, 43% were tetraploid, 3.6% polyploid, and 1.7% triploid. Partial moles had lower pre-evacuation beta-hCG levels than complete moles. Persistent tumor followed 33% of complete moles and 12% of partial moles. Although the numbers were small, no patient with a diploid, tetraploid, aneuploid, or haploid partial mole developed persistent disease. Among complete moles, the pre-evacuation beta-hCG level was not predictive of persistence (P = .15). Subdividing complete moles by ploidy, we found that tetraploid moles were associated with higher pre-evacuation beta-hCG levels than were diploid moles. However, tetraploidy was not associated with increased persistent tumor among complete moles. Although most partial moles were triploid and most complete moles were diploid or tetraploid, there was wider DNA heterogeneity among molar gestations than previously reported. In this series, DNA ploidy was not an independent predictor of persistence in complete moles.


Lipocortin and vasocortin: two species of anti-inflammatory proteins mimicking the effects of glucocorticoids.

The anti-inflammatory effect of glucocorticoids depends, at least in part, on the induction of two regulatory proteins, lipocortin and vasocortin, both preventing the release of inflammatory mediators. Lipocortin inhibits phospholipase A2 (PLA2) and therefore reduces arachidonic acid metabolites formation. Vasocortin inhibits histamine release from mast cells. Lipocortin and vasocortin may be regarded as the first two identified members of the (perhaps greater) family of glucocorticoid-induced proteins.


Should we lift or should we roll? Nursing practice following prosthetic hip surgery.

Following prosthetic hip surgery, patients are at high risk of pressure sores, and must be moved regularly. Although no research has concluded that dislocation is caused by moving patients in bed, lifting is still widely used. A flexible practice adapting to individual needs represents the best approach.


Fc gamma R-dependent regulation of the biosynthesis of complement C3 by murine macrophages: the modulatory effect of IL-6.

The effect of murine IgG isotypes on the gene expression and secretion of the third component of complement (C3) has been studied using the monocytoid cell line P388D1 and oil-elicited mouse peritoneal macrophages. It is demonstrated that the binding of IgG2a and IgG2b but not IgG1 and IgG3 augments the biosynthesis of C3 both in the presence and in the absence of the phorbol ester, phorbol myristate acetate in the case of both cell types. The multifunctional cytokine interleukin-6 (IL-6) alone reveals no effect on the gene expression of C3, but increases the effectiveness of mouse IgG2a and IgG2b. Confirming the role of Fc gamma RII, a strong up-regulation of C3 gene expression and C3 secretion was found when macrophages were cultured with the F(ab')2 fragment of the Fc gamma RII-specific monoclonal antibody 2.4G2.


Insulin secretion from pancreatic B cells caused by L-arginine-derived nitrogen oxides.

L-arginine causes insulin release from pancreatic B cells. Data from three model systems support the hypothesis that L-arginine-derived nitrogen oxides (NOs) mediate insulin release stimulated by L-arginine in the presence of D-glucose and by the hypoglycemic drug tolbutamide. The formation of NO in pancreatic B cells was detected both chemically and by the NO-induced accumulation of guanosine 3',5'-monophosphate. NG-substituted L-arginine analogs inhibited the release of both insulin and NO. Protein immunoblot and histochemical analysis with antiserum to type I NO synthase suggest that the formation of NO in pancreatic B cells is catalyzed by an NADPH- (reduced form of nicotinamide adenine dinucleotide phosphate), Ca2+/calmodulin-dependent type I NO synthase of about 150 kilodaltons.


Renal allograft-infiltrating lymphocytes. A prospective analysis of in vitro growth characteristics and clinical relevance.

One-hundred consecutive human renal allograft Tru-cut needle biopsies were studied for in vitro proliferation of T lymphocytes under restrictive culture conditions containing low-dose recombinant interleukin 2. Each biopsy was entered into a blinded code and evaluated prospectively for visual evidence of growth at 24 hr and for sustained growth. Those T cell populations exhibiting sustained growth were then evaluated for cell surface phenotype by FACS; for allospecific cytotoxicity by 51Cr release; for a proliferative response to alloantigen by incorporation of [3H]-thymidine; and for secretion of IL-2, IL-4, IFN-gamma, and TNF-alpha in response to alloantigenic stimulation by ELISA. All results were compared with clinical diagnosis, immunosuppression at time of biopsy, diagnosis and phenotype by immunopathology, short-term outcome and long-term graft survival. Growth at 24 hr was predictive of acute cellular rejection (P less than 0.0005), unrelated to chronic rejection (P = 0.663) or maintenance immunosuppression (P = 0.911), and inversely correlated with cyclosporine toxicity (P = 0.051) and treatment with OKT3 (P = 0.014). The CD4/CD8 ratio of the sustained T cell populations was unrelated to that seen on histological examination (correlation coefficient = -0.098 and 0.044 for diffuse and aggregate infiltrates, respectively). Cytotoxic specificity for HLA class II was mediated by CD4+ cells and for HLA class I by CD8+ cells. Enhanced secretion of IL-2 in response to alloantigen distinguished those cells associated with irreversible allograft damage from those associated with complete functional recovery (P = 0.01). This study demonstrates that early evaluation of T cell proliferation in vitro identifies activated T cell infiltrates mediating acute cellular allograft rejection in a time frame suitable for clinical diagnostic application. It strengthens the concept that donor-specific cytotoxicity is governed by the stabilization of the alloantigen-T-cell receptor interaction by the accessory molecules CD4 and CD8, but either interaction is equally able to participate in an episode of acute rejection. Irreversible graft injury is associated with infiltrating cells that are capable of amplifying their responsiveness through secretion of IL-2.


