Expression of cell adhesion molecules in posterior uveitis.

Cells in the eye express surface molecules that direct leukocyte migration during ocular inflammation. We studied the expression of intercellular adhesion molecule-1, lymphocyte function-associated antigen-1, and endothelial leukocyte adhesion molecule-1 in six enucleated eyes from patients with uveitis and in seven normal control eyes. Lymphocyte function-associated antigen-1 was expressed on infiltrating lymphocytes and intercellular adhesion molecule-1 was expressed on endothelial cells of retinal and choroidal blood vessels and the retinal pigment epithelium in all uveitic eyes, but in none of the normal control eyes. Ocular inflammatory cells stained strongly positive for tumor necrosis factor alpha in all eyes with uveitis, but four of six uveitic eyes showed mild staining for tumor necrosis factor beta and interferon gamma. Cell adhesion molecules are expressed in eyes with posterior uveitis and may be regulated by cytokines such as tumor necrosis factor alpha.


Roll, roll, roll your leucocyte gently down the vein....

The rapid advances being made in unravelling the biochemistry and function of the three categories of adhesion molecule involved in leucocyte-endothelial-cell interactions, namely selectins, integrins and the Ig super-family molecules, were highlighted at a recent meeting. In this short report, Nancy Hogg describes these developments and the prospects for anti-adhesion molecule therapy in the clinic.


The mechanism of action of cyclosporin A and FK506.

CsA and FK506 are powerful suppressors of the immune system, most notably of T cells. They act at a point in activation that lies between receptor ligation and the transcription of early genes. Here, Stuart Schreiber and Gerald Crabtree review recent findings that indicate CsA and FK506 operate as prodrugs: they bind endogenous intracellular receptors, the immunophilins, and the resulting complex targets the protein phosphatase, calcineurin, to exert the immunosuppressive effect.


Cardiovascular effects of intrathecal administration of substance P in the rat: interactions with serotonergic mechanisms.

Intrathecal (i.th.) administration of substance P (SP, 6.5 nmol) at the Th 8-10 level in conscious rats increased blood pressure (carotid artery), heart rate and plasma catecholamine concentrations. The responses were antagonized by the intravenous (i.v.) but not i.th. pretreatment with the 5-HT2-receptor antagonists ketanserin and ritanserin and intrathecally administered serotonin (5-HT, 10 micrograms). The pressor response and the increase in plasma noradrenaline concentrations were also antagonized by i.v. or i.th. pretreatment with the 5-HT1A-agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT). In contrast the pressor response to SP was facilitated by the 5-HT1A-antagonist 1-pindolol (i.v. or i.th). Pretreatment with SP (i.th) reduced the hypotensive response to i.v. 8-OH-DPAT. These results demonstrate functional interactions between SP and serotonergic mechanisms in the central system, but the precise location and nature were not elucidated.


Evidence for post-transcriptional regulation of cytokeratin gene expression in a rat liver epithelial cell line.

T51B, a cell line of the rat liver nonparenchymal cell compartment, contains a cytokeratin (CK) pair composed of CK8, a CK typical of simple epithelium, and CK14, a CK usually present in proliferative stratified epithelium. T51B-Ni, a subclone selected by prolonged exposure of the parental clone to nickel subsulfide contains CK8 and CK18 (its usual partner in simple epithelium), as well as CK14, at a lower level. The two clones have comparable levels of vimentin. Northern blot analyses of cytoplasmic mRNA demonstrated that the differences in the steady state mRNA levels of each CK paralleled those observed at the protein level, thus showing that the regulatory events occurred prior to translation. The most prominent difference was a 30-fold higher level of CK18 mRNA in T51B-Ni cells. Run-off assays of isolated nuclei revealed that the level of CK8, CK14, and vimentin was regulated primarily at the transcriptional level. However, the large increase in CK18 mRNA levels in T51B-Ni cells did not result from a corresponding increase in the relative level of CK18 gene transcription nor from a change in cytoplasmic CK18 mRNA stability. Comparative Northern blot analyses of nuclear and cytoplasmic mRNAs further suggested that the control of CK18 gene expression in T51B cells is post-transcriptionally mediated by nuclear events.


Intermediate filaments in rat ovarian surface epithelial cells: changes with neoplastic progression in culture.

Interrelationships between neoplastic progression and the expression of intermediate filaments were examined in primary cultures, immortal lines, and Kirsten murine sarcoma virus (KiMSV) transformed lines of rat ovarian surface epithelial (ROSE) cells. Immunofluorescence microscopy revealed abundant keratin filaments in all cells of primary cultures. In immortal, nontumorigenic lines, keratin filaments were detected in fewer cells, in smaller numbers, and in microscopically altered forms. The percentage of keratin-positive cells ranged from 4 to 54%. Its expression was inversely proportional to cell density. Keratin expression was similar in the two immortal lines, although one had retained a monolayered epithelial growth pattern resembling primary cultures, while in the other the growth pattern of the cells was more atypical. The two KiMSV-transformed lines were previously shown to produce tumors in vivo that resemble human ovarian endometrioid stromal sarcomas. In spite of this histologic appearance, the proportion of keratin-positive cells in these cells was increased over the immortal lines. Keratin expression was unrelated to cell density, and keratin in most virally transformed cells was limited to few, fine filaments. In thymidine-labelled immortal and virus-transformed cultures stained for keratin, no correlation was found between keratin expression and proliferative activity. The keratin profiles of primary and immortal cultures were identical on Western blots, with subtypes ranging from 52 to 66 kDa. The two virally transformed lines lacked some of the subtypes. Vimentin networks were faint or absent in primary cultures. In the immortal and the virus-transformed lines, neoplastic progression was associated with increasing vimentin expression but with no changes in filament morphology and distribution. The results show that the abnormalities in intermediate filament expression that accompany immortalization do not preclude the retention of a normal epithelial morphology and growth pattern in this cell type. Furthermore, the number of intermediate filaments and their intracellular distribution appear to be altered at an earlier stage in neoplastic progression than those mechanisms that select for specific keratin subtypes, or those that respond to regulation by cell density. Finally, the presence of keratin in the KiMSV-transformed lines examined in this study supports the hypothesis that human ovarian stromal sarcomas can arise in the OSE.


Structural characterization of the O-polysaccharide of the lipopolysaccharide produced by Salmonella milwaukee O:43 (group U) which possesses human blood group B activity.

The O-polysaccharide of the lipopolysaccharide produced by Salmonella milwaukee O:43 (group U) was shown by composition analysis, methylation, periodate oxidation, and 1H and 13C nuclear magnetic resonance spectroscopic analytical methods to be a polymer of branched pentasaccharide repeating units having the structure: [formula: see text] The blood-group activity of the O-polysaccharide was established by its serological reactivity with a specific monoclonal antibody to human blood group B, using passive hemagglutination and ELISA assays, indicating the common antigenic epitope to be a nonreducing terminal trisaccharide unit composed of L-Fucp and D-Galp (1:2) residues.


Structure and function of L-selectin.

The selectins are a newly described family of carbohydrate-binding adhesion molecules involved in the regulation of leukocyte traffic. Selectins are composed of an N-terminal C-type lectin domain, a single EGF domain, a variable number of short consensus repeat (SCR) domains, a transmembrane region and a cytoplasmic tail. L-selectin (LAM-1/LECAM-1/LECCAM-1) is the only selectin expressed on leukocytes, and mediates a number of leukocyte-endothelial interactions, including the binding of lymphocytes to HEV of peripheral lymph node high endothelial venules (HEV), neutrophil rolling, and leukocyte attachment to cytokine-treated endothelium in vitro. Stable transfectants expressing a series of chimeric selectins and deletion mutants were functionally analyzed in order to determine the molecular basis of adhesion mediated by L-selectin. The specificity of adhesion was found to reside entirely within the lectin domain, suggesting that this domain is the only domain of the protein to interact with the carbohydrate ligand. These results make previous observations that certain mAbs which block function map to each of the extracellular domains difficult to interpret. In addition, deletion of the cytoplasmic tail of L-selectin abolished adhesion, without affecting ligand recognition. Thus, each domain of the selectins has an important, but distinct, role in cell adhesion.


Sympathetic nerve contact alters membrane resistance of cells of the RBL-2H3 mucosal mast cell line.

Indirect evidence links sensory nerves with mast cells (MC) in inflammatory reactions of airway, skin, and intestine. Isolated MC secrete histamine, serotonin, and other inflammatory mediators in response to neuropeptides such as substance P (SP) in vitro. To obtain direct evidence of nerve/MC interactions, we used a tissue culture model involving the co-culture of murine sympathetic neurons and rat basophilic leukemia (RBL) cells (homologous to mucosal MC). An electrophysiologic analysis of the consequences of neuron/RBL cell contacts showed that neurite contact with RBL cells reduced the control input resistance (Ro) of 61.8 +/- 3.2 (n = 110) M omega to 22.4 +/- 4.8 (n = 13) M omega (P less than 0.01) without change in the membrane potential. Time course studies showed that Ro of RBL cells with neurite contact was always lower by 30 to 54% than adjacent RBL cells lacking such contact. This effect was not seen in RBL cells cultured on rat fibroblasts. Direct application of SP, bradykinin, and somatostatin, but not acetylcholine, noradrenaline, or the putative neurotransmitter ATP, could partly mimic the effect of neurite contact. Therefore, neurotransmitter release from sympathetic neurons in contact with RBL cells may decrease RBL cell membrane resistance, possibly leading to activation.


Phylogenetic relationships of three porcine mycoplasmas, Mycoplasma hyopneumoniae, Mycoplasma flocculare, and Mycoplasma hyorhinis, and complete 16S rRNA sequence of M. flocculare.

The nucleotide sequence of the 16S rRNA gene of Mycoplasma flocculare was determined and was compared with the sequence of a related porcine mycoplasma, Mycoplasma hyopneumoniae. While the overall level of DNA-DNA homology was approximately 11%, sequence alignment of the two 16S rRNA genes yielded a homology value of more than 95%, emphasizing the highly conserved nature of the 16S rRNA gene. Multiple sequence alignments with other mollicutes indicated that M. flocculare, M. hyopneumoniae, and Mycoplasma hyorhinis form a subcluster within the fermentans phylogroup, and this subcluster is distinct from the Mycoplasma pneumoniae phylogroup. Thus, the three mycoplasmas isolated from porcine respiratory systems exhibit phylogenetic similarities.


Phylogenetic relationships between the western aster yellows mycoplasmalike organism and other prokaryotes established by 16S rRNA gene sequence.

