Alpha 4/beta 1 integrin (VLA-4) ligands in arthritis. Vascular cell adhesion molecule-1 expression in synovium and on fibroblast-like synoviocytes. Expression of vascular cell adhesion molecule-1 (VCAM-1) in synovial tissue was determined using the immunoperoxidase technique. Normal, rheumatoid arthritis (RA), and osteoarthritis (OA) synovia bound VCAM-1 antibodies in the intimal lining as well as blood vessels. The amount of VCAM-1 was significantly greater in the synovial lining of RA and OA tissues compared with normal synovium (p less than 0.002). There was also a trend toward greater levels of VCAM-1 staining in blood vessels of arthritic tissue (RA greater than OA greater than normal). Because VCAM-1 staining was especially intense in the synovial lining, VCAM-1 expression and regulation was studied on cultured fibroblast-like synoviocytes (FLS) derived from this region. Both VCAM-1 and intercellular adhesion molecule 1 were constitutively expressed on FLS. VCAM-1 expression was further increased by exposure to IL-1 beta, TNF-alpha, IL-4, and IFN-gamma. These cytokines (except for IL-4) also induced intercellular adhesion molecule 1 expression on FLS. ELAM was not detected on resting or cytokine-stimulated FLS. The specificity of VCAM-1 for FLS was demonstrated by the fact that only trace amounts were detected on normal and RA dermal fibroblasts. Cytokines induced intercellular adhesion molecule 1 display on dermal fibroblasts but had minimal effect on VCAM-1 expression. Finally, in adherence assays, Jurkat cell binding to resting FLS monolayers was inhibited by antibody to alpha 4/beta 1 integrin (VLA-4), CS-1 peptide from alternatively spliced fibronectin (which is another VLA-4 ligand), and, to a lesser extent, anti-VCAM-1 antibody. After cytokine stimulation of FLS, Jurkat-binding significantly increased, and this increase was blocked by anti-VCAM-1 antibody. Therefore, both CS-1 and VCAM-1 participate in VLA-4-mediated adherence to resting FLS in vitro, and VCAM-1 is responsible for the increase in Jurkat binding mediated by cytokines.


Characterization of a tolerogenic T cell epitope of type II collagen and its relevance to collagen-induced arthritis. A synthetic peptide representing sequences of type II collagen, (CII 245-270), has previously been used to induce tolerance and suppress arthritis in DBA/1 mice. To determine important residues, a series of peptides, each containing one or two site-directed substitutions, was generated. Mononuclear cells from DBA/1 mice immunized with CII were cultured in the presence of each peptide and the T cell response determined by measuring IFN-gamma in culture supernatant fluids. Substitutions within the region CII 260-270 led to significant decreases in IFN-gamma responses, identifying this sequence as a T cell epitope. To determine the effects of substitutions within this epitope on arthritis, substituted peptides were administered to neonatal mice as tolerogens. Five site-directed substitutions, four of which included the insertion of a residue found in type I collagen to replace its type II counterpart, abrogated the ability of the peptides to induce tolerance and suppress arthritis. These substitutions were located at residues 260, 261, 263, 264, and 266. Two patterns of T cell reactivity were observed. Peptides containing individual substitutions at positions 261, 264, or 266 were capable of generating a significant T lymphokine response, although those containing substitutions at residues 260 or 263 were ineffective Ag. Systematic analysis of the fine structures of T cell determinants important for autoimmune arthritis can lead to strategies for therapeutic intervention.


The immunopathology of acute experimental allergic encephalomyelitis induced with myelin proteolipid protein. T cell receptors in inflammatory lesions. To determine whether there is predominance of T cells expressing a particular TCR V beta chain in the inflammatory lesions of an autoimmune disease model, TCR expression was analyzed in central nervous system (CNS) tissues of mice with experimental allergic encephalomyelitis (EAE). Acute EAE was induced in SJL/J mice either by sensitization with a synthetic peptide corresponding to myelin proteolipid protein residues 139-151 or by adoptive transfer of myelin proteolipid protein peptide 139-151-specific encephalitogenic T cell clones. Mice were killed when they showed clinical signs of EAE or by 40 days after sensitization or T cell transfer. Cryostat CNS and lymphoid tissue sections were immunostained with a panel of mAb to T cell markers and proportions of stained cells were counted in inflammatory foci. In mice with both actively induced and adoptively transferred EAE, infiltrates consisted of many CD3+, TCR alpha beta+, and CD4+ cells, fewer CD8+ cells, and small numbers of TCR gamma delta+ cells. Approximately 30% of CD45+ leukocytes in the inflammatory foci were T cells. Cells expressing TCR V beta 2, 3, 4, 6, 7 and 14 were detected in the infiltrates, whereas TCR V beta 8 and 11, which that are deleted in SJL mice, were absent. When EAE was induced by transfer of T cell clones that use either V beta 2, 6, 10, or 17, there was also a heterogeneous accumulation of T cells in the lesions. Similar proportions of TCR V beta+ and gamma delta+ cells were detected in EAE lesions and in the spleens of the mice. Thus, at the time that clinical signs are present in acute EAE, peripherally derived, heterogeneous TCR V beta+ cells are found in CNS lesions, even when the immune response is initiated to a short peptide Ag or by a T cell clone using a single TCR V beta.


Isolation of sequences from a random-sequence expression library that mimic viral epitopes. We describe the use of random peptide sequences for the mapping of antigenic determinants. An oligonucleotide with a completely degenerate sequence of 17 or 23 nucleotides was inserted into a bacterial expression vector. This resulted in an expression library producing random hexa- or octapeptides attached to a beta-galactosidase hybrid protein. Mimotopes, or antigenic sequences that mimic an epitope, were selected by immunoscreening of colonies with monoclonal antibodies, which were specific for antigenic sites on the spike protein of the coronavirus transmissible gastroenteritis virus. We report one mimotope for antigenic site II, eight for site III and one for site IV. The site III and site IV mimotopes were closely similar to the corresponding linear epitopes, localized previously in the amino acid sequence of the S protein. An alignment of the site II mimotope and the sequence of the S protein around Trp97, which is substituted in escape mutants, suggests that this mimotope mimics a conformational epitope located around residues 97-103. Applications of mimotopes to epitope mapping, serodiagnosis and vaccine development are discussed.


Kinetics of killing by monoclonal cytotoxic T lymphocytes. I. Colorimetric detection of cryptic CTL determinants on adherent target cells and survivorship analysis. Some targets of cell-mediated cytolysis do not efficiently release 51Cr but manifestly are killed in direct viability assays. We characterize and validate an alternative and non-radioactive (colorimetric) method for measuring killing of adherent targets by monoclonal CTL. The method obviates concerns about the effects of trypsinization, is technically simple, quantitative and in some cases more sensitive than conventional 51Cr assays. Target loss obeyed first-order kinetics with respect both to [CTL] and time. These results are consistent with an exponential (Poisson) model of killing and support the use of a single kinetic parameter to describe the lytic activity of monoclonal CTL on adherent targets. When monoclonal CTL are used at appropriate effector:target ratios (less than or equal to 1:1), the residuals obtained after least squares linear regression are homoscedastic and normally distributed, justifying the use of commonly available statistical calculators or programs for the analysis of CTL data.


Analysis of B lymphocyte cross-reactivity at the single cell level. A method is described for assaying whether individual in vivo activated B cells produce antibodies which cross-react with two distinct antigens. The technique utilizes a modification of the ELISA spot assay to determine the isotype and antigenic specificity(s) of these antibody secreting cells. To perform the assay, two plastic slides are coated with different antigens and secured together to form chambers capable of holding a monolayer of Ig secreting lymphocytes. The immunoglobulin secreted during culture binds to one or both sides of the antigen-coated chamber (depending upon the specificity and cross-reactivity of the antibody product). This technique was used to evaluate the cross-reactivity of hybridomas and freshly isolated IgM and IgG secreting B cells.


Detection of rotaviruses in the day care environment by reverse transcriptase polymerase chain reaction. Group A rotavirus is an important cause of morbidity among infants and toddlers in day care centers. Transmission by the fecal-oral route is well established, but fomites and environmental surfaces may also play an important role in transmission. A highly sensitive polymerase chain reaction (PCR) assay was used to detect rotavirus RNA in day care environments. Areas sampled included floors, diaper change areas, toy balls, and other surfaces. In two centers undergoing outbreaks of rotavirus, 7 (39%) of 18 toy balls had detectable rotavirus as did 8 (21%) of 39 swabs from environmental surfaces. By comparison, only 1 (5%) of 21 toy balls and 1 (2%) of 44 environmental surface swabs had detectable rotavirus in centers without rotavirus outbreaks (P = .0001). Thus, rotaviruses are highly prevalent in day care centers during outbreaks of diarrhea. The monitoring of environments by sensitive nucleic acid amplification techniques may lead to strategies for the diminution of disease transmission within the day care environment.


Molecular epidemiology of group B streptococcal infections: use of restriction endonuclease analysis of chromosomal DNA and DNA restriction fragment length polymorphisms of ribosomal RNA genes (ribotyping). Epidemiologic investigation of group B streptococcal (GBS) infections has been limited by the lack of a discriminatory typing system. Therefore, the use of restriction endonuclease analysis of chromosomal DNA (REAC) and DNA restriction fragment length polymorphisms of rRNA genes (ribotyping) to subtype molecularly GBS isolates associated with human invasive disease was investigated. Chromosomal DNA of selected GBS isolates was initially digested with 24 different restriction enzymes. HhaI gave the best discrimination of hybridization banding patterns (ribotypes) and was used with all study isolates. Ribotyping and REAC differentiated among isolates of the same and different serotypes. Nine ribotype patterns were noted among the 76 isolates studied, including 4 among serotype Ia/c and 4 additional ribotypes among serotype III isolates. Epidemiologically related isolates (e.g., mother-infant or twin-twin pairs) had identical REAC and ribotype patterns. Epidemiologically unrelated isolates with the same ribotype usually had different REAC patterns, suggesting that REAC may be a more sensitive technique for strain differentiation. REAC and ribotyping were reproducible and proved to be successful molecular epidemiologic methods for subtyping GBS.