The adverse impact on liver transplantation of using positive cytotoxic crossmatch donors.

Because of the liver graft's ability to resist cytotoxic antibody-mediated rejection, it has become dogma that the conventional transplant crossmatch used to avoid hyperacute rejection of other organs is irrelevant to the liver. We examined this hypothesis in a consecutive series of adult primary liver recipients treated with FK506 and low-dose steroids. Twenty-five of 231 (10.8%) patients received a liver from a cytotoxic-positive crossmatch donor (more than 50% of donor T lymphocytes were killed by dithiothreitol-pretreated recipient serum). The outcome was compared with that of 50 negative crossmatch patients who had their transplantations just before and after the crossmatch positive cases. The one-year graft and patient survivals were 56% and 68%, for positive and 82% and 86% for negative crossmatch patients (P = 0.004, P = 0.03, respectively). The difference between patient and first graft survival was accounted for by retransplantation, which was 4 times more frequent in the positive-crossmatch cases. Histologically, failed allografts obtained at the time of retransplantation revealed a spectrum of pathologic findings related to vascular injury. This study showed a higher difficulty of intraoperative blood product management, a degraded prognosis, and a poorer average quality of ultimate graft function when liver transplantation was performed against positive cytotoxic crossmatches. In such patients for whom crossmatch-negative donors may never be found because of the broad extent and intensity of sensitization, special therapeutic strategies perioperatively must be evolved if results are to improve.


A three-year experience with serum anodal trypsinogen as a biochemical marker for rejection in pancreatic allografts. False positives, tissue biopsy, comparison with other markers, and diagnostic strategies.

Serum values of immunoreactive anodal trypsinogen (sAT) have been claimed to correlate well with rejection occurring in pancreatic allografts. We have studied the behavior of sAT in serial serum samples obtained from 39 type I diabetics undergoing whole-organ pancreas transplantation during the past 3 years. Patients had either received a pancreatic allograft simultaneously with a transplanted kidney (SPK, n = 33) or after a previous kidney transplant (pancreas after kidney [PAK] n = 6). The behavior of sAT was studied in relation to the clinical diagnosis of rejection. Graft amylase output for all 39 patients and serum creatinine for the 33 SPK recipients were also studied. Tissue biopsies were obtained from 11 patients with elevated sAT values and a presumptive diagnosis of rejection. Nine of these patients had SPK grafts and simultaneously elevated creatinine values. Tissue was obtained from the simultaneously transplanted kidney; all specimens revealed rejection. Two of the 11 patients had PAK allografts. Biopsies performed on the graft duodenum were consistent with acute rejection. Three additional patients with unchanged sAT values had biopsies for other reasons; these biopsies failed to demonstrate signs of acute rejection. Thus graft biopsy correlated exactly with sAT behavior in every case in which rejection was suspected. Five patients had elevations of sAT not associated with rejection: one resulted from direct trauma, two had outlet obstruction, and two had clinical diagnoses of graft pancreatitis. The sAT was more sensitive and specific than GAO and as sensitive as creatinine for SPK recipients. These studies confirm that sAT is a reliable, graft-specific biochemical marker for the early diagnosis of pancreatic rejection. The use of sAT should allow for the proper timing of graft biopsies and the judicious use of immunosuppressive agents, which will result in increased allograft survival for PAK and pancreas-alone allografts.


The induction of immunologic hyporesponsiveness by preoperative donor-specific transfusions and cyclosporine in human cadaveric transplants. A preliminary trial.