Restriction fragments containing the 16S rRNA gene of the western aster yellow mycoplasmalike organism (SAY-MLO) were identified in Southern blots probed with cloned fragments of the western X-disease mycoplasmalike organism 16S rRNA gene. Two fragments which contained the entire SAY-MLO 16S rRNA gene and flanking DNA were cloned in M13 and sequenced. The SAY-MLO 16S rRNA gene is approximately 1,535 bp long, has a G+C content of 47 mol%, and has an overall secondary structure similar to that proposed for Escherichia coli. Putative rRNA promoter sequences and sequences involved in processing of the primary rRNA transcript were similar in the SAY-MLO, two Mycoplasma species, and Bacillus subtilis, suggesting that these prokaryotes and the mycoplasmalike organisms may have similar transcriptional and processing enzymes. We identified two tRNA genes, a tRNA(Tyr) (GTA) gene upstream from the 16S rRNA gene and a tRNA(Ile) (GAT) gene in the spacer region between the 16S and 23S rRNA genes. Comparisons of the SAY-MLO 16S rRNA nucleotide sequence with 16S rRNA sequences of other organisms indicated that the SAY-MLO is phylogenetically related most closely to other plant-pathogenic mycoplasmalike organisms, followed by Anaeroplasma species, Acholeplasma species, and some Mycoplasma species.


Mycobacterium confluentis sp. nov.

A new rapidly growing mycobacterium was isolated from human sputum. This organism grew at 22, 31, 37, and 41 degrees C and possessed catalase, acid phosphatase, acetamidase, urease, nicotinamidase, pyrazinamidase, and nitrate reductase activities. It did not produce nicotinic acid, hydrolyze Tween, or have benzamidase, isonicotinamidase, succinidamidase, and arylsulfatase activities. A mycolic acid analysis revealed a simple, unique pattern. The organism is susceptible to antituberculotic drugs. A comparative 16S rRNA sequence analysis placed this organism within the confines of the genus Mycobacterium, most closely related to the thermotolerant rapidly growing species. On the basis of the pattern of enzymatic activities and metabolic properties, as well as the unique 16S rRNA sequence, we propose that our single strain represents a new species, for which we propose the name Mycobacterium confluentis. The type strain is strain 1389/90; a culture of this strain has been deposited in the German Collection of Microorganisms and Cell Cultures as strain DSM 44017.


[How to make slides].

Quality of oral presentation slides is based on content and design. The most common fault is presentation of slides with too much writing, or with type to small to be seen by the audience. With a few design rules and a minimum of photographic equipment, high quality slides are to be obtain. Slides are projected to be read and communicate an idea, make them nice, simple and readable; there is no need for fancy art works. Format and color are important but not the main point, and they will depend on available material, time and money.


Comparative sequence analyses on the 16S rRNA (rDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus and proposal for creation of a new genus, Alicyclobacillus gen. nov.

Comparative 16S rRNA (rDNA) sequence analyses performed on the thermophilic Bacillus species Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus revealed that these organisms are sufficiently different from the traditional Bacillus species to warrant reclassification in a new genus, Alicyclobacillus gen. nov. An analysis of 16S rRNA sequences established that these three thermoacidophiles cluster in a group that differs markedly from both the obligately thermophilic organisms Bacillus stearothermophilus and the facultatively thermophilic organism Bacillus coagulans, as well as many other common mesophilic and thermophilic Bacillus species. The thermoacidophilic Bacillus species B. acidocaldarius, B. acidoterrestris, and B. cycloheptanicus also are unique in that they possess omega-alicylic fatty acid as the major natural membranous lipid component, which is a rare phenotype that has not been found in any other Bacillus species characterized to date. This phenotype, along with the 16S rRNA sequence data, suggests that these thermoacidophiles are biochemically and genetically unique and supports the proposal that they should be reclassified in the new genus Alicyclobacillus.


Phylogenetic analysis of the pathogenic anaerobe Clostridium perfringens using the 16S rRNA nucleotide sequence.

Clostridium perfringens, the first pathogenic clostridium examined, was placed in the nonmycoplasma subgroup of the low-dG+dC-content gram-positive cluster on the basis of the results of a phylogenetic analysis in which we used 16S rRNA comparisons. The closest relative that has been identified to date is Clostridium pasteurianum.


Marrow proliferation and the appearance of giant neutrophils in response to recombinant human granulocyte colony stimulating factor (rhG-CSF).

During a study of recombinant human granulocyte colony stimulating factor (rhG-CSF) administration, 15 patients received twice daily i.v. infusions and nine patients received daily s.c. infusions of rhG-CSF for 5 d prior to cytotoxic therapy, and then a second course subsequent to melphalan administration. There was a striking dose-related neutrophilia and the appearance in the blood of early myeloid cells that express the intercellular adhesion molecule CD54. In addition, giant neutrophils or macropolycytes were observed in the peripheral blood of all patients. These cells were evident on the display of the Technicon H*1 as a population of large peroxidase positive cells, and using Feulgen staining these cells were shown to be tetraploid. Bone marrow kinetics studies performed on Day 4 or 5 indicated an increase in the proportion of bone marrow cells in S phase, G2 and mitosis, reflecting a proliferative response of the marrow. Large myeloid precursors and occasional binucleate promyelocytes were seen in the bone marrows done on Days 14 and 18 but not on Day 5. These findings indicate that administered G-CSF has both quantitative and qualitative effects on myeloid cells in vivo.


Autotransplantation of peripheral blood stem cells mobilized by chemotherapy and recombinant human granulocyte colony-stimulating factor in childhood neuroblastoma and non-Hodgkin's lymphoma.

Seven children with advanced neuroblastoma and non-Hodgkin's lymphoma were treated with myeloablative chemoradiotherapy (180 mg/m2 melphalan plus 12 Gy fractionated total body irradiation), followed by autotransplantation of peripheral blood stem cells (PBSC). Sufficient PBSC to restore bone marrow function were collected by a small number of leukaphereses during haematopoietic recovery after chemotherapy and recombinant human granulocyte colony-stimulating factor (rhG-CSF). Furthermore, rapid recovery of neutrophils was found in all patients by the administration of rhG-CSF following transplantation: median 10 d (range 8-12) to attain more than 0.5 x 10(9)/l neutrophils, and 27 d (range 14-73) to attain more than 50 x 10(9)/l platelets, respectively. Haematopoietic reconstitution has been maintained throughout the follow-up period (median 15 months; range, 6-22). Peripheral blood stem cells mobilized by chemotherapy and rhG-CSF can induce complete haematopoietic reconstitution after myeloablative chemoradiotherapy.


Endogenous platelet fibrinogen: its modulation after surface expression is related to size-selective access to and conformational changes in the bound fibrinogen.

Platelet stimulation results in the release of endogenous platelet fibrinogen which binds to the platelet surface. Previous studies have demonstrated that plasma fibrinogen bound to activated platelets becomes inaccessible to a variety of probes. We have studied endogenous platelet fibrinogen binding to activated platelets by employing an immunopurified polyclonal anti-fibrinogen antibody and F26, a monoclonal anti-fibrinogen antibody, which recognizes fibrinogen only when it is bound to a surface. Employing the Ig or F(ab')2 of the poly- or monoclonal antibody we found a marked decrease of fibrinogen accessibility 30-60 min after platelet activation. In contrast, platelet-bound fibrinogen remains accessible to the Fab fragment of F26 at a constant level for 30 min and increases at 60 min. The reduction of the polyclonal Fab fragment binding at 30 and 60 min is similar to the F26 Ig. These results indicate that the decreased accessibility of bound fibrinogen is related to two mechanisms; (1) that the access route to fibrinogen in size selective for the antibody probes and only small antibody probes, e.g. Fab fragments, can gain access to fibrinogen and (2) fibrinogen undergoes a conformational change(s) after binding which exposes at least one neo-epitope in the D domain of fibrinogen and which may decrease or mask the reactivity of other fibrinogen domains. Only the F26 Fab probe has full access to and identifies fibrinogen present on the platelet surface 60 min after stimulation.


Two distinct patterns of glycosylphosphatidylinositol (GPI) linked protein deficiency in the red cells of patients with paroxysmal nocturnal haemoglobinuria.

We have studied three glycosylphosphatidylinositol (GPI) linked proteins on the erythrocytes of 14 patients with paroxysmal nocturnal haemoglobinuria (PNH). The pattern observed was bimodal in 12 of the patients and trimodal in two. Ten patients had a red cell population with normal CD59 antigen (membrane inhibitor of reactive lysis, MIRL), decay accelerating factor (DAF or CD55) and lymphocyte function-associated antigen (LFA-3 or CD58) and a second abnormal PNH population with absent CD59 antigen, DAF and LFA-3. The other two patients with a bimodal pattern had a red cell population with normal CD59 antigen, DAF and LFA-3 and an abnormal population with reduced, but not absent, CD59 antigen and DAF. The LFA-3 on the abnormal red cells in these two patients appeared to be only slightly reduced. The two patients with a trimodal pattern had a normal population, a population with reduced, not absent, CD59 antigen and DAF, and a population with complete absence of CD59 antigen, DAF and LFA-3. The accuracy of the Ham test in estimating the proportion of red cells with the PNH defect in the two types of PNH was assessed. The case of one patient who appeared to be 'rescued' from severe aplastic anaemia by the development of PNH is described.


Childhood polyclonal T cell lymphocytosis with neutropenia: effects of antilymphocyte globulin and granulocyte colony stimulating factor in vitro and in vivo.

The pathogenesis of the neutropenia that occurs in some patients with chronic T cell lymphocytosis is not well understood. We have investigated a 15-year-old girl with this syndrome. Initial committed bone marrow progenitor numbers (CFUgm) were low but markedly increased in vitro following T cell depletion. Similarly a transient correction of neutropenia was observed following in vivo lymphocyte depletion with antilymphocyte globulin. A sustained neutrophil recovery was achieved with daily therapy using recombinant human granulocyte colony stimulating factor (rhG-CSF) despite persistence of the lymphocytosis; during successful therapy CFUgm numbers remained low, and were not increased by the in vitro addition of rhG-CSF. These observations suggest the possibility of an inhibitory regulatory mechanism specifically acting on neutrophil granulopoiesis.


Rapid identification by denaturing gradient gel electrophoresis of mutations in the gamma-globin gene promoters in non-deletion type HPFH.

We have outlined a fast, non-radioactive strategy to identify point mutations in the 5' flanking region of the gamma-globin genes using denaturing gradient gel electrophoresis (DGGE) of amplified DNA. In a sample of previously characterized carriers of non deletion-type hereditary persistence of fetal haemoglobin (HPFH) the different point mutations in both gamma gene promoters could be easily identified by DGGE of a 327 bp fragment. A 4 bp deletion at position -225 to -222 of the A gamma globin gene was unexpectedly found in several samples and shown to represent a frequent polymorphism. Analysis of a 681 bp fragment specific for the 5' region of the A gamma gene, showed that this can be used to determine the haplotype of the chromosome under study. This technique may be useful in the study of sequence variations associated with high Hb F expression in physiological and pathological conditions.