Epitopes of herpes simplex virus type 1 glycoprotein B that bind type-common neutralizing antibodies elicit type-specific antibody-dependent cellular cytotoxicity. A panel of 45 well-characterized monoclonal antibodies (MAbs) reactive to glycoprotein B (gB) of herpes simplex virus (HSV) type 1 was tested by ELISA and in antiviral functional assays (that included virus neutralization, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent complement-mediated lysis), using type 1 or 2 virus strains. All MAbs were ELISA-reactive. Eleven of the MAbs mediated neutralization and 9 mediated ADCC. All of the ADCC epitopes were contained within the amino-terminal half of the extracellular portion of gB. The ADCC reactions were strictly type 1-specific, whereas 9 of 11 neutralizing MAbs exhibited type-common activity. There was some association between the ADCC and neutralization activities, since of 12 MAbs with functional activity, 8 were positive in both assays. These results suggest that differences in the presentation of gB, and perhaps HSV-1 gB versus HSV-2 gB, on free virus and virus-infected cells determine epitope availability.


Neutrophil oxidative burst in response to blastoconidia and pseudohyphae of Candida albicans: augmentation by granulocyte colony-stimulating factor and interferon-gamma. The effects of granulocyte colony-stimulating factor (G-CSF) and interferon-gamma (IFN-gamma) on the oxidative burst of neutrophils (PMNL) in response to blastoconidia and pseudohyphae of Candida albicans were assessed and compared with those in response to N-FMLP. G-CSF enhanced oxidative burst, as measured by superoxide production, in response to both FMLP and opsonized blastoconidia. The enhancement of oxidative burst in response to FMLP was significantly greater (P = .004) than that in response to blastoconidia (65% and 39%, respectively). G-CSF also enhanced oxidative burst in response to pseudohyphae. IFN-gamma enhanced oxidative burst in response to FMLP and opsonized blastoconidia by 53% and 50%, respectively. Moreover, IFN-gamma significantly enhanced oxidative burst in response to opsonized and nonopsonized hyphae by 86% and 65%, respectively. These results demonstrate that G-CSF and IFN-gamma enhance the oxidative burst of PMNL in response to both blastoconidia and pseudohyphae of C. albicans and suggest an immunomodulatory role of the two cytokines in the host defenses against this fungus.


[Sequential changes of HPV DNA, the expression of EGF-R and CD-14, and histopathological findings in HPV infected benign epithelium and dysplastic lesions of the uterine cervix]. In regard to cases in which HPV DNA was detected in the uterine cervix by the Southern blot method, we studied both changes in HPV DNA and histological diagnosis, and moreover we studied the relationship between CD-14 (monocyte/macrophage), EGF-R, and HPV DNA. 1) Changes in HPV DNA, regardless of the HPV type in benign lesions of the uterine cervix, almost all became negative within 1 year. 2) In 1 year benign lesions, the manifestation frequency of CD-14 was higher from the initial examination and during the subsequent 3 months when HPV DNA was detected. 3) HPV DNA in cases of mild dysplasia was detected after 1 year in 41% (7/17), but it continued for 3 years only in 3 cases. 4) Cases of moderate to severe dysplasia, which are though to have developed cancer at an early period of within 1-1.5 years included 26.7% (4/15), and HPV DNA was continually detected in them. The characteristics of these cases were: (1) The HPV 16 type was the main type. (2) The mean age was 34.8 years, which was younger than in regressed cases with a mean age of 43.1 years. (3) The dysplasia occupation rate in the uterine cervix was higher. 5) In 7 cases of moderate to severe dysplasia which regressed from squamous metaplasia, HPV DNA was detected at 1 year in 71.4% (5/7), but at 2-3 years it became undetectable in all cases, so that it has a close relationship with this histological regression.(ABSTRACT TRUNCATED AT 250 WORDS)


Epidermal growth factor stimulates production of insulin-like growth factor-binding protein-1 in human granulosa-luteal cells. Insulin-like growth factor-I (IGF-I) enhances and epidermal growth factor (EGF) inhibits gonadotrophin-induced aromatization in granulosa cells. Our previous studies have shown that human ovarian granulosa cells synthesize insulin-like growth factor-binding protein-1 (IGFBP-1) which inhibits IGF-stimulated DNA synthesis. The present study addresses the effect of EGF and gonadotrophins in the regulation of IGFBP-1 release by human granulosa cells cultured in serum-free medium. At concentrations of 1-100 micrograms/l EGF was found to stimulate IGFBP-1 secretion. This was not due to cell proliferation, as the viable cell count remained unaffected. Growth hormone and gonadotrophins had no effect on IGFBP-1 secretion when added alone to culture medium. These results suggest that EGF regulates IGFBP-1 secretion in human granulosa-luteal cells.


Radioimmunoassay of insulin-like growth factor-binding protein-6 in human serum and other body fluids. A radioimmunoassay has been established for the insulin-like growth factor-binding protein, IGFBP-6, isolated from a human transformed fibroblast cell-line. The binding proteins IGFBP-I and IGFBP-3 did not cross-react, but both IGF-I and IGF-II markedly inhibited IGFBP-6 tracer binding to antiserum. This inhibition, greater for IGF-II than for IGF-I, was fully reversed by the addition of IGFBP-3 to sequester the IGFs. After fractionation of human serum and follicular fluid samples by gel chromatography, interference in the radioimmunoassay by fractions corresponding to the 150 kDa IGF-IGFBP complex could be eliminated by IGFBP-3. The equivalent fractions from cerebrospinal fluid and amniotic fluid fractionation did not interfere in the assay. The mean IGFBP-6 level in adult human serum was 0.221 +/- 0.110 mg/l, with values significantly higher in men than women, and slightly decreased in pregnancy. Similar values were seen in umbilical cord serum and in amniotic and follicular fluid samples, while the mean level in cerebrospinal fluid was slightly lower, 0.152 +/- 0.049 mg/l. This assay will facilitate studies on the regulation of IGFBP-6 production, and its role as an IGF carrier.


Effects of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, on pituitary hormone release in rats. We have demonstrated that the novel hypothalamic peptide pituitary adenylate cyclase-activating polypeptide (PACAP-38; 0.1-100 nmol/l) caused an increase in the release of GH, ACTH, LH and alpha-subunit and accumulation of intracellular cyclic AMP from dispersed rat anterior pituitary cells in static culture for 24 h. There were no significant effects on TSH or prolactin release over the same time-period. PACAP-38 (10 nmol/l) increased the release of GH by 1.3-fold (P less than 0.05), ACTH by 1.9-fold (P less than 0.05), LH by 3.5-fold (P less than 0.001) and alpha-subunit by 2.0-fold (P less than 0.005) and the accumulation of intracellular cyclic AMP by greater than 2-fold (P less than 0.001) after 24 h. However, the time-course for the effect of PACAP-38 (1 mmol/l) on hormone release and intracellular cyclic AMP levels showed a temporal dissociation. The effect of PACAP-38 on GH and ACTH levels did not reach significance until 24 h whereas the effect of PACAP-38 on LH and alpha-subunit release reached significance after 4 h implying a different mechanism of action for their release. To investigate the PACAP-induced secretion of LH and alpha-subunit further, we examined the effects of PACAP after down-regulation of protein kinase C (PKC). PACAP-38 at a dose maximal for the stimulation of LH and alpha-subunit release (10 nmol/l) added together with the PKC activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 0.1 mumol/l) had no greater effect on LH and alpha-subunit release than TPA alone over a 4 h incubation period.(ABSTRACT TRUNCATED AT 250 WORDS)


Growth hormone treatment increases oestrogen receptor concentration in the guinea-pig uterus. Growth hormone does not act as a body growth-promoting hormone during the postnatal period in the guinea-pig. To determine whether it affects uterine and mammary growth, guinea-pigs of 8-9 weeks of age were treated with either recombinant bovine GH (bGH; 0.5 mg per animal) or vehicle for 10 days. Uterine and mammary weights were not changed by treating the animals with bGH. The amount of available cytosolic oestradiol receptor per unit of uterine weight or DNA content, or in whole uteri was increased in bGH-treated animals (7.3- to 14.0-fold) when compared with controls. The nuclear uterine oestradiol receptor concentration was 3.3- to 6.7-fold higher than in controls. The dissociation constant values did not differ between control and bGH-treated animals, suggesting that uterine oestradiol receptors are regulated by GH through changes in the number of binding sites rather than alteration of their binding affinity. Mammary growth and oestradiol receptor levels were unaffected by bGH treatment. The results of this investigation demonstrate that injected bGH selectively affects the oestradiol receptor level in guinea-pig uterine tissue.


Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens. The lymphocyte activation gene 3 (LAG-3), expressed in human activated T and natural killer (NK) cells, is closely related to CD4 at the gene and protein levels. We report here the initial characterization of the LAG-3-encoded protein. We have generated two monoclonal antibodies after immunization of mice with a 30-amino acid peptide that corresponds to an exposed extra loop region present in the LAG-3 immunoglobulin-like first domain. The reactivity of these reagents is directed against LAG-3 since they recognize both membrane-expressed and soluble recombinant LAG-3 molecules produced in a baculovirus expression system. The two antibodies are likely to react with the same or closely related epitope (termed LAG-3.1) exposed on the LAG-3 first domain extra loop, as assessed in competition experiments on LAG-3-expressing activated lymphocytes. Cellular distribution analysis indicated that the LAG-3.1 epitope is expressed on activated T (both CD4+ and CD8+ subsets) and NK cells, and not on activated B cells or monocytes. In immunoprecipitation experiments performed on activated T and NK cell lysates, a 70-kD protein was detected after SDS-PAGE analysis. 45-kD protein species were also immunoprecipitated. Both the 70- and 45-kD proteins were shown to be N-glycosylated. In Western blot analysis, only the former molecule was recognized by the anti-LAG-3 antibodies, demonstrating that it is LAG-3 encoded. These anti-LAG-3 antibodies were used to investigate whether the LAG-3 protein interacts with the CD4 ligands. By using a high-level expression cellular system based on COS-7 cell transfection with recombinant CDM8 vectors and a quantitative cellular adhesion assay, we demonstrate that rosette formation between LAG-3-transfected COS-7 cells and human leukocyte antigen (HLA) class II-bearing B lymphocytes is specifically dependent on LAG-3/HLA class II interaction. In contrast to CD4, LAG-3 does not bind the human immunodeficiency virus gp120. This initial characterization will guide further studies on the functions of this molecule, which may play an important role in immune responses mediated by T and NK lymphocytes.


In vitro proliferation of primitive hemopoietic stem cells supported by stromal cells: evidence for the presence of a mechanism(s) other than that involving c-kit receptor and its ligand. The preadipose cell line, PA6, can support long-term hemopoiesis. Frequency of the hemopoietic stem cells capable of sustaining hemopoiesis in cocultures of bone marrow cells and PA6 cells for 6 wk was 1/5.3 x 10(4) bone marrow cells. In the group of dishes into which bone marrow cells had been inoculated at 2.5 x 10(4) cells/dish, 3 of 19 dishes (16%) contained stem cells capable of reconstituting erythropoiesis of WBB6F1-W/Wv mice, indicating that PA6 cells can support the proliferation of primitive hemopoietic stem cells. When the cocultures were treated with an antagonistic anti-c-kit monoclonal antibody, ACK2, only a small number of day 12 spleen colony-forming units survived; and hemopoiesis was severely reduced. However, when the cocultures were continued with antibody-free medium, hemopoiesis dramatically recovered. To examine the proliferative properties of the ACK2-resistant stem cells, we developed a colony assay system by modifying our coculture system. Sequential observations of the development of individual colonies and their disappearance demonstrated that the stem cells having higher proliferative capacity preferentially survive the ACK2 treatment. Furthermore, cells of subclones of the PA6 clone that were incapable of supporting long-term hemopoiesis expressed mRNA for the c-kit ligand. These results suggest that a mechanism(s) other than that involving c-kit receptor and its ligand plays an important role in the survival and proliferation of primitive hemopoietic stem cells.


H-2-restricted cytolytic T lymphocytes specific for HLA display T cell receptors of limited diversity. We previously showed that H-2Kd-restricted cytotoxic T lymphocyte (CTL) clones specific for a single nonapeptide derived from the Plasmodium berghei circumsporozoite (PbCS) protein displayed T cell receptors (TCRs) of highly diverse primary structure. We have now analyzed the TCR repertoire of CTLs that recognize a peptide derived from the human class I major histocompatibility complex (MHC) molecule HLA-Cw3 in association with the same murine class I MHC molecule H-2Kd. We first sequenced the TCR alpha and beta genes of the CTL clone Cw3/1.1 and, based on this genomic analysis, the TCR alpha and beta cDNA junctional regions of 23 independent H-2Kd-restricted CTL clones specific for HLA-Cw3. The results show that the TCR chains display very limited heterogeneity, both in terms of V alpha, J alpha, V beta, and J beta segments, and in terms of length and sequence of the CDR3 alpha and beta loops. The TCR repertoire used in vivo was then analyzed by harvesting CTL populations from the peritoneal cavity of immune mice. The peritoneal exudate lymphocytes (PELs) displayed HLA-Cw3-specific cytolytic activity in the absence of any stimulation in vitro. Remarkably, most of these freshly isolated PELs expressed TCRs that shared the same structural features as those from HLA-Cw3-reactive CTL clones. Thus, our results show that a peptide from HLA-Cw3 presented by H-2Kd selects CTLs that bear TCRs of very limited diversity in vivo. When taken together with the high diversity of the TCRs specific for the PbCS peptide, these findings suggest that natural tolerance to self peptides presented by class I MHC molecules may substantially reduce the size of the TCR repertoire of CTLs specific for antigenic peptides homologous to self.


Cytotoxic T lymphocytes (CTL) against a transforming gene product select for transformed cells with point mutations within sequences encoding CTL recognition epitopes. The 94-kD large tumor (T) antigen specified by simian virus 40 (SV40) is sufficient to induce cell transformation. T antigen contains four H-2Db-restricted cytotoxic T lymphocyte (CTL) recognition epitopes that are targets for CTL clones Y-1, Y-2, Y-3, and Y-5. These epitopes have been mapped to T antigen amino acids 207-215 (site I), 223-231 (sites II and III), and 489-497 (site V), respectively. Antigenic site loss variant cells that had lost one or more CTL recognition epitopes were previously selected by coculturing SV40-transformed H-2Db cells with the site-specific Db-restricted CTL clones. The genetic bases for T antigen CTL recognition epitope loss from the variant cells were identified by DNA amplification and direct sequencing of epitope-coding regions from variant cell DNAs. Cells selected for resistance to CTL clone Y-1 (K-1; K-1,4,5; K-3,1) carry deleted SV40 genomes lacking site I, II, and III coding sequences. Point mutations present within the site II/III coding region of Y-2-/Y-3-resistant cell lines specify the substitution of asparagine for lysine as T antigen amino acid 228 (K-2) or phenylalanine for tyrosine at position 230 (K-3). Point mutations identified within independently selected Y-5 resistant populations (K-5 and K-1,4,5) direct the substitution of isoleucine for asparagine at position 496 (K-5) or the substitution of phenylalanine for isoleucine at position 491 (K-1,4,5) of T antigen. Each substitution causes loss of the relevant CTL recognition epitope, apparently by compromising CTL T cell receptor recognition. These experiments identify specific amino acid changes within a transforming protein that facilitate transformed cell escape from site-specific CTL clones while allowing maintenance of cellular transformation. This experimental model system provides unique opportunities for studying mechanisms of transformed cell escape from active immunosurveillance in vivo, and for analysis of differential host immune responses to wild-type and mutant cell-transforming proteins.


Lipopolysaccharide (LPS) partial structures inhibit responses to LPS in a human macrophage cell line without inhibiting LPS uptake by a CD14-mediated pathway. Lipopolysaccharides (LPS) that lack acyloxyacyl groups can antagonize responses to LPS in human cells. Although the site and mechanism of inhibition are not known, it has been proposed that these inhibitory molecules compete with LPS for a common cellular target such as a cell-surface binding receptor. In the present study, we used an in vitro model system to test this hypothesis and to evaluate the role of CD14 in cellular responses to LPS. Cells of the THP-1 human monocyte-macrophage cell line were exposed to 1,25 dihydroxyvitamin D3 to induce adherence to plastic and expression of CD14, a binding receptor for LPS complexed with LPS-binding protein (LBP). The uptake of picograms of [3H]LPS (agonist) and enzymatically deacylated LPS [3H]dLPS (antagonist) was measured by exposing the cells to the radiolabeled ligands for short incubation periods. The amounts of cell-associated LPS and dLPS were then correlated with cellular responses by measuring the induction of nuclear NF-kappa B binding activity and the production of cell-associated interleukin (IL)-1 beta. We found that similar amounts of [3H]LPS or [3H]dLPS were taken up by the cells. The rate of cellular accumulation of the ligands was greatly enhanced by LBP and blocked by a monoclonal antibody to CD14 (mAb 60b), yet no cellular responses were induced by dLPS or dLPS-LBP complexes. In contrast, LPS stimulated marked increases of NF-kappa B binding activity and IL-1 beta. These responses were enhanced by LBP and inhibited by mAb 60b. dLPS and its synthetic lipid A counterpart, LA-14-PP (also known as lipid Ia, lipid IVa, or compound 406) strongly inhibited LPS-induced NF-kappa B and IL-1 beta, yet neither antagonist inhibited the uptake of LPS via CD14. dLPS did not inhibit NF-kappa B responses to tumor necrosis factor (TNF) alpha or phorbol ester. Our results indicate that (a) both stimulatory and nonstimulatory ligands can bind to CD14 in the presence of LBP; (b) the mechanism of inhibition by dLPS is LPS-specific, yet does not involve blockade of LPS binding to CD14; and (c) in keeping with previous results of others, large concentrations of LPS can stimulate the cells in the absence of detectable binding to CD14. The findings indicate that the site of dLPS inhibition is distal to CD14 binding in the LPS signal pathway in THP-1 cells, and suggest that molecules other than CD14 are important in LPS signaling.