A prospective randomized preliminary trial was performed in patients undergoing cadaveric renal transplantation to determine the potential benefits, disadvantages, and logistic problems associated with the administration of donor-specific transfusions and cyclosporine initiated 24 hr before transplantation. Ten patients received DST followed by continuous intravenous CsA approximately 24 hr before cadaveric renal transplantation from the same donor. Twelve patients receiving sequential therapy with Minnesota antilymphoblast globulin, azathioprine, and steroids with subsequent conversion to CsA served as controls. Patient demographics and the donor characteristics were evenly matched in the two groups. While the study group had longer cold ischemia time and more evidence of renal dysfunction within the first two weeks, subsequent renal function was identical in the groups and there were fewer episodes of severe rejection requiring treatment with OKT3 within the first six months in the DST group (5 vs. 0, P less than 0.05), which also had less reactivity in mixed lymphocyte cultures against preserved donor-specific lymphocytes than did the control group (stimulation index 9.0 +/- 3.0 vs. 25.3 +/- 6.0, respectively, P less than 0.05). The need for dialysis, incidence of infections and other complications, and subsequent immunosuppressive therapy were not different in the two groups. It is concluded that DSTs and intravenous CsA initiated 24 hr prior to transplantation are capable of inducing reduced immunologic responsiveness against the specific donor. Patients treated with this therapy should receive organs from "ideal" donors without risk factors and cold ischemia time should not exceed 30 hr. Further clinical studies of this approach are warranted.


[Causes of pain and treatment effect in patients with cancer referred to a multidisciplinary pain clinic].

The causes of pain were analysed in 200 patients referred to a multidisciplinary pain clinic for cancer patients. In 158 patients, pain caused directly by tumour growth was found, 116 patients had pain secondary to the cancer disease or treatment while 33 patients had pain caused by factors unrelated to the cancer disease. The patients had many different combinations of causes of pain and the majority had more than one cause of pain. At the first contact and after treatment for 1-2 weeks, the patients were asked whether they had pain on movement, at rest or pain which interrupted sleep. After treatment for 1-2 weeks and after treatment for more than two weeks, the patients assessed the relief of pain obtained (none, slight, moderate, considerable, complete). The majority of patients achieved relief of pain at rest and during sleep while movement was still accompanied by pain in a number of patients. The majority of patients considered that the relief of pain obtained was moderate or considerable. Treatment consisted of adjustment of medication, blockades and epidural opioids supplemented by psychological intervention and help from social workers in selected patients.


Cytokeratin 20 in human carcinomas. A new histodiagnostic marker detected by monoclonal antibodies.

The authors have recently identified a new cytokeratin (CK) polypeptide, CK 20, whose expression is almost entirely confined to the gastric and intestinal epithelium, urothelium, and Merkel cells. Seven monoclonal antibodies (MAbs) specific for CK 20 were raised and characterized by applying immunoblotting and immunocytochemical screening. All of them reacted on frozen tissue sections. A further MAb, IT-Ks20.8, recognized CK 20 in sections of formalin-fixed, paraffin-embedded tissue samples. A total of 711 cases of primary and metastatic cancer, mostly carcinomas, were analyzed immunohistochemically for CK-20 expression, using CK-20 specific guinea-pig antibodies and MAbs. The expression spectrum of CK 20 in carcinomas resembled that seen in the corresponding normal epithelia of origin. CK-20 positivity was seen in the vast majority of adenocarcinomas of the colon (89/93 cases), mucinous ovarian tumors, transitional-cell and Merkel-cell carcinomas and frequently also in adenocarcinomas of the stomach, bile system, and pancreas. Most squamous cell carcinomas in general and most adenocarcinomas from other sites (breast, lung, endometrium), nonmucinous tumors of the ovary, and small-cell lung carcinomas were essentially or completely negative. The authors propose to use CK 20 as a diagnostic marker valuable in distinguishing different types of carcinomas, notably when presenting as metastases.


Changes in distribution, morphology, and tumor necrosis factor-alpha secretion of alveolar macrophage subpopulations during the development of bleomycin-induced pulmonary fibrosis.

Previous studies indicate that heterogeneous alveolar macrophages (AM) play a pivotal role in events associated with bleomycin-induced pulmonary fibrosis. A critical role has been suggested for tumor necrosis factor-alpha (TNF-alpha), a product of activated macrophages, in this fibrotic process. The present study examined whether the characteristics and function (TNF-alpha secretion) of rat AM subpopulations were altered during the development of bleomycin-induced fibrosis. After intratracheal bleomycin treatment, AM were separated into 18 density-defined subpopulations. Bleomycin treatment altered the distribution and morphology of AM subpopulations of densities 1.017 to 1.061 g/ml at all time points studied (4, 14, and 28 days). Subpopulations of densities 1.090 to 1.141 g/ml were affected only at 4 days after bleomycin treatment. Tumor necrosis factor-alpha secretion increased with time in 14- and 28-day samples of bleomycin-treated rats, particularly in subpopulations of densities 1.075 to 1.097 g/ml. These data indicate that alterations in the distribution, morphology, and function of AM subpopulations accompany the development of bleomycin-induced pulmonary fibrosis. When coupled with previous studies suggesting that TNF-alpha plays a role in the fibrotic process in this disease model, these data indicate that AM of densities 1.075 to 1.097 g/ml are responsible for the production of TNF-alpha associated with bleomycin-induced pulmonary fibrosis.