Human immunodeficiency virus reverse transcriptase: steady-state and pre-steady-state kinetics of nucleotide incorporation.

Steady-state and pre-steady-state kinetic constants were determined for reverse transcriptase catalyzed incorporation of nucleotides and nucleotide analogues into defined-sequence DNA primed-RNA templates. 3'-Azido-3'-deoxythymidine 5'-triphosphate (AZTTP) was almost as efficient a substrate (kcat/Km) as dTTP for the enzyme. In contrast, the four 2',3'-dideoxynucleoside 5'-triphosphates and 3'-deoxy-2',3'-didehydrothymidine 5'-triphosphate (d4TTP) were 6-30-fold less efficient substrates of the enzyme. The kcat values for all nucleotide analogues were similar, consistent with a kinetic model in which the steady-state rate-limiting step was dissociation of the template-primer from the enzyme [Reardon, J. E., & Miller, W. H. (1990) J. Biol. Chem. 265, 20302-20307]. The pre-steady-state kinetics of single-nucleotide incorporation were consistent with the kinetic model: [formula: see text] where E, TP, and dNTP represent reverse transcriptase, a defined-sequence DNA primed-RNA template, and 2'-deoxynucleoside 5'-triphosphate (or analogue), respectively. The dissociation constant (Kd1) for template-primer binding was 10 nM, and the estimated rate constants for association and dissociation of the enzyme.template-primer complex were 4 x 10(6) M-1 s-1 and 0.04 s-1, respectively. The dissociation constants (Kd2) for dTTP, AZTTP, and 3'-deoxythymidine 5'-triphosphate (ddTTP) were 9, 11, and 4.6 microM, respectively. Thus, the differences in steady-state Km values were not due to differences in binding of the nucleotide analogues to the enzyme. In contrast, the rate-limiting step during single-nucleotide incorporation (kp) was sensitive to the structure of the nucleotide substrate.(ABSTRACT TRUNCATED AT 250 WORDS)


Distinct alpha-subunit structures of human insulin receptor A and B variants determine differences in tyrosine kinase activities.

Human insulin receptor isoforms (HIR-A and -B) differ in their alpha-subunit structures which result from alternatively spliced precursor mRNAs. This structural difference causes distinct binding affinities for insulin. To determine the impact of the structural difference on receptor signaling, we characterized the tyrosine kinase activity of HIR-A and HIR-B in vitro and determined the insulin stimulated beta-subunit phosphorylation and tyrosine kinase activation in the intact cell. When 32P incorporation in beta-subunits of equal amounts of isolated HIR-A and HIR-B was measured, an increased 32P incorporation in tyrosine residues of the beta-subunit of HIR-B (2.5-fold) compared to that of HIR-A was found after in vitro insulin stimulation. This was paralleled by an increased rate of phosphorylation (2.0-fold) or poly(GluNa,Tyr 4:1). In vitro analysis of Km values for ATP were similar for HIR-A (Km = 14.3 microM +/- 3.8) and HIR-B (Km = 20.2 microM +/- 8.6), whereas the Vmax of HIR-B was significantly increased (HIR-A Vmax = 5.5 mumol/60 min micrograms-1 +/- 1.4, HIR-B Vmax = 42.5 mumol/60 min micrograms-1 +/- 19.2). HPLC analysis of tryptic beta-subunit phosphopeptides revealed identical patterns, suggesting that the difference in kinase activities is not due to an alteration of the phosphorylation-activation cascade within the beta-subunit. However, when cleavage of the alpha-subunit by short-time trypsinization was used to activate the tyrosine kinase, the differences in 32P incorporation between HIR-A and HIR-B were abolished.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of membrane potential on band 3 conformation in the human erythrocyte membrane detected by triplet state quenching experiments.

The triplet lifetime and absorption anisotropy decay of eosin-labeled band 3 was measured in resealed erythrocyte ghosts. Membrane potentials were generated by the addition of valinomycin in the presence of a K+ gradient. Neither negative nor positive membrane potentials had any detectable effect on the rotational diffusion of band 3 nor on the eosin triplet lifetime. The membrane potential did, however, affect quenching of the eosin triplet state by I- and TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl). Quenching was enhanced by a negative membrane potential (negative inside) and reduced by a positive membrane potential. In addition, it was found that a negative membrane potential enhanced the efficiency of eosin labeling of band 3 in intact erythrocytes. A positive membrane potential had the opposite effect. These results indicate that the eosin binding site on band 3 becomes more accessible to the extracellular aqueous phase in the presence of a negative membrane potential and less accessible in the presence of a positive membrane potential. Quenching by I- and TEMPO of the triplet state of eosin-labeled band 3 was further investigated as a function of pH. Quenching by TEMPO and its dependence on membrane potential were relatively insensitive to pH. In contrast, the rate of quenching by I- showed a marked decrease over the range pH 5.5-9.5. Moreover, the effect of a negative membrane potential on I- quenching also varied with pH. These results are discussed on the supposition that the eosin probe is located in the anion access channel of band 3.(ABSTRACT TRUNCATED AT 250 WORDS)


Comparison of hydrogen exchange rates for bovine pancreatic trypsin inhibitor in crystals and in solution.

Hydrogen exchange rate constants for the 17 slowest exchanging amide NH groups in bovine pancreatic trypsin inhibitor (BPTI) were measured in solution and in form II and form III crystals. All 17 amide hydrogens are buried and intramolecularly hydrogen bonded in the crystal structure, except Lys 41 which is buried and hydrogen bonded to a buried water. Large-scale crystallization procedures were developed for these experiments, and rate constants for both crystal and solution exchange were measured by 1H NMR spectroscopy of exchange-quenched samples in solution. Two conditions of pH and temperature, pH 9.8 and 35 degrees C, and pH 9.4 and 25 degrees C, bring two groups of hydrogens into the experimental time window (minutes to weeks). One consists of the 10 slowest exchanging hydrogens, all of which are associated with the central beta-sheet of BPTI. The second group consists of seven more rapidly exchanging hydrogens, which are distributed throughout the molecule, primarily in a loop or turn. In both groups, most hydrogens exchange more slowly in crystals, but there is considerable variation in the degree to which the exchange is depressed in crystals. Many differences observed for the more rapidly exchanging hydrogens can be attributed to local surface effects arising from intermolecular contacts in the crystal lattice. Within the slower group, however, a very large effect on exchange of Ile 18 and Tyr 35 appears to be selectively transmitted through the matrix of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of intracellular signals on Na+/K(+)-ATPase pump activity in the frog skin epithelium.

The effects of intracellular signals (pHi, Na+i, Ca2+i, and the electrical membrane potential), on Na+ transport mediated by the Na+/K+ pump were investigated in the isolated Rana esculenta frog skin. In particular we focussed on pHi sensitivity since protons act as an intrinsic regulator of transepithelial Na+ transport (JNa) by a simultaneous control of the apical membrane Na+ conductance (gNa) and the basolateral membrane K+ conductance (gK). pHi changes which modify JNa, gNa and gK, do not affect the Na+ transport mediated by the pump as shown by kinetic and electrophysiological studies. In addition, no changes were observed in the number of 3H-ouabain binding sites in acid-loaded epithelia. Our attempts to modify cellular Ca2+ (by using Ca(2+)-free/EGTA Ringer solution or A23187 addition) also failed to produce any significant effects in the Na+ pump turnover rate or the number of 3H-ouabain binding sites. The Na+ pump current was found to be sensitive to the basolateral membrane potential, saturating for very positive (cell) potentials and a reversal potential of -160 mV was calculated from I-V relationships of the pump. Changes in Na+i considerably affected the Na+ pump rate. A saturating relationship was found between pump rate and Nai+ with maximal activation at Nai+ greater than 40 mmol/l; a high dependence of the pump rate and of the number of 3H-ouabain binding sites was observed in the physiological range of Nai+. We conclude that protons (in the physiological pH range) which act directly and simultaneously on the passive transport pathways (gNa and gK), have no direct effect on the Na+/K+ pump rate. After an acid load, the inhibition of JNa is primarily due to the reduction of gNa. This results in a reduction of Nai and the pump turnover rate then becomes dependent on other pathways of Na+ entry such as the basolateral membrane Na+/H+ exchanger.


Monte Carlo studies of a model for lipid-gramicidin A bilayers.

This paper presents results of Monte Carlo simulations of a full bilayer of 200 lipid chains and one gramicidin A dimer. Simulations are described for systems with lipid chains of 14, 16, and 18 carbons, respectively. Using accepted potential functions to calculate interactions between all non-hydrogen atoms a Monte Carlo configuration sampling is generated from which order parameter profiles are calculated and specific configurations are displayed. Results are compared with experimental data for lipid-gramicidin bilayers.


The stability and functional properties of proteoliposomes mixed with dextran derivatives bearing hydrophobic anchor groups.

Liposomes composed of Escherichia coli phospholipid were coated with polysaccharides bearing hydrophobic palmitoyl anchors. The effect on the stability of liposomes without or with integral membrane proteins was investigated. A high concentration of hydrophobized dextrans protected the liposomes against detergent degradation, decreased the fluidity of the membranes, prevented fusion of the liposomes and enhanced their stability. Proteoliposomes containing beef heart cytochrome-c oxidase and the lactose transport carrier of E. coli were similarly affected by coating with the dextrans. Under these conditions both membrane proteins were still active. Long-term stability of the coated liposomes was obtained only in the absence of the integral membrane proteins.


Properties of plasma membrane-induced amylase release from rat parotid secretory granules: effects of Ca2+ and Mg-ATP.

A secretory granular fraction (SG) and a plasma membrane rich fraction (PM) have been isolated from rat parotid gland by differential and Percoll gradient centrifugation. With these two fractions, a cell-free interaction system has been reconstituted to clarify the exocytotic interaction between the secretory granules and plasma membranes, and the conditions of amylase release from SG have been characterized in vitro. The addition of PM into this assay system induced a rapid and transient release of amylase from SG. Some other membranes such as erythrocyte ghosts also mimicked the effect of PM. This release was increased by Ca2+, but was not completely blocked by EGTA. Simultaneous addition of 1 mM ATP with 1 mM MgCl2 (Mg-ATP) in the presence of Ca2+ reduced this release. However, in spite of the existence of Mg-ATP, the stimulation of PM-induced amylase release was caused by Ca2+ in a concentration-dependent manner (10(-7)-10(-3) M). These results suggest that Ca2+ and Mg-ATP should participate as important regulators in the exocytotic interaction between secretory granules and plasma membranes in this system. Furthermore, the differences between our system and intact cells are also discussed.