Cytokine RANTES released by thrombin-stimulated platelets is a potent attractant for human eosinophils. Thrombin stimulation of human platelets results in the release of a preformed proteinaceous human eosinophil (Eo)-chemotactic activity. By the use of different high-performance liquid chromatography techniques, two Eo-chemotactic polypeptides (EoCPs), tentatively termed EoCP-1 and EoCP-2, were purified to homogeneity. Upon SDS-PAGE analysis, these chemotaxins showed molecular masses near 8 kD. NH2-terminal amino acid sequence analysis revealed identical sequences for both EoCP-1 and EoCP-2, which are also identical to that of RANTES, a cytokine that structurally belongs to the interleukin 8 superfamily of leukocyte selective attractants, and that is known to be a "memory-type" T lymphocyte-selective attractant. In the major Eo chemotaxin, EoCP-1, the residues 4 and 5, which in EoCP-2 were found to be serine residues, could not be identified. Electrospray mass spectrometry (ESP-MS) of EoCPs revealed for EoCP-2 a molecular mass of 7,862.8 +/- 1.1 daltons, which is 15.8 mass units higher than the calculated value of RANTES, indicating that EoCP-2 is identical to the full-length cytokine, and oxygenation, probably at methionine residue number 64, has taken place. Upon ESP-MS, EoCP-1 showed an average molecular mass of 8,355 +/- 10 daltons, suggesting O-glycosylation at these serine residues. Both natural forms of RANTES showed strong Eo-chemotactic activity (ED50 = 2 nM) with optimal chemotactic migration at concentrations near 10 nM, however, there were no significant migratory responses with human neutrophils. Chemotactic activity of RANTES for human Eos could be confirmed using recombinant material, which has been found to be as active as the natural forms. Since RANTES gene expression has been detected in activated T lymphocytes, and recombinant RANTES was shown to be a "memory" T lymphocyte-selective attractant, it is now tempting to speculate about an important role of RANTES in clinical situations such as allergene-induced late-phase skin reactions in atopic subjects or asthma, where in affected tissues both memory T cells and Eos are characteristic.


Calmodulin is a subunit of nitric oxide synthase from macrophages. A central issue in nitric oxide (NO) research is to understand how NO can act in some settings as a servoregulator and in others as a cytotoxin. To answer this, we have sought a molecular basis for the differential regulation of the two known types of NO synthase (NOS). Constitutive NOS's in endothelium and neurons are activated by agonist-induced elevation of Ca2+ and resultant binding of calmodulin (CaM). In contrast, NOS in macrophages does not require added Ca2+ or CaM, but is regulated instead by transcription. We show here that macrophage NOS contains, as a tightly bound subunit, a molecule with the immunologic reactivity, high performance liquid chromatography retention time, tryptic map, partial amino acid sequence, and exact molecular mass of CaM. In contrast to most CaM-dependent enzymes, macrophage NOS binds CaM tightly without a requirement for elevated Ca2+. This may explain why NOS that is independent of Ca2+ and elevated CaM appears to be activated simply by being synthesized.


A polyalanine peptide with only five native myelin basic protein residues induces autoimmune encephalomyelitis. The minimum structural requirements for peptide interactions with major histocompatibility complex (MHC) class II molecules and with T cell receptors (TCRs) were examined. In this report we show that substituting alanines at all but five amino acids in the myelin basic protein (MBP) peptide Ac1-11 does not alter its ability to bind A alpha uA beta u (MHC class II molecules), to stimulate specific T cells and, surprisingly, to induce experimental autoimmune encephalomyelitis (EAE) in (PL/J x SJL/J)F1 mice. Most other amino acid side chains in the Ac1-11 peptide are essentially irrelevant for T cell stimulation and for disease induction. Further analysis revealed that binding to A alpha uA beta u occurred with a peptide that consists mainly of alanines and only three of the original residues of Ac1-11. Moreover, when used as a coimmunogen with MBP Ac1-11, this peptide inhibited EAE. The finding that a specific in vivo response can be generated by a peptide containing only five native residues provides evidence that disease-inducing TCRs recognize only a very short sequence of the MHC-bound peptide.


Identification of insulin-like growth factor-binding proteins in the circulation of four teleost fish species. Insulin-like growth factor-binding proteins (IGF-BPs) were demonstrated in the circulation of four teleost fish species. In the coho salmon (Oncorhynchus kisutch), serum binding of 125I-labelled human IGF-I (125I-hIGF-I) was competitively inhibited by addition of excess recombinant bovine IGF-I (rbIGF-I) in a manner similar to that when rat serum was used. Western-ligand blot procedure using the same labelled hormone identified at least three major forms of IGF-BPs in the plasma of all four teleost species investigated: coho salmon, striped bass (Morone saxatilis), tilapia (Oreochromis mossambicus), and longjawed mudsucker (Gillichthys mirabilis). The first form is around 40-50 kDa, may be regulated by growth hormone (GH), and seems to be a good candidate for the fish version of mammalian IGF-BP3 (which is in the same size range and is GH-regulated). The second and third forms are 29 kDa and 31 kDa and are good candidates for the fish versions of mammalian IGF-BP1 and IGF-BP2, respectively, as they appear to be regulated by insulin and are in the same size range as their mammalian counterparts. Functionally different classes of circulating IGF-BPs may be conserved between fish and mammal.


Isoelectric focusing of human hair keratins: correlation with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns and effect of cosmetic treatments. A new isoelectric focusing (IEF) technique in polyacrylamide gels with 6M urea and 1.5% Nonidet P40 has been developed to characterize human hair samples. The phenotypes demonstrated with this procedure has been correlated with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns described by other authors. The method described can be applied in the forensic science analysis of a single human hair. Using the same IEF technique we have studied the changes in electrophoretic patterns of cosmetically treated hair. The characteristics of the modifications observed and its utility in forensic science work are also discussed in this paper.


Actions of ethanol on gamma-aminobutyric acid-activated chloride channels. Ethanol appears to exert its intoxicating effects, at least partly, by potentiating gamma-aminobutyric acid (GABA) mediated inhibitory synaptic transmission in the brain. Work over the past decade using mouse lines selected for different ethanol sensitivities has implicated GABA-activated chloride channels as a potential target of ethanol. More recently, molecular biological techniques have identified a subunit (gamma 2L) of the GABA channel which appears to be required for the effects of ethanol on GABA channels. The presence of this subunit, however, cannot account for the full degree of potentiation of GABA responses observed in native GABA channels. Ongoing research is directed at identifying other factors which might be involved in the effects of ethanol on GABA channels and in understanding ethanol's molecular mechanism of action. Such information could be vital for designing drugs to combat ethanol intoxication.


Calbindin immunoreactivity in a subset of cat thalamic reticular neurons. Recent studies have shown that the thalamic reticular nucleus of cats is made up of several cytoarchitectonically distinct subdivisions and that the nucleus contains accurate topographical maps of the cortical sheet and of the dorsal thalamus. The present study describes immunocytochemically demonstrable heterogeneity in the reticular nucleus of cats, with an antibody to calbindin D28k. The striking feature of calbindin immunoreactivity in the reticular nucleus of cats is that the immunoreactive neurones are located in the caudal half of the nucleus only. In these regions, labelled cells form a small proportion of the total population of reticular cells only and are not distinct in somal size or shape from neighbouring non-labelled reticular cells. Double labelling shows that the calbindin-immunoreactive cells are also immunoreactive to parvalbumin and GABA. There is a distinct tendency for the calbindin-immunoreactive cells to be more numerous ventrally than dorsally in the caudal half of the nucleus, which receives afferents from the somatosensory and auditory systems.


Immunohistochemical study of the distribution of S-100 protein in pleomorphic adenomas of minor salivary glands. Twelve pleomorphic adenomas of minor salivary gland origin were examined for the distribution of S-100 protein, detected using the peroxidase-antiperoxidase (PAP) method. Strong S-100 protein immunoreactivity was noted in areas containing plasmacytoid cells, stellate and spindle cells against a myxochondroid or hyalinous stroma, and solid epithelial areas. Tubular and duct-like structures showed variable stainability. Stromal tissue and normal salivary glands were generally negative for S-100 protein. These findings were compared with those reported elsewhere.


Initial characterization of the metabolism of intervertebral disc cells encapsulated in microspheres. Adult, canine intervertebral disc cells were isolated with a sequential digestion of pronase and bacterial collagenase. The nonchondrodystrophoid nucleus pulposus exhibits two populations of cells: large notochordal cells and smaller chondrocyte-like cells. The cells from the transition zone and anulus fibrosus are uniform in size, ranging from 17 to 21 microns. The isolated cells were encapsulated in alginate beads and cultured in Ham's F-12 medium containing 5% heat-inactivated fetal bovine serum. Alginate bead formation requires calcium ions and can be reversed with a suitable chelator, thus releasing viable cells. We observed that 58% of the newly synthesized proteoglycans formed large-molecular-weight aggregates with hyaluronic acid. The proteoglycans contained low amounts of keratan sulfate (KS) (less than 5% of the total glycosaminoglycans synthesized). The chondroitin sulfates (CS) consisted of 51-67% as 6-O-sulfate and 29-39% as 4-O-sulfate, with the remainder (4-10%) present as 4,6-sulfate for all three zones of the disc. The majority of cells synthesized significant amounts of matrix as evidenced by Alcian Blue staining. By immunohistochemical analysis, the matrix contained chondroitin 6-sulfate as demonstrated by monoclonal antibodies to the unsaturated disaccharides remaining on the proteoglycan core after chondroitinase ABC digestion. Keratan sulfate was also present in the majority of the matrices around cells. These results emphasize the similarity of the newly synthesized proteoglycans secreted by cells grown in alginate beads to those synthesized by the neonate disc. These experiments also demonstrate the usefulness of this method as a microculture technique for disc cells.


Recombinant human granulocyte colony-stimulating factor promotes wound healing in a patient with congenital neutropenia. We report a patient with congenital neutropenia or Kostmann's Syndrome who suffered many complications after presenting with Clostridium septicum enterocolitis, including absence of wound healing. Because of several reports of the use of granulocyte colony-stimulating factor (G-CSF) in patients with various complications of neutropenia, we treated this patient with recombinant human (rh) G-CSF. We found that once rhG-CSF restored neutrophil counts to normal, progressive wound healing followed. Thus, rhG-CSF therapy may be useful in treating neutropenic patients with wound complications.