Choice of palliation for malignant hilar biliary obstruction.

Clinical data from 50 consecutive patients with unresectable hilar tumors situated at or proximal to the common hepatic duct were retrospectively analyzed to aid in the selection of appropriate palliative measures. Thirty-four patients had cholangioenteric bypass (CEB) to either left (28 patients), right (3 patients), or both (3 patients) intrahepatic ductal systems. Sixteen patients had nonoperative drainage (NOD) established either endoscopically (4 patients), percutaneously (9 patients), or using a combined endoscopic-percutaneous approach (3 patients). When compared with patients with CEB, patients with NOD had more frequent medical problems (p less than 0.03) and lower serum albumin levels on admission (p less than 0.03). While comparable postprocedural complications (13 CEB patients versus 4 NOD patients) were observed, patients with NOD had a significantly higher hospital mortality (9 CEB patients versus 9 NOD patients, p less than 0.05). Excluding the 12 patients (6 CEB patients versus 6 NOD patients) who died within 30 days after drainage, the quality of survival of the remaining 38 patients was analyzed with reference to 6 objective parameters. Although patients with NOD had significantly more frequent admissions relating to their catheters (p less than 0.02), there was no qualitative difference in the survival rate between the two groups of patients. For selected high-risk patients with limited life expectancy, NOD should be offered. However, additional prospective studies are required to decide the best choice of palliation for patients who are not at such high risk.


Relationship between pathologic prognostic factors and abnormal levels of des-gamma-carboxy prothrombin and alpha-fetoprotein in hepatocellular carcinoma.

The relationship between pathologic prognostic factors and abnormal levels of des-gamma-carboxy prothrombin and alpha-fetoprotein was investigated in 42 patients with resectable hepatocellular carcinoma. The frequencies of macroscopic massive type, intrahepatic metastasis, and portal vein tumor thrombus were significantly higher in patients with positive des-gamma-carboxy prothrombin (p less than 0.05) but not with alpha-fetoprotein. Other histologic factors in tumorous and nontumorous tissues were not significantly different irrespective of the positivity of these markers. In patients with tumors not more than 6 cm in diameter, the frequency of intrahepatic metastasis was positively correlated with the positivity of des-gamma-carboxy prothrombin (p less than 0.05) and inversely with that of alpha-fetoprotein (p less than 0.05). Furthermore, intrahepatic metastasis was most frequently observed in patients with positive des-gamma-carboxy prothrombin and negative alpha-fetoprotein (eight of nine) and least frequently in cases with negative des-gamma-carboxy prothrombin and positive alpha-fetoprotein (one of eight). These findings indicated that both des-gamma-carboxy prothrombin and alpha-fetoprotein might be useful markers for the prediction of intrahepatic spread of hepatocellular carcinoma.


Endoscopic laser therapy for neoplastic lesions of the colorectum.

Surgical resection is the therapy of choice for most colorectal neoplasms. Endoscopic laser therapy (ELT) is a recently developed alternative for treatment of colorectal neoplasms and is applicable in a variety of clinical circumstances in which nonoperative treatment is preferable. The experience with ELT using the Nd:YAG (neodynium:yttrium-aluminum-garnet) laser in 42 patients was analyzed. The diagnosis was colorectal adenocarcinoma in 32 patients (76%) and neoplastic polyps in 10 (24%). ELT was undertaken either as a palliative treatment for malignant disease (60%), with curative intent for benign disease (26%), or as a temporizing measure (14%) in a total of 84 treatment sessions. Successful palliation or cure was achieved in 40 patients (95%) with 4 minor complications (9%) and no procedure-related deaths. This experience confirms ELT as an effective alternative to surgical therapy in the palliative, curative, or interim treatment of certain colorectal neoplasms in patients with prohibitive operative risk, limited anticipated survival, incurability, or diffidence toward operation.


In vitro activation of bronchoalveolar lavage cells by house dust mite allergens.