Peroral pharyngeal block for placement of esophageal endoprostheses.

The placement of plastic peroral endoprostheses frequently is done in the United States as a palliation for esophageal cancer. However, the combination of topical local anesthetics and sedatives, the most commonly used means to achieve anesthesia, can cause complications and often does not adequately suppress the gag reflex. The purpose of this study was to compare sedation requirements in patients receiving the standard topical local anesthetic versus patients receiving peroral pharyngeal plexus block. From December 1987 through April 1991, 11 patients underwent endoscopic esophageal stent placement. The first six patients received topical 10% lidocaine spray, the other five patients received pharyngeal plexus blocks. Supplemental sedation was given until the patient closed their eyes but were responsive to verbal stimuli. Completeness of block was evaluated by stimulating the posterior oropharynx. Total sedative requirements were recorded for each patient. Patients receiving pharyngeal plexus block had profound anesthesia and suppression of the gag reflex, as determined by examination and the patient's tolerance of the procedure. Patients receiving only topical anesthesia and intravenous sedation tolerated the procedure poorly and required a greater amount of intravenous sedation than those in the blocked group (p less than 0.01). There were no anesthetic complications in patients receiving pharyngeal blocks. Our experience indicates that the endoscopic placement of esophageal endoprostheses is optimally performed with the aid of pharyngeal plexus block. This block provides profound anesthesia with minimal risk in debilitated, high-risk patients. The neuroanatomy of the oropharynx is also reviewed.


Effects of analogs of the DNA minor groove binder Hoechst 33258 on topoisomerase II and I mediated activities.

By contrast with other DNA minor groove binders, Hoechst 33258 inhibited topoisomerase-mediated activity in intact cells. To determine whether specific structural alterations could modify the topoisomerase reactivity of this drug, a series of analogs of Hoechst 33258 (compound 1) was examined. When the relative DNA binding affinities (Ka) of these agents were determined, compound 1 had the highest Ka while agents with substitutions in either of the benzimidazole moieties showed reduced affinity. Whether these changes in DNA binding correlated with topoisomerase inhibitory potency was next examined. In isolated nuclei, 25 microM of agents 1, 5 and 7 reduced VM-26 induced cross-links by 64, 65 and 83%, compared with 15 to 25% reductions by agents 2, 3, 4 and 6, respectively. The structural modification common to the less active compounds was the substitution of an oxygen for nitrogen at either position 1 or 2. On the basis of these results, agents 1, 2, 3 and 7, representing a range of inhibitory potency, were chosen for further analyses. Cross-link induction by m-AMSA and camptothecin in isolated nuclei, as well as by VM-26 in intact cells, was inhibited to a greater extent by agents 1 and 7 than 2 or 3. Additionally, all four drugs inhibited relaxation of pBR 322 DNA induced by both topoisomerases, although topoisomerase I was 2 to 5-fold more sensitive than topoisomerase II. A linear correlation was observed between the logarithms of the Ka value of compounds 1, 2 and 3 and their IC25 values for both topoisomerases, suggesting a strong dependence on DNA binding affinity for enzyme inhibition. Nevertheless, agent 7, despite having less affinity for calf thymus DNA than 1, was the most potent topoisomerase inhibitor tested in intact cells and in isolated enzyme systems. Thus, retention of nitrogen at positions 1 and 2 as well as the addition of nitrogen at position 16 was associated with increased topoisomerase inhibitory potency.


Analysis of a gene cluster coding for the Xenopus laevis U7 snRNA.

A cluster of Xenopus laevis U7 snRNA genes has been isolated and sequenced. The gene structure is more compact than, but otherwise comparable to, the major U snRNA genes since the distal sequence element (DSE) is located only 4 nt upstream of the PSE. The corresponding RNA is present in the oocyte and accumulates early in oogenesis.


Molecular cloning of substance P receptor cDNA from guinea-pig uterus.

A cDNA encoding guinea-pig uterine substance P (SP) receptor has been isolated using the homology screening approach. Northern blot analysis reveals that the corresponding mRNA, of approx. 4.8 kb, is expressed in all tissues tested, but predominantly in the uteri of non-pregnant animals; during pregnancy its expression is reduced. The guinea-pig SP receptor was expressed in COS-7 cells and demonstrated relative ligand affinity in the order: SP much greater than neurokinin A greater than neurokinin B.


Recovery kinetics after chemotherapy and circulating mononuclear cells expressing the CD34 antigen in pediatric cancer patients.

Hematopoietic progenitor cells collected from the peripheral blood are capable of restoring hematopoiesis after myeloablative therapy. The numbers of circulating mononuclear cells expressing the CD34 antigen were calculated and the colony-forming capacity was determined in 26 blood samples, which were drawn during rapid rise of leukocytes after chemotherapy cycles that were followed by aplasia. Culture assay after 14 days revealed a median 507 (210-2029) myeloid progenitors (CFU-GEMM/GM) per 10(5) nucleated cells (NC) in 13 CD34-positive samples, and only a median 76 (9-224) in 13 CD34-negative ones (p less than 0.001). Median 343 (175-2450) erythroid burst-forming units (BFU-E) per 10(5) NC were detected in the CD34-positive samples, whereas only 72 (10-315) per 10(5) NC were found in the negative ones (p less than 0.01). The percentage of CD34-positive cells clearly correlated with the growth of CFU-GEMM/GM and BFU-E (p less than 0.01). The content of CD34-positive cells in circulation was determined within 120 min by FACS analysis and predicted colony-forming capacity of circulating mononuclear cells. These observations will help to select the optimal individual days for leukaphereses.


Developmental regulation of granulocyte-macrophage colony-stimulating factor production during human monocyte-to-macrophage maturation.

Cells of the macrophage lineage are a major source of various cytokines and hematopoietic growth factors. With regard to the growth factors acting on cells of their own lineage, macrophage colony-stimulating factor (M-CSF) has been proven to be secreted by monocytes (MO) and macrophages (MAC), whereas the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by human MO/MAC is under debate. Here we report that in elutriation-purified MO, as well as in MAC derived from cultured MO, GM-CSF m-RNA was regularly induced by LPS. In MO the GM-CSF message was still detectable 18 h after stimulation under serum-free conditions, but in contrast was already lost at this time point in MAC. Secreted GM-CSF protein was detected in the culture medium using a sandwich ELISA. Furthermore, a factor-dependent cell line (M-07) was used for a biological assay. Here, a neutralizing anti GM-CSF antibody specifically blocked the proliferation-inducing activity of MO/MAC supernatants. Whereas only small amounts of GM-CSF were detected in MO, its secretion increased severalfold upon MO-to-MAC differentiation in vitro. A similar increase upon in vitro maturation of MO was observed for the production of granulocyte colony-stimulating factor. The highest amounts of GM-CSF (up to 2.8 ng/10(6) cells) were produced by MAC that had been derived from MO cultured under serum-free conditions in the presence of 0.5 mg/ml albumin as the only medium supplement.


Vibrational CD of the amide II band in some model polypeptides and proteins.

The amide II vibrational CD (VCD) spectra of poly (L-glutamic acid) and poly (L-lysine) in various conformational forms and those of several proteins in H2O have been measured. Characteristic VCD patterns have been observed in the amide II region due to helix, beta-sheet, and coil conformations in polypeptides. Based on their x-ray crystal structures, the proteins studied have been assigned to six categories. Proteins in the same category give rise to similar amide II VCD. While the protein conformational type is indicated using the amide II VCD, discrimination between types is less characteristic than with the previously studied amide I' VCD in D2O.


Ultrastructure of alpha 2-macroglobulins.

New results concerning the ultrastructure of human alpha 2-macroglobulin (alpha 2M) molecules are presented in connection and comparison with the historical, the current and our own most recent, even unpublished results on the structure and function of alpha 2M and related proteins. The electron microscopic approach uses classical negative staining, combined with the new imaging mode "Electron Energy Loss Spectroscopy", which provides unusual contrast, resolution and readability of the electron micrographs. Immuno- and cryoelectron microscopy, as well as image processing has provided new data necessary to the building of tentative 3D models of the molecule. A model for the native tetrameric alpha 2M is described for the first time, and tries to explain and gather the various observations, sometimes contradictory, taken from different laboratories. A revised version for a model of the methylamine- and proteinase-transformed forms of alpha 2M is also shown. The probable positions of the bait regions and the thiol esters are given on both models. We confirm that alpha 2M is a twin trap capable of inactivating one or two proteinases by partial immobilization. Preliminary results on the production of crystals of alpha 2M-chymotrypsin complexes are also presented. A critical analysis of our models is presented in comparison with others. The technical limitations reached with some techniques and some possible extensions of future research in the field are also presented.


Auditory-visual word identification test materials: computer application with children.

The purpose of this investigation was to obtain performance data from normal-hearing children using the written word portion of existing auditory-visual word identification materials, for example, the Picture Identification Task (Wilson and Antablin, 1980). The response foils, consisting of four written words (target and alternative words) were entered into computer memory so that responses were made by pointing to a computer monitor. Subjects were 24 fourth graders with normal hearing who repeated auditory-stimulus words in noise in oral conditions, and pointed to written words representing the stimulus words in pointing conditions. Mean percent correct performance scores were higher in the pointing conditions than in the oral conditions, reflecting closed- versus open-set tasks. The results indicated that the written word portion of the Picture Identification Task can be utilized to assess the word identification performance of children who read at a fourth grade level or above.


Reorganization of the ependyma during axolotl spinal cord regeneration: changes in intermediate filament and fibronectin expression.

Changes in intermediate filament content and extracellular matrix material showed that the injury response of ependymal cells in lesioned axolotl spinal cord involves an epithelial-to-mesenchymal transformation, and that fibrous astrocytes are excluded from the remodeling lesion site. Antibody localization was used to visualize cytokeratin-, vimentin-, and glial fibrillary acidic protein- (GFAP-) containing intermediate filaments, as well as the adhesive glycoprotein fibronectin. In normal axolotl spinal cord cytokeratins were found near the apical surface of the ependymal cells. Transmission electron microscopic examination suggested that these cytokeratins were in tonofilaments. Cytokeratin expression was lost and vimentin production was initiated in ependymal cells 2-3 weeks following spinal cord injury. There was a period of approximately 1-2 weeks when cytokeratins and vimentin were co-expressed in vivo. This co-expression was maintained in vitro by culture on a fibronectin-coated substratum. As the central canal reformed, vimentin expression was lost. Ependymal cells lacked GFAP intermediate filaments, but GFAP was present in fibrous astrocytes of the neuropil and white matter. Following injury, GFAP localization showed that fibrous astrocytes disappeared from the remodeling lesion site and reappeared only after the ependymal epithelium reformed and newly myelinated axons were found. Fibronectin expression closely followed the expression of vimentin during mesenchymal ependymal cell outgrowth. These results suggest that the ependymal cell outgrowth requires changes in cell shape followed by changes in production of extracellular matrix.