Immunotargeting chemotherapy for AFP-producing pediatric liver cancer using the conjugates of anti-AFP antibody and anti-tumor agents. The effect of immunotargeting chemotherapy for hepatoblastoma (HB) and hepatocellular carcinoma (HCC) following the application of adriamycin (ADM) or cis-platinum conjugated with anti-alpha-fetoprotein (AFP) antibody was evaluated experimentally and clinically. The conjugate was made from mouse monoclonal antihuman AFP antibody linked to ADM or CDDP, with a weight ratio of 2.5:1 via a dextran bridge. Experimentally, AFP-producing human HCC transplanted subsequently on nude mice was used. A mixture of the antibody and ADM or CDDP was prepared with the same ratio. Each drug was injected intraperitoneally, three times at the total dose of 14.4 mg/kg as ADM and one time at the dose of 8 mg/kg as CDDP. Tumor growth was inhibited significantly in the conjugate group compared with the other mixture group, the ADM or CDDP group, and the control group. Clinically, the conjugates were administered intraarterially in 4 cases (2 HBs and 2 HCCs) and intravenously in one case (1 HB). ADM and CDDP conjugated with anti-AFP antibody were used in 2 cases and 3 cases, respectively. Antitumor effects from the viewpoint of volume suppression rate showed partial response in 2 cases and no change in 3 cases. The immunotargeting chemotherapy using anti-AFP monoclonal antibodies may be a promising method for treatment of malignant epithelial liver cancer in children.


High-performance liquid chromatographic analysis of docusate sodium in soft gelatin capsules. A rapid and simple high-performance liquid chromatographic (HPLC) method for the analysis of docusate sodium in soft gelatin capsules was developed with progesterone as the internal standard. The method requires a reversed-phase column and a paired-ion technique to separate docusate sodium from other components. A C22 column was used with a mobile phase of acetonitrile:water (70:30) containing 0.005 M tetrabutylammonium phosphate. The flow rate was 1.8 mL/min, and the effluent was monitored at 214 nm. Docusate sodium and progesterone had retention times of 4.5 and 6.8 min, respectively. The proposed HPLC method is linear, accurate, and precise. A mean assay value of 99.6% was obtained by the proposed method when five samples, each containing a composite of 10 capsules, were analyzed. The results obtained by the proposed HPLC, tetra-n-butylammonium iodide titration, and USP XXII HPLC methods are compared.


Serum-induced sensitization of cyclic AMP accumulation in C62B rat glioma cells. Exposure of C62B rat glioma cells to fresh medium containing fetal bovine serum induced a sensitization of the subsequent ability of isoproterenol and forskolin to stimulate cyclic AMP accumulation, compared to cells exposed to fresh medium without serum. Isoproterenol stimulation was typically increased by 2- to 4-fold and forskolin stimulation by 3- to 5-fold. Sensitization occurred rapidly, was rapidly reversible and appeared to result from an increase in maximal stimulation. A commercial preparation of albumin, purified chromatographically so as to retain bound lipids and other factors, was able to mimic the effect of serum. In contrast to the effects of serum, exposure of cells to phorbol 12-myristate, 13-acetate induced little or no change in forskolin stimulation but a marked desensitization of isoproterenol stimulation that was due primarily to a decrease in potency. Neither the protein kinase C inhibitor staurosporine or overnight exposure to phorbol 12-myristate, 13-acetate to down-regulate protein kinase C prevented serum-induced sensitization. Pertussis toxin almost completely blocked serum-induced sensitization, suggesting involvement of a pertussis toxin-sensitive guanine nucleotide-binding protein in mediating the effects of serum. Sensitization was poorly retained in membrane adenylate cyclase assays. Studies with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, direct assays of cyclic AMP degradation by intact cells and assays of phosphodiesterase activity in cell lysates all indicated that degradation of cyclic AMP was decreased in serum-pretreated cells. Thus, both increased cyclic AMP synthesis and decreased cyclic AMP degradation may contribute to sensitization in these cells.


Comparison of ethanol sensitivity of rat brain kainate, DL-alpha-amino-3-hydroxy-5-methyl-4-isoxalone proprionic acid and N-methyl-D-aspartate receptors expressed in Xenopus oocytes. The effect of ethanol (EtOH) on kainate (KA), DL-alpha-amino-3-hydroxy-5-methyl-4-isoxalone proprionic acid and N-methyl-D-aspartate (NMDA) receptor-operated channels was examined electrophysiologically in Xenopus laevis oocytes expressing mRNA from rat hippocampus and cerebellum. EtOH (50, 100 mM) inhibited KA-induced currents but did not alter the EC50 for KA (approximately 78 microM). For a series of n-alcohols, potency for inhibition of KA responses was related to chain length. 6,7-dinitroquinoxaline-2,3-dione inhibited maximum KA responses with an IC50 of approximately 1 microM; EtOH (50, 100 mM) did not alter the IC50 for 6,7-dinitroquinoxaline-2,3-dione but did not produce further inhibition of KA-induced currents. Despite the apparent noncompetitive inhibition produced by EtOH on KA receptor-mediated responses, the EtOH inhibition increased as the KA concentration decreased in hippocampal and cerebellar mRNA expressing oocytes. This differential inhibition was not due to the different current amplitudes stimulated by low vs. high KA concentrations. In contrast, oocytes expressing NMDA channels demonstrated a constant percent inhibition by EtOH in the presence of 25 to 200 microM NMDA. Altering the extracellular Ca++ concentration did not affect the ability of EtOH to inhibit NMDA responses. Maximal NMDA-stimulated currents were inhibited by 100 mM EtOH to a lesser extent (31%) in oocytes injected with rat cerebellar mRNA than oocytes expressing rat hippocampal mRNA (47%), suggesting brain regional differences in NMDA channel inhibition by EtOH.(ABSTRACT TRUNCATED AT 250 WORDS)


Antinociceptive properties of two alkylating derivatives of morphinone: 14 beta-(thioglycolamido)-7,8-dihydromorphinone (TAMO) and 14 beta-(bromoacetamido)-7,8-dihydromorphinone (H2BAMO). This study investigated the antinociceptive properties of two alkylating derivatives of morphinone, 14 beta-(thioglycolamido)-7,8- dihydromorphinone (TAMO) and 14 beta-(bromoacetamido)-7,8-dihydromorphinone (H2BAMO) in the mouse tail-flick assay. Intracerebroventricular administration of either TAMO or H2BAMO produced short-term antinociception. Both TAMO and H2BAMO were 11.6-fold more potent than an i.c.v. administration of morphine. These effects were antagonized by the mu-selective antagonist, beta-funaltrexamine, but not by the delta-selective antagonist, N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH. TAMO pretreatment from 8 to 48 hr produced a time-related, dose-dependent antagonism of morphine-induced antinociception without showing any agonistic effect. Pretreatment with TAMO for 24 hr antagonized antinociception produced by both H2BAMO and morphine, as well as TAMO itself, but not that of the delta-selective agonist [D-Pen2,D-Pen5]enkephalin (DPDPE) or U50,488, a kappa-selective agonist. In order to distinguish this antagonistic effect from cross-tolerance between TAMO and morphine, two mu agonists, [D-Ala2,N(Me)Phe4,Gly-ol]enkephalin (DAMGO) and H2BAMO, were chosen for comparison. A single i.c.v. pretreatment of DAMGO or H2BAMO, at a dose that had equivalent analgesic effects as TAMO, attenuated morphine-induced antinociception, reaching a maximal effect at the time of the disappearance of agonistic effects of DAMGO and H2BAMO and lasting up to 24 hr. Additionally, a 16-hr pretreatment with TAMO, but not DAMGO or H2BAMO, reduced the development of physical dependence to morphine at 24 hr after morphine pellet implantation. Therefore, this study demonstrated that both TAMO and H2BAMO act as mu opioid agonists to produce short-term antinociception.(ABSTRACT TRUNCATED AT 250 WORDS)


Endothelins constrict guinea pig tracheas by multiple mechanisms. When tracheal rings isolated from guinea pigs were treated with endothelins (ETs), a dose-dependent constriction was observed and measured isometrically. ET potencies were ET-1 = ET-2 greater than ET-3. Dose-response curves of epithelium-denuded tracheas were shifted to the left by approximately one order of magnitude. Lung tissue contained saturable binding sites to 125I-labeled ET-1. These binding sites were replaced by ET-1 = ET-2 greater than ET-3. Because the airway tissue contained primarily ET-1 as measured by immunoassay after high-performance liquid chromatography separation, we investigated the mechanism of ET-1-induced tracheal constriction. Tracheal constriction induced by ET-1 required Ca++ in the medium. Because suppression of the contractile response by nicardipine and diltiazem was small, Ca++ entry appeared to be gated primarily through Ca++ channels rather than via voltage-dependent ones. FPL55712, SC47014A, indomethacin and OKY046 suppressed the dose-response curves, suggesting that lipid mediators are formed in response to ET-1. Diphenhydramine also altered dose-response curves, suggesting that histamine release from mast cells was partially responsible for the constriction. Pretreatment of tracheas with compound 48/80 resulted in suppression of the contractile responses. Furthermore, the combination of indomethacin, FPL55712 and diphenhydramine gave essentially identical effects. Our observations suggest that ET-1 provokes the contractile response of guinea pig tracheas not only by direct actions on smooth muscle but also by indirect actions through production of chemical mediators in mast cells and other inflammatory cells.