Mast cells represent a small but important proportion of bronchoalveolar lavage cells and are directly exposed to environmental triggers including allergens. Histamine and PGD2 are mediators released during the activation of mast cells. Fourteen patients allergic to Dermatophagoides pteronyssinus were studied. After bronchoalveolar lavage the unfractionated cell pellet containing metachromatic cells was submitted to allergen challenge using three concentrations of a standardized Dermatophagoides pteronyssinus extract and one concentration of A23187 (2.5 microM). The release of histamine was measured by radioimmunoassay using a monoclonal antibody against acylated histamine and PGD2 was measured by enzyme immunoassay using a polyclonal antibody against methoxamine-PGD2. Histamine was released in 13/14 patients following stimulation of the cells with A23187 and 12/14 patients after stimulation with Dermatophagoides pteronyssinus extract. The release of histamine was 3.5-fold greater when cells were stimulated by the Dermatophagoides pteronyssinus extract than with A23187. PGD2 was released in 10/12 patients when cells were stimulated with A23187 and 6/14 patients in the case of Dermatophagoides pteronyssinus extract stimulation. In the latter case, the mean release was not significantly greater than baseline. For histamine, the maximum release usually occurred with the more concentrated extract whereas in the experiments where PGD2 was released, maximal generation usually occurred with the lowest concentration used. There was no correlation between the severity of asthma and the release of mediators. This study confirms the activation of metachromatic cells by allergen and shows some heterogeneity in the release of granule and membrane-derived mediators.


Effect of dehydration on cardiovascular responses and electrolytes after hypertonic saline/dextran treatment for moderate hemorrhage.

To determine if hypertonic saline/dextran (HSD) is effective in treating hemorrhage in the presence of dehydration. After surgical preparation, swine were euhydrated or dehydrated for 24 or 48 hours. Animals were bled 25 mL/kg over 60 minutes and treated with HSD. Laboratory. Seventeen immature Yorkshire pigs. 4 mL/kg HSD (7.5% NaCl in 6% dextran-70) administered over one minute. All euhydrated animals survived; 100% of the pigs survived 180 minutes after treatment. Two animals dehydrated for 24 hours and three animals dehydrated for 48 hours died within three hours of HSD treatment. In all groups, plasma potassium was reduced significantly and equally; cardiac output was increased; mean arterial pressure rose rapidly within first five minutes, but was sustained only in euhydrated animals; hematocrit, hemoglobin, and plasma total protein levels were reduced; and plasma glucose increased with persistent between-group differences. HSD immediately rectified the decreases in mean arterial pressure and cardiac output incurred during hemorrhage; over time, however, the improvement in pressure was not sustained in dehydrated pigs. Parallel increases in plasma osmolality and sodium concentrations were offset by the initial group differences resulting from dehydration. Dehydration does not compromise the efficacy of HSD as a resuscitation treatment for hemorrhagic shock.


Treatment of progressive membranous glomerulopathy. A randomized trial comparing cyclophosphamide and corticosteroids with corticosteroids alone. The Glomerular Disease Collaborative Network.

To determine if deterioration in renal function could be ameliorated by adding cyclophosphamide to corticosteroid therapy in patients with progressive membranous glomerulopathy. Randomized, controlled treatment trial. Patients were followed for a mean of 29.2 +/- 17.1 months. Collaborative network of 120 university and private-practice nephrologists. Patients with membranous glomerulopathy whose renal function deteriorated (as evidenced by doubling of the serum creatinine level, a 50% fall in the glomerular filtration rate, or a sustained serum creatinine level of greater than 2.0 mg/dL [reciprocal creatinine value, 0.5], or whose nephrotic range proteinuria persisted in association with morbid complications. Of 156 patients with biopsy-proven membranous glomerulopathy, 36 became eligible for randomization. Twenty-six of these 36 patients were randomly assigned to receive one of the two treatments. Pulse methylprednisolone, oral corticosteroids, and 6 months of intravenous cyclophosphamide or alternate-day corticosteroid therapy alone. At entry, no statistical differences were found between the treatment groups in duration of renal disease, age, gender, serum creatinine level, 24-hour urine protein excretion, or biopsy stage. The groups showed no difference in mean arterial blood pressure during follow-up. Four of the 13 patients receiving corticosteroids alone and 4 of the 13 patients receiving corticosteroids plus intravenous cyclophosphamide progressed to end-stage renal disease during follow-up. Reciprocal creatinine values tested at 6-month intervals showed no statistical differences between treatment groups at any time point. The log of the 24-hour protein excretion values showed no statistical differences between treatment groups after treatment. The power to detect a substantial improvement in renal function, defined as a doubling of the reciprocal of the serum creatinine, at the 0.05 significance level was 0.92. Combination therapy with intravenous cyclophosphamide and corticosteroids, when compared with corticosteroid therapy alone, does not improve renal function in patients with progressive membranous glomerulopathy.


Studies on complement deposits in epidermolysis bullosa acquisita and bullous pemphigoid.