Alkylation of DNA with aziridine produced during the hydrolysis of N,N',N''-triethylenethiophosphoramide.

A reaction pathway by which thiotepa (N,N',N''-triethylenethiophosphoramide) and tepa (N,N',N''-triethylenethiophosphoramide), its major metabolite in humans, alkylate and depurinate DNA involves hydrolysis to aziridine (ethylene imine), a highly reactive monofunctional alkylating agent. Hydrolytic cleavage of an N-P bond of thiotepa releases aziridine which reacts with DNA, resulting in depurination and formation of the stable N-7 adduct 7-(2-aminoethyl)guanine and an aminoethyl adduct of adenine. Chromatographically identical alkylated products were observed in the reaction of thiotepa and tepa with individual nucleosides. Adducts with deoxycytidine or thymidine were not detected. Aziridine was measured by HPLC after derivatization with 1,2-naphthoquinone 4-sulfate. On the basis of the identity of the DNA adducts and the rate of formation of aziridine by hydrolysis in vitro, thiotepa is concluded to be a lipophilic, stabilized form of aziridine which serves as a cell-penetrating carrier of aziridine.


The role of hyaluronan-binding protein in assembly of pericellular matrices.

Hyaluronan-dependent pericellular matrices or "coats" are expressed by a variety of cell types in culture and modulation of their expression may be important in regulation of cell interactions in vivo during development. Monoclonal antibody IVd4, which recognizes hyaluronan-binding protein with the properties of a hyaluronan receptor, was shown to block formation of these coats by a variety of cells. Using rat fibrosarcoma cells, it was found that the antibody not only blocked initial formation of the coats but also caused their loss when added subsequent to formation. The loss of preformed coats in the presence of antibody occurred at 4 degrees and 37 degrees, implying that the function of hyaluronan-binding protein in coat formation is not in mediating metabolic processes. The antibody also had no significant effect on hyaluronan production by the fibrosarcoma cells. In addition, hyaluronan hexasaccharide, a competitive inhibitor of the interaction between polymeric hyaluronan and its cell surface receptor, was found to inhibit coat formation. Thus it is concluded that a hyaluronan-binding protein with the properties of a hyaluronan receptor is required for pericellular matrix formation.


Comparative study of Langerhans cells in normal and pathological human scars. I. Atrophic scars.

In the present study, we investigated Langerhans cells (LCs) in the epidermal component of human atrophic scars, comparing them with those in control skin and normotrophic scars. A preliminary analysis of the histological features was first carried out on vertical serial sections, stained with hematoxylin and eosin. The total epidermal thickness and the thickness of the single epidermal layers were then measured, by means of a digitizing tablet and a morphometric program run on an Apple IIe computer. These parameters were found to be significantly lower (40%) in atrophic scars, if compared to control skin and normotrophic scars (p less than 0.05). CDla-positive and HLA-DR-positive LCs were marked by indirect immunofluorescence. Their position among the epidermal layers, their dimensions, their density and their morphology were examined. In atrophic scars, LCs were densely and evenly distributed in all the epidermal layers. Their density was increased (about 1200 cells/mm2 of epidermal area), if compared to control skin and normotrophic scars (both 300-400 cells/mm2 of epidermal area; p less than 0.001). The CDla-positive definite cell bodies, exhibiting an unstained nucleus, were as large as those evidentiated in the normotrophic scars and twice as much the control skin values (p less than 0.001). The present results provide morphological data that distinguish atrophic scars from control skin and normotrophic scars, and suggest an involvement of the Langerhans cells in this particular case of pathological scarring.


Comparative study of Langerhans cells in normal and pathological human scars. II. Hypertrophic scars.

Langerhans cells (LCs) seem to play a crucial role in the immune system of the skin. Changes in their density, distribution, phenotype and/or morphology have been described in a number of skin diseases, mostly immunologically mediated. For this reason, we investigated LCs in human hypertrophic scars, since these scars are presently believed to have an immunological basis. A preliminary analysis of the histological features was carried out on vertical serial sections, stained with hematoxylin and eosin. Both epidermal and dermal components of hypertrophic scar biopsies were examined. The total epidermal thickness and the thickness of the single epidermal layers were also measured; the values obtained were similar to those of control skin and normotrophic scars. Subsequently, CDla-positive LCs, revealed by indirect immunofluorescence and immunoperoxidase techniques, were studied to determine their position among the epidermal layers and within the dermis, their dimensions, their density and their morphology. According to these observations, two main types of hypertrophic scars were identified. In the first type (7 scars), LCs were widely clustered within both the whole epidermis and the dermis. Their density was increased (about 750 cells/mm2 of epidermal area), if compared to control skin and normotrophic scars (both about 400 cells/mm2 of epidermal area; p less than 0.001). The epidermal cell profiles, nearly three times larger than those of control skin, exhibited a dense network of interconnected dendrites. Further analysis for the presence of HLA-DR molecules revealed an anomalous expression of these antigens on keratinocytes. In the second type (3 scars), LCs density within the stratum Malpighii was unchanged, relative to control skin and normal scars, while CDla-positive cell bodies remained numerous in basal position and within the subpapillary corion. Epidermal LCs, only slightly larger than those evidentiated in control skin, displayed short and retracted dendritic projections. The aberrant expression of HLA-DR antigens on keratinocytes was very weak and sparse. The present results strongly suggest an immunologically activated state of the tissues examined; they provide morphological data that support the involvement of the immune system in hypertrophic scarring.


A novel class of nuclear RNA (nRNA) with a long life span as a gene repressor candidate.

Different systemic organs of fetal mice were continuously labelled with 5-3H-uridine during the organogenesis periods, and chased for various lengths of time after birth. In the autoradiographs made on paraffin-embedded sections of the organs taken after the chase for longer periods than 1 week, including a 12-months chase, specific labels were present exclusively in all the nuclei. The specific nuclear labels were resistant to RNase A or H digestion and to acid hydrolysis with 1 N HCl at 60 degrees C for 5 min, but were completely abolished by DNase digestion or prolonged acid hydrolysis for 10 min, the optimum condition for the Feulgen reaction to stain DNA. Electrophoretic analysis of the total nucleic acids extracted from the different organs chased for 3 or 12 months showed all the tritium radioactivity to be present in the DNA fraction before digestion with DNase or RNase A, and to be completely absent from the DNA fraction and shifted to the RNA fraction, or to be largely destroyed by degradation, after each digestion, respectively. By HPLC analysis of the total nucleic acid extract after further successive digestions with nuclease P1 and alkaline phosphatase into the constituent nucleotides, it was shown that all tritium activity was incorporated in uridine, without any detectable label in other nucleotides. By the simultaneous labeling of human peripheral lymphocytes at the late S-phase with 5-3H-uridine and BrdU, it was demonstrated that the autoradiographic labels, which, this time, were labile to RNase A digestion, were present in the G-bands of the spread chromosomes as identified by BrdU immunohistochemistry. The findings strongly indicate the presence of a novel class of nuclear RNA (nRNA). This type of RNA (a) may be localized in the nucleus in close association or hybridization with nuclear DNA, (b) have a life span as long as that of the cell, and (c) be duplicated in the late phase of DNA replication. The nRNA may play a fundamental role as gene repressor existing in the G-bands of metaphase chromosomes in the process of ontogeny and cytodifferentiation.


Mallory stain may indicate differential rates of RNA synthesis: I. A seasonal cycle in the harderian gland of the green frog (Rana esculenta).

When Mallory's trichrome stain is used, acinar nuclei of the Harderian gland of Rana esculenta display different affinities for the dye. Some of the orangiophilic nuclei show affinity for aniline blue (blue nuclei). In the Harderian gland of Rana esculenta their number and the intensity of staining with aniline blue may vary during the year. The affinity for aniline blue disappears following digestion of paraffin sections with RNAase, but not with DNAase or trypsin. Furthermore, in vitro incubation with [5, 6-3H]-Uridine shows a selective incorporation by the majority of blue nuclei. Therefore, the affinity for aniline blue is likely due to increased RNA synthesis. The increment of nuclear RNA shown by these methods is supported by the quantitative determination of total RNAs during the resumption (October) and enhancement (May) of secretory activity, when the percentage of blue nuclei of the acinar cells is at its highest levels of the year. The affinity of RNA-rich nuclei for aniline blue, while others are strictly orangiophil, is discussed on the basis of molecular structure of the dyes used in the staining mixture. Mallory's trichrome stain appears to be an useful tool for detecting changes in cell nuclear status.


Apoptosis of rat thymocytes triggered by prednisolone, camptothecin, or teniposide is selective to G0 cells and is prevented by inhibitors of proteases.

Rat thymocytes were treated in culture with prednisolone or the DNA topoisomerase I or II inhibitors, camptothecin (CAM) or teniposide (TN), and proportions of cells in different phases of the cell cycle were estimated by flow cytometry using a staining methodology which makes it possible to discriminate between G0 and G1 cells, as well as to recognize the cells which undergo apoptosis. The appearance of apoptotic cells in cultures treated with pharmacological concentrations of these drugs, observed as early as 3-6 hr after treatment, coincided with the selective loss of G0 cells in these cultures, while no significant changes in the proportion of S or G2+M cells were apparent. Agarose gel electrophoresis of DNA isolated from the treated cells indicated degradation of the internucleosomal spacer sections, typical of the endonucleolytic activity which accompanies apoptotic cell death. The data indicate that G0 thymocytes were particularly sensitive to agents that induce apoptosis while cells progressing through the cell cycle were resistant. This suggests that under in vivo conditions (immunological response), the selective death of G0 cells may promote the clonal expansion of stimulated thymocytes which enter the cell cycle. Together with our earlier studies on the effects of CAM and TN on MOLT-4 and HL-60 leukemic cell lines, these data indicate that both, phenotypic- and and cell cycle phase specific- factors modify the ability of cells to respond to toxic agents, including chemotherapeutics by apoptosis.(ABSTRACT TRUNCATED AT 250 WORDS)


A simple method for insertion of an intraprostatic coil.

A simplified method of insertion of a urethral coil for treatment of benign prostatic obstruction has been tested in 25 patients. The prostatic urethra is measured either by abdominal/transrectal ultrasound scanning or by rigid or flexible endoscopy. The coil is inserted into the prostatic urethra using simple measurements taken from a Foley catheter; 21 coils were correctly positioned at the first attempt using this new method--a success rate equivalent to that of ultrasound-guided insertion.


Circulating prostate specific antigen-positive cells correlate with metastatic prostate cancer.