Stereoselective modulation of ryanodine-sensitive calcium channels by the delta isomer of hexachlorocyclohexane (delta-HCH). delta-Hexachlorocyclohexane (delta-HCH) is shown to be 30-fold more potent as a positive inotropic agent with rat atrial strips compared with lindane (gamma-HCH). Threshold and ED50 values for enhanced contractile force at a pacing frequency of 0.5 Hz are less than 1 microM and 2.2 microM for delta-HCH and 40 microM and 63 microM for gamma-HCH, respectively. Contracture developed in atria exposed to greater than 4 microM delta-HCH (ED50 = 11 microM) but not in atria exposed to gamma-HCH. Uptake and release of Ca++ measured from actively loaded cardiac sarcoplasmic reticulum (SR) vesicles is measured with antipyrylazo III. Although delta-HCH (30 microM) decreases Ca(++)-dependent ATPase by 20%, it does not significantly alter Ca++ loading in the presence of ruthenium red. Addition of delta-HCH (5-50 microM) after loading is complete causes rapid, dose-dependent release of Ca++ from SR. Ca++ release induced by delta-HCH is markedly stereoselective. Compared with gamma-HCH (50 microM), delta-HCH (50 microM) induces a nearly 20-fold higher initial rate of Ca++ release (4.3 nmol of Ca++/mg/sec). Studies with [3H]ryanodine demonstrate that delta-HCH sharply inhibits Ca(++)- or daunorubicin-activated radioligand binding (IC50 = 37 and 25 microM, respectively, logit slope = 2). Inhibition of [3H]ryanodine-binding by delta-HCH is stereoselective inasmuch as IC50 values for alpha, beta and gamma isomers are greater than 100 microM. The delta-HCH modified Ca++ channel appears to proceed by a noncompetitive mechanism (reducing Bmax in equilibrium experiments) with respect to the conformationally sensitive binding site for [3H]ryanodine.(ABSTRACT TRUNCATED AT 250 WORDS)


Stimulation by risperidone of rat prolactin secretion in vivo and in cultured pituitary cells in vitro. Risperidone, a new antipsychotic agent which antagonizes both 5-hydroxytryptamine-2 (5-HT2) and dopamine-2 (D2) receptors, was evaluated for its effects on prolactin release. One hr after either p.o. or i.p. dosing, risperidone was 3 to 5 times more potent than the classical D2 receptor antagonist haloperidol in stimulating rat prolactin levels in vivo. This result was unexpected because haloperidol is a more potent D2 receptor antagonist than risperidone in vitro. Mechanisms which might explain these observations were investigated. Compared to haloperidol, risperidone was 0.34 (95% confidence interval: 0.23, 0.48) times as potent in reversing the suppression by dopamine of rat anterior pituitary cell prolactin release in vitro, which is consistent with the compounds' striatal D2 receptor binding potencies in vitro. Administration of ketanserin, a 5-HT2 receptor antagonist, along with haloperidol did not modify haloperidol's activity on either in vitro pituitary cell prolactin release (up to 1000 nM ketanserin) or in vivo prolactin concentrations (5 mg/kg ketanserin, i.p.). In vitro incubation of haloperidol and risperidone with a liver homogenate supernatant (S-9) led to extensive (greater than 86%) metabolism of each compound. S-9 treatment abolished haloperidol's effects on pituitary cell prolactin release. Risperidone's ability to increase prolactin release was unchanged after S-9 treatment, and 9-hydroxy-risperidone, identified as a major metabolite in the S-9 conditioned media, was equipotent to risperidone in modulating prolactin release in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)


N-methyl-D-aspartate receptor antagonism alters substance P and met5-enkephalin biosynthesis in neurons of the rat striatum. A reduction of striatal excitatory amino acids or of corticostriatal axons alters substance P (SP) and met5-enkephalin (ME) biosynthesis in striatal neurons of the rat. To determine the role of the N-methyl-D-aspartate (NMDA) receptor in this effect, adult rats were treated acutely with a single i.c.v. injection or chronically by 7 days of continuous infusion of an NMDA antagonist. The striatal content of preprotachykinin (PPT) and preproenkephalin (PPE) mRNA was assessed by in situ hybridization histochemistry while the content of SP and ME in, respectively, the substantia nigra and globus pallidus was measured by quantitative radioimmunocytochemistry. Eight hours after a single injection, striatal PPT and PPE mRNA levels were significantly reduced. At 24 hr, the level of PPE had returned to control level whereas that of PPT mRNA remained depressed. Nigral SP and pallidal ME levels were not acutely changed. Chronically, the effect of NMDA antagonist at low doses was to increase the striatal content of PPE mRNA. However, at higher concentrations, the effect was to reduce in a dose-dependent manner the striatal content of PPT and PPE mRNA and the level of pallidal ME. The nigral level of SP did not change significantly at any dose. The results suggest that excitatory amino acid transmission mediated by the NMDA receptor serves as a tonic signal to stimulate neuroactive peptide biosynthesis.


Hemi-gap-junction channels in solitary horizontal cells of the catfish retina. 1. Solitary horizontal cells were isolated from catfish retinas and their membrane current was recorded with a whole-cell voltage clamp. Reducing the extracellular Ca2+ concentration produced a current that could be suppressed by dopamine. This Ca(2+)- and dopamine-sensitive current is hereafter termed I gamma. The voltage dependence, cytoplasmic regulation, and permeability of the I gamma channel suggest that it is half of a gap-junction channel. 2. I gamma was voltage and time dependent. In the steady state, the current-voltage relation displayed outward rectification at voltages more depolarized than 0 mV and a negative resistance region at voltages more hyperpolarized than -15 mV. The reversal potential was 3.3 +/- 1.5 mV when NaCl was the predominant extracellular salt and potassium-D-aspartate was the predominant intracellular salt. 3. The size of I gamma depended on the extracellular Ca2+ concentration. I gamma was maximal at external Ca2+ concentrations below 10 microM, half-maximal at 220 microM-Ca2+, and reduced to less than 4% of its maximum amplitude at external Ca2+ concentrations above 1 mM. Increasing the extracellular Ca2+ concentration reduced the amplitude of I gamma without changing the shape of the current-voltage relation or the kinetics of inactivation. Thus, rectification does not result from a voltage-dependent block by extracellular Ca2+. 4. Patches of cell membrane were voltage clamped in both the cell-attached and excised-patch configurations. In the cell-attached configuration, the addition of dopamine to the solution outside the patch pipette blocked the opening of channels within the membrane patch. Thus, dopamine closes I gamma channels by initiating an intracellular messenger cascade. In the excised-patch configuration, a maximum conductance of 145 pS was measured while Cs+ and tetraethylammonium+ (TEA+) were the only monovalent cations on both sides of the membrane. 5. The ability of dopamine to suppress I gamma was blocked by introducing an inhibitor of the cyclic AMP-dependent protein kinase, PKI5-24, into the cytoplasm. Thus, the action of dopamine is mediated by a pathway that includes the activation of a cyclic AMP-dependent kinase. 6. I gamma was suppressed by nitroprusside, an agent which activates guanylate cyclase and increases the intracellular cyclic GMP concentration. The effect of nitroprusside was not altered by the intracellular application of PKI5-24. Thus, nitroprusside suppresses I gamma through a pathway that does not include the activation of a cyclic AMP-dependent kinase.(ABSTRACT TRUNCATED AT 400 WORDS)


Modulation of potassium channels in intact human T lymphocytes. 1. A voltage-dependent K+ channel called the 'n' type (for 'normal') is the most prevalent ion channel found in whole-cell recordings from T lymphocytes. In whole-cell patch-clamp recordings activity of the n-type channel is affected by mitogenic agents, pH, Ca2+ and temperature but not by cyclic nucleotides. Because channel properties and regulation can depend on cytoplasmic components we sought to reassess the properties of K+ channels in intact, normal human T lymphocytes using cell-attached, patch-clamp recordings. In the present study, we show that the predominant K+ channel in resting, intact cells is the n type and is affected by voltage, temperature and Ca2+ in ways similar to the disrupted cell. Moreover, K+ channels are activated by agents that raise cyclic AMP in intact cells. 2. In cell-attached recordings, we found voltage-activated K+ channels in about 60% of patches at room temperature. The channel was K+ selective as judged from the reversal potential under different Ka(+)-K+ gradients and at different resting membrane potentials. Some patches were subsequently excised and the selectivity further confirmed. The current-voltage relation was inwardly rectifying under symmetrical K+ concentrations and had a slope conductance of 9.4 pS at 50 mV depolarized and 23.8 pS at 50 mV hyperpolarized from the resting potential. From the reversal potentials under various conditions the cell resting potential was -51 +/- 1 mV in normal NaCl saline and about 0 mV when the bath contained 150 mM-KCl saline. Two other types of K+ channel were seen in resting, intact cells, but were much less common (less than 5% and 11% of patches). A large-conductance K+ channel was seen in less than 1% of inside-out patches. 3. The predominant K+ channel in intact, resting T lymphocytes was confirmed as the n type underlying the whole-cell K+ current evoked by voltage steps. In cell-attached patches there was a low, steady-state level of activity at the resting potential but activity was greatly increased by depolarizing voltage jumps. Steady-state inactivation could be removed by a hyperpolarizing pre-pulse. Ensemble currents constructed by summing channel openings during repeated voltage jumps showed sigmoid kinetics of current activation and a monoexponential decay phase. These kinetics were well fitted by a Hodgkin-Huxley-type n4j kinetic model with time constants very similar to the whole-cell current of disrupted cells. Moreover, the kinetics depended on the external K+ concentration as previous research has shown.(ABSTRACT TRUNCATED AT 400 WORDS)