Epidermolysis bullosa acquisita (EBA) is an inflammatory subepidermal blistering disease characterized by circulating and tissue-bound autoantibodies specific for type VII collagen of the basement membrane zone. The antibodies consist of both complement- and noncomplement-binding populations and belong to all four subclasses of IgG. We investigated the presence of the membrane attack complex C3b, C5, and S protein in EBA and compared C3b and C5 in EBA and bullous pemphigoid. In 10 patients with EBA, these components were detected at the basement membrane zone as follows: membrane attack complex, 90%; S protein, 90%; direct C5, 90%; C3b, 100%; and C5 binding, 90%. In the patients with bullous pemphigoid, the results were as follows: direct C5, 58%; C3b, 33%; and C5 binding, 19%. These results provide additional evidence for complement activation at the basement membrane zone in EBA, show that complement activation in EBA proceeds to activation of terminal complement components, and suggest that EBA antibodies are more potent activators of C5 than are bullous pemphigoid antibodies.


Synthesis and characterization of 7-nitrobenzo-2-oxa-1,3-diazole (NBD)-labeled fluorescent opioids.

Alkylation of sarcosine with 4-chloro-nitrobenzo-2-oxa-1,3-diazole (NBD-chloride) furnished a fluorescent tag that was coupled with a tetrahydrothebaine derivative and beta-naltrexamine, respectively, to yield the fluorescent opioids 7 alpha-(1R)-1-hydroxy-1-methyl-3-(4-hydroxyphenyl)-propyl]-6,14- endoethenotetrahydrothebaine NBD-sarcosinate (ASM-5-10) and N-cyclopropylmethyl-3-hydroxy-14 beta-hydroxy-6 beta-(NBD sarcosinyl)-amino-epoxymorphinan (ASM-5-67). The fluorescence intensity of the novel opioids allowed their detection at subnanomolar concentrations, and was dependent on the polarity of the solvent. Maximum quantum yield was obtained in ethyl acetate and ethanol, and minimal fluorescence in heptane and water. Compounds ASM-5-10 and ASM-5-67 displaced the opioid receptor binding of [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol in monkey brain membranes with IC50 values of 8.4 and 1.5 nM, respectively. Whereas ASM-5-67 bound to mu, delta, and kappa receptors with comparable affinities, ASM-5-10 was mu-selective, with selectivity indices (ratio of respective IC50 values) of 0.04 for both mu/delta and mu/kappa. The sodium response ratio in binding revealed a pronounced agonist property of ASM-5-10. Both opioids were lipophilic, with octanol-water partition coefficients (log Papp) of 2.8 (ASM-5-10) and 1.0 (ASM-5-67). ASM-5-10 exhibited particularly strong membrane retention that was not reversible by four washes. Their favorable characteristics in fluorescence, receptor binding, and membrane interaction make these newly developed ligands useful molecular probes to study opioid receptor mechanisms.


Comparison of growth kinetics of producing and nonproducing hybridoma cells in batch culture.

Several clones of nonproducing cells were isolated from a continuous culture of hybridoma cells, which were originally producing antibody. Their behavior was compared to that of the producing cells in batch culture. The growth kinetics of five out of six clones exhibited higher specific growth rate, higher yield of cell mass on glutamine, and lower yields of lactate and ammonium. The implications of the comparisons for growth of hybridoma cultures are discussed.


Social skills and their correlates: preschoolers with developmental delays.

Fifteen preschool-age children with mild mental retardation (developmental delays) from mainstreamed schools were observed during two structured play sessions with matched peers without mental retardation. Children with developmental delays spent more time alone and when they played, showed less social play. The two groups did not differ on communication behaviors that maintained play or in negative affect; however, the children with developmental delays evidenced more disruptive entry, more regressive behaviors, and less positive affect. Families were interviewed concerning their attitudes about, and teaching of, social skills. For the children without mental retardation, level of social play was positively related to the family's teaching and the child's communication abilities. For the children with delays, social play related to developmental age and communication ability but not to family teaching.


The role of nitric oxide in hippocampal long-term potentiation.

Long-term potentiation is a long-lasting, use-dependent increase in the strength of synaptic connections. We investigated the role of nitric oxide (NO) in determining the duration of potentiation induced by high frequency stimulation of afferents in the CA1 region of the rat hippocampus. The calcium/calmodulin-dependent production of NO can be initiated by activation of excitatory amino acid receptors and results in increased levels of cGMP in target cells. Here we report that only a relatively short-term potentiation can be induced in the presence of nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor. The effects of L-NAME on the duration of potentiation are partially reversed by coadministration of L-arginine, a precursor of neuronal NO, and by dibutyryl cGMP. Hemoglobin, which binds extracellular NO, also shortens the duration of stimulus-induced potentiation. The results suggest a role for NO in the maintenance of activity-dependent synaptic enhancements, possibly via the generation of cGMP.


Cloning of a putative glutamate receptor: a low affinity kainate-binding subunit.