Analytical flow cytometry was used to study circulating prostate specific antigen (PSA)-positive cells in 40 consecutive patients with newly diagnosed, untreated prostate cancer; 25 patients (63%) had metastatic disease confirmed by a positive bone scan. Cell suspensions were prepared for each patient from both the primary tumour and peripheral blood samples. The cells were stained with a monoclonal antibody against PSA, and analysed by flow cytometry; PSA-positive cells were sorted according to their immunofluorescence and light scatter properties. The cellular deoxyribonucleic acid (DNA) content of each specimen was also analysed to establish ploidy status. PSA-positive cells were detected in the peripheral blood of 33 patients (83%). The presence of these cells in the circulation showed a higher degree of sensitivity and specificity in predicting positive bone scans than did serum PSA levels. Circulating PSA-positive cells may represent either a subpopulation of tumour cells with distinct metastatic properties or, alternatively, host immunocytes which take up PSA in an active or passive manner.


[The relationship between weights of fetuses with autosomal trisomies and low maternal serum alpha-fetoprotein].

An association between low maternal serum alpha-fetoprotein (MSAFP) and fetal trisomy has now been thoroughly documented. The mechanisms resulting in low MSAFP are still unclear. In order to determine whether the low values of MSAFP in autosomal trisomies are associated with smaller fetal weights, we compared 11 fetuses with Down syndrome (trisomy 21), 4 with trisomy 18, with 45 normal fetuses. All of them are aborted in the second trimester of pregnancy. No significant difference in the weight distribution between fetuses with Down syndrome and control fetuses was found. In contrast, as compared with the control fetuses, fetuses with trisomy 18 had a significant lower weight distribution. The mean value of MSAFP was 0.72 +/- 0.26 MoM for those with Down syndrome and 0.51 +/- 0.33 MoM for fetuses with trisomy 18; both results being significantly lower than that of normal control (1.01 +/- 0.28). The mean value of amniotic fluid AFP was 0.63 +/- 0.23 MoM for fetuses with Down syndrome. This value was significantly lower than those of the fetuses with trisomy 18 and normal controls (1.02 +/- 0.09 and 1.02 +/- 0.30). A linear relationship between MSAFP and fetal weight was found in normal fetuses at a given gestational age but was not found in trisomy pregnancy. Fetal weight cannot be used to explain the reason for low MSAFP in Down syndrome pregnancy but may partially account for the lower levels noted in fetuses with trisomy 18.


Mast cell ionic channels: significance for stimulus-secretion coupling.

The activation of mast cells (MC) due to immunological stimulation causes an immediate and dramatic inflammatory response. We review current evidence indicating that the membrane permeabilities for calcium, chloride, sodium, and potassium have a significant role in the activation of these cells, and in some cases, specific ionic channels have been identified. Moreover, a number of intracellular mechanisms controlling these channels are pointed out, including different classes of G proteins, intracellular calcium, cAMP, and products of phosphoinositol breakdown. However, the interplay between factors controlling membrane conductances for different ions is not currently understood. The diversity of ionic effects on MC activation is depicted, illustrating that the ionic mechanisms of MC activation are specific for different MC types. Since nerve/mast cell interaction is a key element in the burgeoning field of neuroimmunology, we discuss the role of ionic channels as targets of neurotransmitter action in MC activation.


In vivo circumvention of human colon carcinoma resistance to bleomycin.

Metabolic inactivation of bleomycin (BLM) by cysteine proteinase-like enzymes is thought to be a major mechanism of BLM tumor resistance. We now report that the human colon carcinoma COLO-205 is highly resistant to BLM and that E-64, a cysteine proteinase inhibitor, sensitizes COLO-205 to BLM. Treatment of COLO-205-bearing nude mice with either E-64 (40 mg/kg) or BLM (10 mg/kg) alone did not inhibit COLO-205 growth. However, pretreatment with E-64 prior to BLM prevented these xenografts from growing. Analysis by high performance liquid chromatography of in vivo BLM metabolism following [3H]BLM A2 treatment of COLO-205-bearing nude mice showed a different metabolic profile among the various organs and the tumor. Whereas [3H]BLM A2 was the only major radioactive peak detected in sera and tumors, several metabolites, including deamido-BLM A2, were found in kidney, liver, and lung as early as 15 min. Pretreatment of mice with E-64 inhibited tumor, kidney, and lung BLM A2 metabolism. Furthermore, pretreatment with E-64 increased BLM A2 accumulation in tumors (6.1-fold), kidney (4.0-fold), lung (2.8-fold), liver (1.8-fold), and serum (1.7-fold). E-64 pretreatment did not enhance the major toxicity of BLM, pulmonary fibrosis, as determined by both lung hydroxyproline levels and histopathology. Thus, the cysteine proteinase inhibitor E-64 affects the metabolic fate and the levels of accumulation of BLM in vivo. These results demonstrate that resistance of human COLO-205 tumors to BLM can be circumvented by E-64 without enhancement of the major side effect of BLM, suggesting a possible clinical use of this combination therapy.


A phenotypically and karyotypically stable human thyroid epithelial line conditionally immortalized by SV40 large T antigen.

Primary cultures of normal human neonatal thyroid follicular cells were transfected with a plasmid expressing a temperature-sensitive (tsA58) mutant of SV40 large T antigen. An epithelial cell line, designated B-thy-ts.1, was obtained which showed tight temperature-dependent growth. In sharp contrast to previous such lines, which were derived from adult thyroid, B-thy-ts.1 has retained a well-differentiated phenotype as reflected in its morphology and cytokeratin expression pattern. In addition to phenotypic stability the line also displays an unusually stable karyotype, lacking the usual clastogenic effects of SV40, which we speculate to result from a greater DNA repair capacity of its cell of origin. B-thy-ts.1 should be a particularly useful tool with which to study the effects of activated oncogenes on epithelial growth and differentiation.


Three discourse profiles of closed-head-injury speakers: theoretical and clinical implications.

This study examined the variation in narrative discourse production noted in an earlier report of 11 closed-head-injury (CHI) patients and 21 normal speakers. Measures of productivity (fluency), content, and cohesion for two narratives (one elicited visually and the other auditorily) were analysed for each CHI speaker. Three distinct discourse profiles emerged, categorized as follows: confused, impoverished, and inefficient. It is suggested that these data, although involving few subjects in each profile, have important theoretical and clinical implications. Most particularly, a treatment approach based on each discourse profile is presented.


Communication disorders following closed head injury: new approaches to assessment and rehabilitation.

In this paper communication disorders following closed head injury are described and various forms of language assessment techniques are reviewed. The usefulness of conventional, pragmatic and cognitive approaches is considered. A new cognitive-pragmatic approach is then advocated which is an amalgam of the last two approaches. From the examples given it is argued that such an approach is practical in terms of: 1) incorporating knowledge of co-existing cognitive deficits; and 2) focusing on normal everyday communication practices. It is also suggested that the use of a cognitive-pragmatic framework has potential in refining remediation and management strategies with the closed-head-injured population.


Expression of N-myc, c-myc, and MDR-1 proteins in newly established neuroblastoma cell lines: a study by immunofluorescence staining and flow cytometry.

A methodology for rapid isolation of neuroblastoma cells from marrow with metastatic neuroblastoma cells was developed using a cocktail of five antibodies and magnetic microspheres coated with secondary antibodies. Cells bound to microspheres were released by brief exposure to chymopapain, followed by repeated culture of released cells in serum-supplemented Dulbecco's modified Eagle's medium and selection for adherent cells. Using this methodology, over 35 primary cell lines were obtained free of contaminating normal cells. Detailed analyses of over 14 cell lines revealed gross differences in cell phenotype, size, morphology development of neurite processes, and doubling time (40 to 80 h). All cell lines expressed the M(r) 145,000 neurofilament, and a few expressed the M(r) 200,000 neurofilament, with very little or no expression of the M(r) 68,000 neurofilament. Eight % of all cells lines had near-diploid DNA content. High expression of the MDR-1 protein was detected in six of the 22 cell lines tested. Great heterogeneity was observed in the expression of N-myc oncoprotein, with ten of 13 patients overexpressing the protein. c-myc oncoprotein was also expressed in all cell lines; however, the level of expression was 4- to 10-fold lower than the N-myc oncoprotein. Localization studies of c-myc and N-myc oncoproteins on the level of light microscopy and electron microscopy revealed exclusive nuclear localization of c-myc, whereas N-myc was localized to the nucleus and to the cytoplasm.


Genetics of cystic fibrosis.

The cystic fibrosis gene specifies a membrane transport protein and over 100 disease-causing mutations have now been identified. Although at least some of these determine the incidence of pancreatic disease, there is no association between a particular mutation and the severity of lung and liver disease.


Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways.

The receptor for platelet-derived growth factor (PDGF) binds two proteins containing SH2 domains, GTPase activating protein (GAP) and phosphatidylinositol 3-kinase (PI3-kinase). The sites on the receptor that mediate this interaction were identified by using phosphotyrosine-containing peptides representing receptor sequences to block specifically binding of either PI3-kinase or GAP. These results suggested that PI3-kinase binds two phosphotyrosine residues, each located in a 5 aa motif with an essential methionine at the fourth position C-terminal to the tyrosine. Point mutations at these sites caused a selective elimination of PI3-kinase binding and loss of PDGF-stimulated DNA synthesis. Mutation of the binding site for GAP prevented the receptor from associating with or phosphorylating GAP, but had no effect on PI3-kinase binding and little effect on DNA synthesis. Therefore, GAP and PI3-kinase interact with the receptor by binding to different phosphotyrosine-containing sequence motifs.


Influenza virus M2 protein has ion channel activity.

The influenza virus M2 protein was expressed in Xenopus laevis oocytes and shown to have an associated ion channel activity selective for monovalent ions. The anti-influenza virus drug amantadine hydrochloride significantly attenuated the inward current induced by hyperpolarization of oocyte membranes. Mutations in the M2 membrane-spanning domain that confer viral resistance to amantadine produced currents that were resistant to the drug. Analysis of the currents of these altered M2 proteins suggests that the channel pore is formed by the transmembrane domain of the M2 protein. The wild-type M2 channel was found to be regulated by pH. The wild-type M2 ion channel activity is proposed to have a pivotal role in the biology of influenza virus infection.


Molecular cloning and nucleic acid binding properties of the GAP-associated tyrosine phosphoprotein p62.

p62 is a tyrosine phosphoprotein that associates with p21ras GTPase-activating protein (GAP). Purification and cDNA cloning of p62 reveal extensive sequence similarity to a putative hnRNP protein, GRP33. Recombinant human p62 purified from insect Sf9 cells binds to DNA and to mRNA and, like many proteins involved in mRNA processing, recombinant p62 is modified by dimethylation on multiple arginine residues. p62 also binds tightly to p21ras GAP in vitro: this binding depends on phosphorylation of p62 on tyrosine residues and occurs through SH2 regions of GAP. These data suggest that p120-GAP and p62 play a role in some aspect of mRNA processing or utilization and that this role may be regulated by tyrosine phosphorylation, and indirectly, by p21ras.