Release of intracellular calcium and modulation of membrane currents by caffeine in bull-frog sympathetic neurones. 1. Calcium release and sequestration were studied in whole-cell voltage-clamped bull-frog sympathetic neurones by image analysis of Fura-2 signals. 2. Application of caffeine (10 mM) to cells voltage clamped at -38 mV caused a rapid increase in intracellular calcium concentration ([Ca2+]i) to a mean value of 352 +/- 33 nM, which activated an outward current. In the continued presence of caffeine the rise in [Ca2+]i slowly declined to a sustained plateau of 196 +/- 20 nM (112 nM above control levels), while the outward current rapidly decayed. Peak calcium release was highest at the edge of the cell. 3. The caffeine-evoked intracellular calcium increase was reduced by two inhibitors of calcium-induced calcium release, ryanodine and procaine. The residual non-suppressible increase in [Ca2+]i may indicate that caffeine can release calcium from two pharmacologically distinct intracellular stores. 4. Inhibition of the caffeine-evoked release of calcium by ryanodine was both concentration and 'use dependent' so that the full inhibitory effect was only observed when caffeine was applied for the second time in the presence of ryanodine. In contrast, the action of procaine did not show any 'use dependence' and unlike ryanodine was fully reversible. 5. The outward current was sensitive to blockers of the large conductance calcium-activated potassium current, Ic. Analysis of variance from this current indicated that it arose at least partly from summation of spontaneous miniature outward currents. 6. The magnitude and duration of calcium release by caffeine was dependent on the resting level of intracellular calcium and the caffeine exposure time. This, together with the pharmacology of the release, suggests that caffeine increases intracellular calcium by sensitizing calcium-induced calcium release. 7. The evoked [Ca2+]i increase was enhanced in amplitude by intracellular application of Ruthenium Red. This effect was mimicked by extracellular application of the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) but not by internal application of FCCP or other inhibitors of mitochondrial Ca2+ uptake. This suggests that the evoked increase in [Ca2+]i is predominantly buffered by a Ruthenium Red-sensitive sequestration process which is not mitochondrial.


Involvement of sarcoplasmic reticulum 'Ca2+ release channels' in excitation-contraction coupling in vertebrate skeletal muscle. 1. Pharmacological blockers of calcium-induced calcium release from isolated skeletal sarcoplasmic reticulum (SR) vesicles have been introduced into frog skeletal muscle fibres to determine their effects on excitation-contraction coupling. 2. Among the blockers tested, Ruthenium Red, neomycin, gentamicin and 9-aminoacridine inhibited the SR Ca2+ release associated with excitation-contraction (E-C) coupling as much as they inhibited caffeine potentiation of that release. Protamine, certain of its derivatives, and spermine were ineffective in both in situ tests. 3. Alternative sites of polyamine action on the contractile proteins, SR Ca2+ uptake or charge movements were ruled out. 4. All polyamines tested required considerably higher concentrations to inhibit excitation-contraction coupling than to block Ca2+ release from isolated SR vesicles. 5. The quantitative pharmacological difference in sensitivity between isolated and intact systems serves as a reminder that results on isolated systems cannot generally be used to predict results of the same substances on more physiological systems. 6. Since caffeine is known to open the SR 'Ca2+ release channels' (the ryanodine receptors that mediate Ca(2+)-induced Ca2+ release), the equal effectiveness of these blockers at inhibiting excitation-contraction (E-C) coupling and its potentiation by caffeine suggests that the SR 'Ca2+ release channels' are indeed involved in excitation-concentration coupling in skeletal muscle, although the results do not indicate how the channel is gated open during E-C coupling.


Morphometric study of nucleolar organizer regions in human oral normal mucosa, papilloma and squamous cell carcinoma. A morphometric study of nucleolar organizer regions (NOR) was performed to analyze their distribution, volume, number and shape in the different strata of human normal oral mucosa epithelium and papilloma and in squamous cell carcinoma employing microphotographs of silver-stained paraffin sections. The different NOR-related parameters evidenced significant differences between normal mucosa, papilloma and squamous cell carcinoma. The functional polarity of normal mucosa epithelium and of papilloma is also evidenced in terms of NOR-related parameters. The discriminative value of certain NOR parameters was demonstrated.


Ion selectivity of colicin E1: II. Permeability to organic cations. Channels formed by colicin E1 in planar lipid bilayers have large diameters and conduct both cations and anions. The rates at which ions are transported, however, are relatively slow, and the relative anion-to-cation selectivity is modulated over a wide range by the pH of the bathing solutions. We have examined the permeability of these channels to cationic probes having a variety of sizes, shapes, and charge distributions. All of the monovalent probes were found to be permeant, establishing a minimum diameter at the narrowest part of the pore of approximately 9 A. In contrast to this behavior, all of the polyvalent organic cations were shown to be impermeant. This simple exclusionary rule is interpreted as evidence that, when steric restrictions require partial dehydration of an ion, the structure of the channel is able to provide a substitute electrostatic environment for only one charged group at time.


Cl- channels in basolateral renal medullary vesicles: V. Comparison of basolateral mTALH Cl- channels with apical Cl- channels from jejunum and trachea. Cl- channels from basolaterally-enriched rabbit outer renal medullary membranes are activated either by increases in intracellular Cl- activity or by intracellular protein kinase A (PKA). Phosphorylation by PKA, however, is not obligatory for channel activity since channels can be activated by intracellular Cl- in the absence of PKA. The PKA requirement for activation of Cl- channels in certain secretory epithelia is, in contrast, obligatory. In the present studies, we examined the effects of PKA and intracellular Cl- concentrations on the properties of Cl- channels obtained either from basolaterally-enriched vesicles derived from highly purified suspensions of mouse medullary thick ascending limb (mTALH) segments, or from apical membrane vesicles obtained from two secretory epithelia, bovine trachea and rabbit small intestine. Our results indicate that the Cl- channels from mTALH suspensions were virtually identical to those previously described from rabbit outer renal medulla. In particular, an increase in intracellular (trans) Cl- concentration from 2 to 50 mM increased both channel activity (Po) and channel conductance (gCl, pS). Likewise, trans PKA increased mTALH Cl- channel activity by increasing the activity of individual channels when the trans solutions were 2 mM Cl. Under the latter circumstance, PKA did not activate quiescent channels, nor did it affect gCl. Moreover, when mTALH Cl- channels were inactivated by reducing cis Cl- concentrations to 50 mM, cis PKA addition did not affect Po. These results are consistent with the view that these Cl- channels originated from basolateral membranes of the mTALH. Cl- channels from apical vesicles from trachea and small intestine were completely insensitive to alterations in trans Cl- concentrations and demonstrated markedly different responses to PKA. In the absence of PKA, tracheal Cl- channels inactivated spontaneously after a mean time of 8 min; addition of PKA to trans solutions reactivated these channels. The intestinal Cl- channels did not inactivate with time. Trans PKA addition activated new channels with no effect on basal channel activity. Thus the regulation of Cl- channel activity by both intracellular Cl- and by PKA differ in basolateral mTALH Cl- channels compared to apical Cl- channels from either the tracheal or small intestine.


Modification of gating of an airway epithelial chloride channel by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) has been shown to produce a reduction in channel open probability in a number of epithelial chloride channels. We have investigated its action on a calcium-dependent airway epithelial chloride channel, which shows voltage-dependent gating following incorporation into planar phospholipid bilayers. Since the channel demonstrates a distinct subconductance state at approximately one-third of the fully open level, both 50 and 20% threshold analysis (TA) have been used to describe channel kinetics. Lifetime analysis using 50% TA in the absence of NPPB showed that two open and three closed lifetimes provided the optimal fit. Similar analysis using 20% TA required only two closed lifetimes and allowed for tentative assignment of tau values to either substate or fully closed events. NPPB (10-100 microM) produced a concentration-related shift of the normal voltage dependence of gating, with more negative holding potentials being required to produce a given channel open probability than in the absence of NPPB. Ten microM NPPB produced a similar shift in lifetime values. However, addition of 50 microM NPPB produced a unique, single open lifetime. No evidence was found for NPPB acting as a direct voltage-dependent blocker of chloride conductance.


Localization of epitopes for antibodies that differentially label sodium sodium channels in skeletal muscle surface and T-tubular membranes. We previously characterized two monoclonal antibodies, A/B2 and L/D3, that bind to the amino-terminus of the sodium channel but produce distinct immunocytochemical patterns in innervated adult skeletal muscle. Because these findings suggested the presence of several channel isoforms, we sought to define the epitopes for each antibody. Five peptides encompassing the amino-terminal 126 residues of the adult skeletal muscle sodium channel were synthesized and tested by radioimmunoassay against each antibody. Both monoclonals bound only to a peptide comprising residues 1-30 (I1-30). A nested set of peptides within this region was then synthesized and used to compete for antibody binding to I1-30. L/D3 binding was quantitatively inhibited by oligopeptides 1-30, 7-30, 13-30, and 19-30 but not 25-30, while binding of A/B2 was blocked only by the intact I1-30 peptide. This data implies that the epitope for L/D3 lies within residues 19-25 while the epitope for A/B2 is contained within residues 1-6. These tentative epitope localizations were confirmed using both proteolytic cleavage of I1-30 and immunoreactivity of a peptide corresponding to residues 1-12 with A/B2 but not L/D3. Therefore, epitopes for each monoclonal antibody are present in the SkM-1 sequence and are in close proximity in the amino-terminus of the protein. Their characteristic immunocytochemical labeling patterns may reflect differing accessibility of the epitopes in various membrane environments.


Soluble glycoprotein D blocks herpes simplex virus type 1 infection of rat eyes. Herpes simplex virus type 1 (HSV-1) ocular infection in rats was blocked by treating the eyes with UV-inactivated virions containing glycoprotein D (gD) prior to ocular challenge. In contrast, rats treated with UV-inactivated virions lacking gD were not protected. A soluble, truncated form of HSV-2 gD (gD-2t) also protected against ocular infection. Treatment with gD-2t not only reduced mortality but also restricted progression of pathology and reduced the amount of viral antigen in the cornea. Host antibody or alpha/beta interferon responses to the gD-2t treatment were not detected. These results are similar to those observed in cell culture (D. C. Johnson, R. L. Burke, and T. Gregory, J. Virol. 64:2569-2576, 1990). The in vivo effect of exogenous gD is consistent with blocking of a cell surface gD receptor or with an inhibitory interaction of gD with virions.