Kainate, a glutamate receptor agonist, is a potent neuroexcitatory agent that produces epileptiform activity and selective neuronal degeneration. Binding studies using neuronal membrane homogenates or brain sections have identified sites having either high or low affinity for [3H]kainate. Here we report the cloning of a gene, GluR7, with approximately 75% sequence identity with the previously cloned GluR5 and GluR6 subunit genes. Transcripts of the GluR7 gene are evident in brain areas that bind [3H]kainate and are susceptible to kainate-induced neurotoxicity. We have performed ligand binding studies with membranes of transfected HeLa cells expressing GluR6 or GluR7 subunits. Our data show that the GluR6 and GluR7 subunits have a rank order of agonist affinity (domoate greater than kainate much greater than L-glutamate, quisqualate much greater than AMPA, NMDA) and a dissociation constant for kainate (95 and 77 nM, respectively) characteristic of the low affinity kainate-binding sites described in the brain.


Loss of extrasynaptic channel modulation by protein kinase C underlies the selection of serotonin responses in an identified leech neuron.

Pressure-sensitive (P) neurons contacted by serotonergic Retzius (R) neurons of the leech in culture selectively reduce a protein kinase C (PKC)-dependent cation response to serotonin and are innervated by the inhibitory, Cl(-)-dependent synapse seen in vivo. We have examined whether the reduction of extrasynaptic cation channel modulation is due to changes in sensitivity of the channels to second messenger. In inside-out membrane patches from single, uncontacted P cells in culture, cation channel activity was increased by rat brain PKC and cofactors. In contrast, the activity of cation channels in patches isolated from P cells paired with R cells was unaffected by PKC. These results demonstrate the loss of extrasynaptic channel modulation by PKC during synapse formation.


Isoform-selective deficit of glycine receptors in the mouse mutant spastic.

The mutant mouse spastic (spa) develops a characteristic motor disorder about 2 weeks after birth, with symptoms resembling sublethal poisoning by the glycinergic antagonist strychnine. Correspondingly, adult homozygotic mutants (spa/spa) exhibit a severe reduction of inhibitory glycine receptors in spinal cord and brain. Here we show that the spastic mutation selectively interferes with the postnatal accumulation of the adult isoform of the glycine receptor protein, whereas perinatal expression of the neonatal receptor isoform is not detectably affected. Heterologous expression in X. laevis oocytes of poly(A)+ RNA and Northern blot analysis indicate normal levels of glycine receptor alpha 1 subunit transcripts in spinal cord of adult spastic mutants. Thus, the age-dependent manifestation of spastic symptoms after birth reflects a selective effect of the mutation on the developmental expression of the adult glycine receptor isoform.


Temporal and spatial modulation of a cytoskeletal antigen during peripheral axonal pathfinding.

A neuron-specific cytoskeletal antigen (5E10), whose expression pattern during initial motoneuron outgrowth into the chick limb suggests that it is playing a role in axon guidance, is described. This antigen, which was shown to be a phosphorylated epitope, probably of the intermediate weight neurofilament protein (NF-M), exhibits a highly stereotyped and spatially heterogeneous pattern of expression. The point of onset of expression, which was abrupt and occurred in the distal axon and base of the growth cone, differed between groups of neurons that projected to different targets. Specifically, expression occurred from positions where previous perturbation experiments suggested that the axons in question would begin responding to specific guidance cues, and it remained high along the axon from this point to the target. Expression of this antigen could also be induced in cultured motoneurons by activating several second messenger systems.


QT dispersion in sinus beats and ventricular extrasystoles in normal hearts.

Recent studies have suggested that QT interlead variability (dispersion) on the surface electrocardiogram may have potential as a measure of recovery time dispersion. To test this hypothesis further QT dispersion occurring in sinus beats was compared with that in ventricular extrasystoles. Simultaneous electrocardiograms were recorded at 50 mm/s during sinus rhythm in a drug free state while ventricular extrastimuli were introduced by programmed right ventricular stimulation at different coupling intervals. QT dispersion, defined as the difference between the maximum and minimum QT, was calculated separately for the extrasystoles and preceding and following sinus complexes. To correct for the influence of the number of measurable leads on QT dispersion, an "adjusted" QT dispersion calculated as QT dispersion/square root of the number of measurable leads, was used to compare sinus complexes and extrasystoles. Nine patients were studied who were undergoing electrophysiological study for investigation of palpitation and were found to have electrically normal ventricles. At all coupling intervals tested "adjusted" QT dispersion was significantly greater in the ventricular extrasystoles than in either the preceding or following sinus complexes. For the coupling interval 350 ms, the 95% confidence intervals for the difference between means was 52 to 78 ms (preceding sinus complex) and 56 to 82 ms (following sinus complex) (p less than 0.00001). There was no correlation between the coupling interval and the magnitude of the "adjusted" QT dispersion. These results accord fully with expected differences in ventricular recovery time dispersion and offer further support for the hypothesis that QT dispersion reflects regional variation in ventricular recovery. If substantiated by invasive studies, these findings have wide implications for both the usefulness and the method of QT measurement.