Primary culture of liver cancer tissues with or without transcatheter arterial embolization and establishment of a cell strain.

More epithelial-like cells are found in primary cultures of transcathetel arterial embolization (TAE)-untreated human liver cancer tissues than in those of TAE-treated tissues. A new cell strain, HUH-33, established from the former, grows slowly and is untransplantable into nude mice and secretes alpha-fetoprotein, albumin and some other tumor markers. Chromosome numbers are widely distributed. HUH-33 expresses hepatocyte type keratin and contains desmosome- or intermediate filament bundle-like structures.


Pentafraction reduces the lung lymph response after endotoxin administration in the ovine model.

For the past half-century, several high molecular weight compounds have been used for volume expansion during cardiopulmonary resuscitation. However, the effectiveness and side effects of these different expanders are varied. We have compared plasma, pentastarch, and a new product, pentafraction, for effective plasma volume expansion before and after tissue injury with endotoxin administration. In each group, eight range ewes instrumented with a Swan-Ganz, arterial, and venous catheters, and lung and flank lymphatic cannulas were compared. Each group received 15 ml/kg of either 6% pentafraction, 6% pentastarch, or plasma followed two hours later by 1.5 micrograms/kg/0.5 hr E. Coli endotoxin over 30 min. Data were collected for an additional 24 hr after endotoxin administration. Our results indicated a plasma volume expansion in all three groups. However, the prior administration of pentafraction significantly attenuated the increase in the lung lymph flow and early evaluation of systemic vascular resistance noted with endotoxin in comparison to the other two groups.


Profile of renal permselectivity by simultaneous enzyme-linked immunosorbent assay of albumin, transferrin, IgG, and alpha 1-microglobulin with a new microplate reader.

A competitive enzyme-linked immunosorbent assay (ELISA) is described for determining a renal permselectivity profile involving the urinary proteins albumin, transferrin, IgG, and alpha 1-microglobulin (alpha 1-m). The ELISA reader uses a computer-controlled array of multiplexed light-emitting diode (LED)-photodiode pairs for rapid measurements of absorbance on microplates. A 3,3'-dimethylnaphthidine reagent adapts the 3,5,3',5'-tetramethylbenzidine chromophore to monochromatic LED emission at 550 nm. We applied this ELISA to the determination of renal permselectivity in healthy children and young adults and in children with insulin-dependent diabetes mellitus. The geometric means (and SD) of protein excretion rates in a group of 85 normal subjects were as follows: albumin, 3.5 micrograms/min (1.83); transferrin, 173 ng/min (2.76); IgG, 1.11 micrograms/min (2.22), and alpha 1-m, 0.98 microgram/min (2.36).


Ultrasensitive radioimmunoassay of prostate-specific antigen.

We describe a modification of the Yang Pros-check radioimmunoassay for prostate-specific antigen (PSA) that increases the analytical sensitivity of the assay approximately threefold (from a working range of 0.3-50 to 0.1-1.2 micrograms/L). It can detect PSA added to zero-concentration diluent (bovine serum albumin solution) at 0.10 microgram/L or added to zero-concentration control female serum at 0.20 microgram/L (P less than 0.05). In 26 patients tested after cystoprostatectomy for bladder cancer (who had normal prostates without cancer on histologic examination), PSA values by this ultrasensitive assay were all less than 0.10 microgram/L. Therefore, we propose this value as the upper limit of the 95% reference interval. In a retrospective study of two patients who developed recurrent prostate cancer, serum PSA values increased above the 0.1 microgram/L detection limit 175 and 581 days before increasing above the 0.3 microgram/L detection limit of the standard Yang assay. This ultrasensitive radioimmunoassay of PSA should prove more useful than current methods for detecting early recurrence of prostate cancer.


Glomerular deposition of complex-forming glycoprotein heterogenous in charge (protein HC) in IgA nephropathy.

IgA immune complexes and polymeric IgA are presumed to play important roles in the development and progression of IgA nephropathy. Complex-forming glycoprotein heterogenous in charge (protein HC), being inhibitors of neutrophilic chemotaxis, has been reported as binding to IgA. As a working hypothesis it was assumed that complexes of protein HC and IgA are present in glomeruli from IgA nephropathy patient in stable state. In this study, we examined the glomerular deposition of protein HC in 40 patients with IgA nephropathy and in 10 patients with non-IgA nephropathy. We used highly specific antibody against protein HC, that does not cross-react with alpha-1-microglobulin. An immunofluorescent study revealed that 10 out of the 40 patients (25%) showed an intensity of 1+, 16 (40%) showed weak positive (+/-), and the other 14 (35%) were negative. There was no deposition of protein HC in non-IgA nephropathy patients. Histopathological analysis demonstrated a significant correlation between the intensity of glomerular-deposited protein HC and pathological activity (p less than 0.005); the latter was defined as having either crescents in more than 15% of the remaining glomeruli (excluding global sclerotic glomeruli), or segmental necrosis or sclerosis in more than 30% of the remaining glomeruli. A significant correlation was observed between pathological activity and the intensity of deposited IgG, IgA and IgM (p = 0.01), and lambda chain (p less than 0.005). Considering anti-inflammatory activity of protein HC, these results suggest that protein HC cannot protect sufficiently acute inflammation or tissue damages due to co-deposited IgG and IgM and/or other factors.(ABSTRACT TRUNCATED AT 250 WORDS)


Redistribution of natural killer (NK) cell frequency and NK cytotoxic activity in primary IgA nephropathy.

Recent findings have indicated an imbalance of immune responsiveness in primary IgA nephropathy (IgAN). Thus natural killer (NK) cell frequency and NK cytotoxicity were evaluated in fifteen IgAN patients. CD8+, CD11+, CD56+ and CD57+ lymphocyte percentages in IgAN individuals fell within normal values, while a significant decrease of CD16+ cells was observed in the same group of patients. In contrast, NK activity overlapped that seen in controls as assessed by an agarose-single cell cytotoxic assay. To further investigate the discrepancy between CD16+ cell level and NK cytotoxic activity in IgAN, the proportion of CD11+ CD57+, CD56+ CD16+ and CD57+ CD16+ lymphocytes was determined. In spite of the unaffected CD56+ CD16+ cell frequency, IgAN subjects exhibited a significant decrease of CD11+ CD57+ and CD57+ CD16+ lymphocyte percentages in comparison to controls. It is suggested that a redistribution of NK lymphocyte subsets occurs in IgAN. This may have an important role in the impairment of the immunoregulatory network.


Effect of caffeine and isobutyl-methyl-xanthine on production of binucleate cells and on post-replicative repair.

Caffeine (CAF) but not 3-isobutyl 1-methyl-xanthine (IBMX) displayed a strong DNA anti-repair effect in G2 prophase, as estimated by the frequency of abnormal chromosomes in anaphase-telophase found shortly after treatment. IBMX is more effective than CAF in inhibiting cytokinesis, while the binucleate cells formed by a short treatment with any of these two xanthines have similar cycle kinetics.


Effects of verapamil on postextrasystolic potentiation.

To assess the effects of verapamil on postextrasystolic potentiation (PESP), contrast left ventriculography was performed in ten healthy anesthetized dogs before and after the intravenous administration of verapamil, 0.1 mg/kg. During the contrast ventriculography, a single atrial premature stimulus was introduced. Ejection fractions of a control beat and postpremature beat were measured before and after verapamil. Before verapamil, the mean ejection fractions of the control beat and the postpremature beat were 60 +/- 10 percent and 67 +/- 10 percent, respectively (p less than 0.05). Following the administration of verapamil, the mean ejection fraction of the control beat decreased from 60 +/- 10 percent to 55 +/- 11 percent (p less than 0.05). However, the mean ejection fraction of the postpremature beats increased when compared with the control beats following intravenous verapamil (65 +/- 8 percent and 55 +/- 11 percent, respectively; p less than 0.05). These results suggest that PESP is not inhibited by the administration of intravenous verapamil.


Intra-articular injection pentosanpolysulphate results in increased hyaluronan molecular weight in joint fluid.

The influence of an oversulphated glycosaminoglycan, pentosanpolysulphate, on hyaluronan metabolism of the synovial lining cell was studied in vivo in human volunteers. Significant increases in the mean degree of polymerisation of the hyaluronan chains were observed after a series of four to six intra-articular injections of this glycosaminoglycan. No increases in hyaluronan synthesis rates were observed. Repeated administration of the drug did not cause any inflammation or bleeding in the joint cavity.


Characterization and specificity controls of murine monoclonal antibodies against serogroup C1 Salmonella.

Two IgG3 murine monoclonal antibodies, Cl-1 and Cl-2, that showed serologic specificities for the O antigens of serogroup C1 (0:6,7) Salmonella were established. The epitopes for the antibodies were found to reside on the repeating units of the serogroup C1 Salmonella lipopolysaccharide and were labile to sodium metaperiodate oxidation. Serologic reactivities of Cl-1 and Cl-2 were not inhibited by commercial monospecific antiserum to O antigen 7, but were inhibited to various degrees by anti-[O:6,7] serum. Both antibodies reacted strongly with all strains of serogroup C1 Salmonella that have either O:6(1),7, O:6(2),7, or O:6(1,2),7 antigens. Reactivities of Cl-1 and Cl-2 with the phage-14 lysogenized C1 strains that bear the phage-modified O antigen (O:6,7----O:6,7,14) were detected by slide agglutination method only and not by whole-cell enzyme-linked immunosorbent assay. Both Cl-1 and Cl-2 antibodies did not react with other O serogroups of salmonellae, nor with other Gram-negative or Gram-positive bacteria. The diagnostic value of these monoclonal antibodies together with a previously described monoclonal antibody against the serogroup C2 Salmonella was demonstrated using the slide agglutination method with monoclonal antibodies ascitic fluids.


Endogenous event-related potentials as indices of dementia in multiple sclerosis patients.

Auditory event-related potentials (ERPs) were recorded in a "double oddball" paradigm requiring an easy and a hard pitch discrimination from multiple sclerosis (MS) patients with and without dementia, and a group of age and sex matched normal subjects. Cognitive function was assessed by a short battery of neuropsychologic (NP) tests, and the two groups of MS patients were selected on the basis of substantial non-overlapping degrees of cognitive deficit in the demented as compared to the non-demented group. The N100, P200 and P300 ERP components were longer in latency in the demented patients, and the N100-P300 interval was prolonged as well, compared to the non-demented patients, whose ERP latencies did not differ from those of the normal subjects. Increased P300 latency was associated with poorer performance on the NP tests, especially those sensitive to impairment of learning and retrieval from memory. The reaction times of both patient groups were prolonged as compared to the controls, whereas the accuracy of the demented patients was significantly poorer than that of the non-demented patients.