Antinuclear antibodies: clinical correlations and biologic significance.

Several trends become evident from the foregoing discussion. As the different ANA antigenic specificities have been identified, they have often been found to be highly conserved polypeptides that subserve very basic cellular functions that are carried out in the nucleus, nucleolus, and ribosomes. The reasons why only 30 or so basic cellular proteins become the targets of an autoimmune response in patients with connective tissue disease at the exclusions of the other 10,000 macromolecules that exist inside cells remain a mystery. However, some insight into this enigma might be provided by the mechanism of molecular mimicry (Table 9). The possibility that highly conserved immunogenic molecules that are expressed by infectious pathogens can trigger an immune response in a genetically predisposed human host that cross-reacts with cellular autoantigens is a well documented phenomenon in disorders such as rheumatic fever. This mechanism is now being mentioned with increasing frequency in discussions pertaining to the pathogenesis of autoimmune connective tissue diseases. Another trend relates to the increasing sensitivity of the newer assays that have been developed to detect ANA. When highly purified or recombinant autoantigens are used in versatile assays such as ELISA, radioimmunoassays, or immunoprecipitation, the frequency with which certain autoantibodies can be detected in patient subgroups can go up significantly. For example, with classical immunodiffusion, anti-Ro/SS-A antibodies can be detected in 25% of unselected patients with SLE, whereas with an ELISA based on affinity purified Ro/SS-A antigen, 50% of patients with SLE are found to have elevated levels of this autoantibody specificity. As is often the case, we pay for increased sensitivity in a laboratory test with decreased specificity. With immunodiffusion, virtually no normal individuals have anti-Ro/SS-A antibodies, but with the ELISA as many as 10% of normals have elevated anti-Ro/SS-A binding levels. Thus, the incremental diagnostic value of this newer anti-Ro/SS-A assay could be questioned. The true clinical value of this new laboratory technology will become more evident when these more sophisticated ANA assays are used together in a panel-like fashion to profile a given patient's autoimmune response at the very onset of his illness. Preliminary work has already begun in this area. This approach, if well standardized, could have significant diagnostic and prognostic value. Another benefit of this newer technology will be the ability to measure antibody binding levels to individual autoepitopes--limited portions of an autoantigen's amino acid sequence that represent single antibody binding sites. It is possible that certain patterns of clinical disease could be linked to autoantibody production against individual autoepitopes rather than whole autoantigenic molecules. This area is only now beginning to be explored.


Dual appearance of fluorescein staining in vivo of diseased human corneal epithelium. A non-contact photomicrographic study.

Adherence of fluorescein sodium dye to diseased epithelial cells, a hitherto unreported phenomenon, was captured in photomicrographs in severe herpes zoster and keratoconjunctivitis sicca keratopathies. It is notable that this phenomenon differs completely from the well known fluorescent property of the dye penetrating into defective corneal epithelium, and that the staining pattern shown by adherent fluorescein correlates well with the staining pattern shown by rose bengal dye.


Controlled trial of laser photocoagulation of pigment epithelial detachments in the elderly: 4 year review.

Patients who were enrolled in a controlled treatment trial of laser grid photocoagulation for retinal pigment epithelial detachment as part of age-related maculopathy were reviewed 4 years after entry into the trial. The data imply that the original conclusion that this form of treatment did not improve the visual prognosis at 18 months was also justified at 4 years. It has become clear that lesions with evidence of subpigment epithelial new vessels were included in the trial. In a retrospective study the lesions were separated into those in which there was evidence of subretinal neovascularisation and those in which no such evidence existed. A difference was identified in the behaviour of the treated and untreated lesions designated avascular in that the treated eyes had a poorer visual outcome. These cases accounted for the different behaviour between two management groups in the initial study such that the original conclusion that grid photocoagulation of avascular pigment epithelial detachments in the elderly does not improve the visual prognosis is justified.


Detection of subpigment epithelial neovascularisation in cases of retinal pigment epithelial detachments: a review of the Moorfields treatment trial.

The entry angiograms of 42 eyes with detachment of the retinal pigment epithelium in a treatment trial of laser photocoagulation were reviewed in a masked fashion by three observers in order to assess the possible presence of subpigment epithelial neovascularisation. Vascularity or avascularity was designated with reference to a list of clues believed to imply the presence of subpigment epithelial neovascularisation. As a predictor of outcome the initial assessment achieved a sensitivity and specificity of 77% and 82% respectively. Despite notable parity of the degree of sensitivity and specificity among the three observers, full agreement on the initial assessments was reached in only 23 eyes (55%), 10 with vascular and 13 with avascular outcome. Of these, only one eye which developed new vessels after 4 years had an outcome which differed from that predicted by classification of the entry angiograms.