An event-related potential study of controlled and automatic processes in 6-8-year-old boys with attention deficit hyperactivity disorder.

Event-related potentials (ERPs) were recorded from 2 groups (attention deficit hyperactivity disorder (ADHD) and normal control) of 12 boys aged from 6 to 8 years. Subjects were submitted to 2 different types of categorization tasks (with rare targets and frequent standards) implying either the use of a verbal class or that of an ordered series. Each type of task was performed twice, the first with reading and the second without. Amplitudes and latencies of a fronto-central N150-P250 complex, a parieto-occipital N250-P350 complex and a parieto-occipital P500 were measured. Regardless of the task, hyperactive children showed larger fronto-central P250, larger parieto-occipital N250 and smaller parieto-occipital P350s and P500s; moreover, the latencies of their parieto-occipital P350s were shortened. When the categorization depended on the use of a verbal class, ERP reading effects were significantly smaller in hyperactives than in normal controls for the parieto-occipital waves only. Alternatively, the target effects were significantly larger in hyperactive children but for the fronto-central P250 only. These results suggest that in ADHD automatic processes were enhanced when higher-order controlled processes were inadequate.


Intermodal selective attention. I. Effects on event-related potentials to lateralized auditory and visual stimuli.

The effects of intermodal selective attention on event-related brain potentials (ERPs) were examined in 2 experiments. In experiment 1, auditory ERPs were compared (1) when subjects responded to easy and difficult-to-detect target tones in sequences of tone bursts; and (2) when they ignored the same auditory sequences and played a demanding video game. In experiment 2, auditory ERPs to tone bursts and visual ERPs to vertical line gratings were compared as subjects responded to difficult-to-detect targets in one modality or the other. Attention to auditory stimuli resulted in biphasic enhancements in auditory ERPs, the Nda (negative auditory difference wave, latency 120-160 msec) and the Pda (positive auditory difference wave, latency 200-240 msec) waves. These had longer latencies and somewhat different scalp distributions than N1 and P2 components evoked by non-attended tones. The Nda and Pda could be contrasted with the monophasic processing negativities typically found in dichotic selective attention tasks. Nda amplitudes were larger for difficult-to-detect targets (closely resembling standards) than for standards themselves, but no Ndas were recorded to highly deviant targets. Deviant auditory stimuli evoked mismatch negativities (MMNs) that persisted during visual attention. MMN amplitudes to difficult-to-detect deviants were enlarged with attention, but no change was found in MMN amplitudes to easy-to-detect deviants. In experiment 2 intermodal attention was associated with biphasic changes in visual ERPs over the posterior scalp: the occipital Pdv (100-130 msec), and contralateral-temporal Ndv (120-320 msec) deflections. Deviant visual stimuli also elicited mismatch negativity/N2b components, largest over the inferotemporal cortex contralateral to the stimulated visual field. Like the auditory MMN, the MMN increased in amplitude with attention, but it was also evident during attend auditory conditions. The results suggest that sustained, intermodal attention depends primarily in processing modulations in modality-specific cortex. We found no evidence of the participation of modality non-specific cortex. This excludes the possibility that intermodal attention depends on a single, supramodal attention system. The relatively long latency of intermodal effects suggests that they may depend on the reafferent (top down) modulation, and do not index "template matching" operations.


Intermodal selective attention. II. Effects of attentional load on processing of auditory and visual stimuli in central space.

The effect of processing load on event-related brain potentials (ERPs) was investigated in an intermodal selective attention task in which subjects attended selectively to auditory or visual stimuli. Processing load was manipulated by requiring subjects to detect either difficult-to-detect (deviant) or easy-to-detect (DEVIANT) targets in separate blocks of trials. Attention to auditory stimuli was associated with negative (Nda, 90-170 msec) and positive (Pda, 190-270 msec) enhancements in the ERPs to auditory stimuli. The Nda increased in amplitude with increasing processing load. Deviant auditory stimuli occurring among auditory standard stimuli elicited frontally distributed mismatch negativities (MMNs). The MMN persisted during visual attention and was unaffected by visual processing load. However, the MMN to deviants but not DEVIANTS was enhanced in amplitude with auditory attention. Attention to visual stimuli resulted in positive (Pdv, latency 70-130 msec) and negative (Ndv, 170-270 msec) modulations of visual ERPs, that increased with increasing processing load. Prominent visual deviance-related negativities were observed at occipital and infero-temporal scalp sites (latencies 90-290 msec), but only to DEVIANT visual stimuli. The early MMN-like portion of the visual deviance-related negativity was independent of attention, with equal amplitudes during different auditory and visual conditions.


ERP components related to stimulus selection processes.

Event-related potential (ERP) components associated with target stimulus selection in a double discrimination visual task were studied. The experimental paradigm consisted in the presentation of low intensity stimuli that varied according to two physical features: geometrical form (squares and circles) and location (a spot in different positions inside the stimulus). Subjects performed 3 tasks on these stimuli: control task in which they looked passively at the stimuli, and 2 discrimination tasks, in which they had to respond to a certain stimulus (a specific conjunction of form and spot location). The early components (P1 and N1) obtained in the control and discrimination tasks were associated with sensory analysis of simple stimulus features. Relevance of a particular feature modified the latency and/or the area of these components. The longer-latency components (N2 and P3) were elicited only in the discrimination tasks. N2 was associated with target stimulus selection because its area was significantly larger for target stimuli and because its "offset" latency correlated with choice reaction time. Results are discussed and contrasted with various models of target selection.


Vestibular short latency responses to pulsed linear acceleration in unanesthetized animals.

Linear acceleration transients were used to elicit vestibular compound action potentials in non-invasively prepared, unanesthetized animals for the first time (chicks, Gallus domesticus, n = 33). Responses were composed of a series of up to 8 dominant peaks occurring within 8 msec of the stimulus. Response amplitudes for 1.0 g stimulus ranged from 1 to 10 microV. A late, slow, triphasic, anesthesia-labile component was identified as a dominant response feature in unanesthetized animals. Amplitudes increased and latencies decreased as stimulus intensity was increased (MANOVA P less than 0.05). Linear regression slope ranges were: amplitudes = 1.0-5.0 microV/g; latencies = -300 to -1100 microseconds/g. Thresholds for single polarity stimuli (0.035 +/- 0.022 g, n = 11) were significantly lower than those of alternating polarity (0.074 +/- 0.028 g, n = 18, P less than 0.001). Bilateral labyrinthectomy eliminated responses whereas bilateral extirpation of cochleae did not significantly change response thresholds. Intense acoustic masking (100/104 dB SL) produced no effect in 2 animals, but did produce small to moderate effects on response amplitudes in 7 others. Changes were attributed to effects on vestibular end organs. Results of unilateral labyrinth blockade (tetrodotoxin) suggest that P1 and N1 preferentially reflect ipsilateral eighth nerve compound action potentials whereas components beyond approximately 2 msec reflect activity from vestibular neurons that depend on both labyrinths. The results demonstrate that short latency vestibular compound action potentials can be measured in unanesthetized, non-invasively prepared animals.


Relationship between vertex potentials and magnitude of pre-pain and pain sensations evoked by electrical skin stimuli.

Vertex potentials evoked by painful and non-painful electrical skin stimuli were recorded in 10 healthy male volunteers. Peak-to-peak amplitudes of the major vertex potential components were compared with subjective rating of the stimuli. While a significant correlation was observed between peak-to-peak amplitudes and stimulus rating following non-painful stimuli, generally no such correlation was present following stimulations above the pain threshold. These findings suggest that vertex potentials elicited by electrical skin stimuli may reflect central processing of non-noxious afferent information rather than of pain-related information.


A simple format for exchange of digitized polygraphic recordings.

A simple digital format supporting the technical aspects of exchange and storage of polygraphic signals has been specified. Implementation of the format is simple and independent of hard- or software environments. It allows for any local montages, transducers, prefiltering, sampling frequencies, etc. At present, 7 laboratories in various countries have used the format for exchanging sleep-wake recordings. These exchanges have made it possible to create a common database of sleep records, to compare the analysis algorithms local to the various laboratories to each other by applying these algorithms to identical signals, and to set up a computer-aided interlaboratory evaluation of manual and automatic analysis methods.


A chloroplast gene is required for the light-independent accumulation of chlorophyll in Chlamydomonas reinhardtii.

The light-independent pathway of chlorophyll synthesis which occurs in some lower plants and algae is still largely unknown. We have characterized a chloroplast mutant, H13, of Chlamydomonas reinhardtii which is unable to synthesize chlorophyll in the dark and is also photosystem I deficient. The mutant has a 2.8 kb deletion as well as other rearrangements of its chloroplast genome. By performing particle gun mediated chloroplast transformation of H13 with defined wild-type chloroplast DNA fragments, we have identified a new chloroplast gene, chlN, coding for a 545 amino acid protein which is involved in the light-independent accumulation of chlorophyll, probably at the step of reduction of protochlorophyllide to chlorophyllide. The chlN gene is also found in the chloroplast genomes of liverwort and pine, but is absent from the chloroplast genomes of tobacco and rice.


Identification of the gene(s) coding for the trans-sialidase of Trypanosoma cruzi.

The gene(s) encoding the Trypanosoma cruzi shed-acute-phase-antigen (SAPA) has a 5' end encoding a region containing two totally and two partially conserved Ser-X-Asp-X-Gly-X-Thr-Trp motifs which are present in bacterial neuraminidases, and a 3' end encoding tandemly repeated units of 12 amino acids. It is now reported that 54-87% of the total neuraminidase activity present in the parasite could be immunoprecipitated with polyclonal or monoclonal antibodies against the repeated amino acid units of SAPA. These immunoprecipitates also had greater than 80% of the trans-sialidase activity of the parasite. SAPA used sialyllactose, fetuin and 4-methylumbelliferyl-sialic acid as substrate donors. In the presence of a suitable acceptor molecule (lactose) the sialic acid residues were transferred to the disaccharide, whereas in the absence of acceptors the residues were transferred to water. If relatively inefficient acceptors (maltose or cellobiose) were added to the incubation mixtures, the sialic acid units were transferred both to the disaccharides and to water. It is concluded that a major T. cruzi antigen has both the trans-sialidase and the neuraminidase activities of the parasite. Both activities are probably located on the N-terminus of SAPA since antibodies directed against the C-terminus, which contains the repeated amino acid units, do not affect the enzymatic activities.