Examination of sera from human immunodeficiency virus type 1 (HIV-1)-infected individuals for antibodies reactive with peptides corresponding to the principal neutralizing determinant of HIV-1 gp120 and for in vitro neutralizing activity. Sera from human immunodeficiency virus type 1 (HIV-1)-infected individuals from the United States and Tanzania were examined for antibody reactivity to four synthetic peptides which corresponded to the principal neutralizing determinant from the V3 region of HIV-1 gp120. We observed that the majority of sera from both countries contained antibodies reactive with a V3 peptide whose sequence is based on that of the HIV-1 MN isolate. We were unable to establish a relationship between the presence of V3-reactive antibodies, as measured by enzyme-linked immunosorbent assay and neutralization of homologous HIV-1 isolates, in sera from either the United States or Tanzania. We observed that some sera which contained high antibody titers to the V3 peptides failed to neutralize HIV-1, while others with no antibody reactivity to the panel of V3 peptides exhibited in vitro neutralizing activity. These results suggest that neutralizing epitopes exist outside the V3 loop and that the presence of V3-reactive antibodies in sera does not imply in vitro neutralization of the homologous HIV-1 isolate. In addition, it appears that the V3 loop may consist of both neutralizing and nonneutralizing epitopes. The identification of neutralizing as well as nonneutralizing epitopes will be important for the design of potential HIV-1 vaccines.


Interactions of polyomavirus middle T with the SH2 domains of the pp85 subunit of phosphatidylinositol-3-kinase. The binding of phosphatidylinositol-3-kinase to the polyomavirus middle T antigen is facilitated by tyrosine phosphorylation of middle T on residue 315. The pp85 subunit of phosphatidylinositol-3-kinase contains two SH2 domains, one in the middle of the molecule and one at the C terminus. When assayed by blotting with phosphorylated middle T, the more N-terminal SH2 domain is responsible for binding to middle T. When assayed in solution with glutathione S transferase fusions, both SH2s are capable of binding phosphorylated middle T. While both SH2 fusions can compete with intact pp85 for binding to middle T, the C-terminal SH2 is the more efficient of the two. Interaction between pp85 or its SH2 domains and middle T can be blocked by a synthetic peptide comprising the tyrosine phosphorylation sequence around middle T residue 315. Despite the fact that middle T can interact with both SH2s, these domains are not equivalent. Only the C-terminal SH2-middle T interaction was blocked by anti-SH2 antibody; the two SH2 fusions also interact with different cellular proteins.


Group C rotavirus requires sialic acid for erythrocyte and cell receptor binding. Current immunological and biochemical information regarding the hemagglutinin and virus-cell interactions of rotavirus is obtained exclusively from studies with group A rotaviruses. In this study, I report that the immunologically and genetically distinct group C rotavirus also possesses a hemagglutinin. The viral hemagglutinin was identified on a cultivable porcine group C rotavirus strain (strain AmC-1) by using agglutinated human and guinea pig erythrocytes. Neuraminidase treatment of fresh human erythrocytes or blocking with glycophorin A or fetuin prevented hemagglutination. Infection of swine testicular cells with group C AmC-1 virus was also prevented by glycophorin A, fetuin, and neuraminidase treatment, suggesting that sialic acid constitutes an essential part of the cell receptor.


Purification of immature cores of mouse mammary tumor virus and immunolocalization of protein domains. The immature capsids of the mouse mammary tumor virus (MMTV), known as intracytoplasmic A particles, have been isolated from murine L1210 leukemia cells. The diameter of the isolated particles was 80 nm as determined by negative staining. Two polypeptides of 77 and 110 kDa were found to be their major polypeptide components, in agreement with the expected sizes of the Gag and Gag-Pro precursor polypeptides of the mature MMTV proteins. Both polypeptides were recognized by antibodies directed toward the matrix (p10) and capsid (p27) proteins of MMTV. Immunogold labeling of p10 on isolated A particles, visualized by negative staining, showed that this protein is located at the surface of the immature capsids, whereas p27 can be detected only in broken or disrupted particles, suggesting that it has an internal location. These observations were confirmed by immunolabeling of both proteins on thin sections of A particle-producing cells. In addition, the viral protease had a more internal position than p27. Since the sequential order of the viral proteins in the Gag precursor is p10-pp21-p27-p14 and that in Gag-Pro is p10-pp21-p27-p30-protease, our results demonstrate the radial organization of the polypeptide precursors forming the intracytoplasmic A particles.


Persistence of azidothymidine-resistant human immunodeficiency virus type 1 RNA genotypes in posttreatment sera. The rate of reversion from azidothymidine (zidovudine; AZT) resistance was studied by direct sequencing of human immunodeficiency virus type 1 (HIV-1) virion RNA in sera from four patients who discontinued long-term treatment. Before cessation of treatment, all four patients harbored HIV-1 with multiple mutations reported to confer AZT resistance. In three patients, slow reversions of these mutations starting after 9, 9, and 18 months were detected. The slow reversions indicate that AZT-resistant HIV-1 variants are likely to have an unaltered replicative capacity and pathogenic potential. Furthermore, there were discrepancies between the in vivo RNA sequences and the sequences of virus isolates, indicating that the isolation procedure may select for nonrepresentative virus variants.


Discontinuous, conserved neutralization epitopes overlapping the CD4-binding region of human immunodeficiency virus type 1 gp120 envelope glycoprotein. Monoclonal antibodies have been isolated from human immunodeficiency virus type 1 (HIV-1)-infected patients that recognize discontinuous epitopes on the gp120 envelope glycoprotein, that block gp120 interaction with the CD4 receptor, and that neutralize a variety of HIV-1 isolates. Using a panel of HIV-1 gp120 mutants, we identified amino acids important for precipitation of the gp120 glycoprotein by three different monoclonal antibodies with these properties. These amino acids are located within seven discontinuous, conserved regions of the gp120 glycoprotein, four of which overlap those regions previously shown to be important for CD4 recognition. The pattern of sensitivity to amino acid change in these seven regions differed for each antibody and also differed from that of the CD4 glycoprotein. These results indicate that the CD4 receptor and this group of broadly neutralizing antibodies recognize distinct but overlapping gp120 determinants.


[Inhibitory effect of somatostatin analog, SMS 201-995, on exocrine secretion from isolated rat pancreatic acini]. In vitro effect of somatostatin analog, SMS 201-995 (SMS), on pancreatic exocrine secretion was investigated using isolated rat pancreatic acini. SMS had no effect on basal, cholecystokinin octapeptide (CCK-8)- or secretin-stimulated amylase release. SMS inhibited pancreatic amylase release in response to simultaneous stimulation with secretin and CCK-8 in a dose-dependent manner. Significant inhibition was observed with 10 nM SMS and maximal inhibition with 0.1-1 microM SMS. Amylase release in response to the combination of 100 pM CCK-8, 1 nM secretin and 0.1-1 microM SMS was similar to that to 100 pM CCK-8 alone. Secretin significantly increased acinar cell cAMP content. SMS partially inhibited an increase in cAMP content induced by secretin. The present study has demonstrated, therefore, that SMS directly inhibits the potentiating effect of secretin on exocrine secretion in part by inhibiting an increase in secretin-induced cAMP accumulation in rat pancreatic acinar cells.


Indirect measurement of lymphatic absorption in CAPD patients is not influenced by trapping. The disappearance rate of intraperitoneally administered macromolecules is often used to calculate lymphatic absorption during CAPD. The possible contribution of local accumulation (trapping) of such solutes in the tissues surrounding the peritoneal cavity, leading to overestimation of lymphatic flow, was investigated in eight CAPD patients. They were studied twice during a four hour dwell, glucose 1.36%, to which polydisperse neutral dextran 70 1 g/liter had been added for measurement of lymphatic flow. After the test on day 1 dextran 130 mg/kg was given intravenously and also dextran 1 g/liter was added to every following dialysis bag until the second test on day 3. This was done to saturate the tissues surrounding the peritoneal cavity and thereby to create a steady state condition. In one patient the dextran administration was continued until a third study was done on day 5. Dextran in serum during day 3 was 1.3 +/- 0.5 g/liter (mean +/- SD). No difference in peritoneal clearance of dextran was found between day 1 and day 3 (1.11 +/- 0.56 versus 0.97 +/- 0.41 ml/min). Also no difference was found between day 1 (0.32), day 3 (0.62), and day 5 (0.42 ml/min). Trapping would have influenced the first but not the second test, as the second time all tissues were saturated with dextran. As the dextran absorption rate remained the same, this indicates that trapping is of no importance and that lymphatic absorption can be measured by the disappearance of a macromolecular marker.


Experimental chronic renal ischemia: morphologic and immunologic studies. Although unilateral clamping of the renal artery to induce chronic ischemia of the kidney tissue has been utilized in several animal species, the resultant morphologic, ultrastructural and immunologic changes have never been well characterized. Moreover, the pathogenesis of these changes, as well as their roles in causing or facilitating the development of chronic tubulointerstitial nephritis have not been known. To examine some of these issues, male Sprague-Dawley rats were subjected to unilateral stenosis of the left main renal artery for 28 days. Stenotic and contralateral kidneys of experimental animals and kidneys from sham-operated controls were subjected to: (1) light microscopic, electron microscopic and immunofluorescent studies; (2) morphometric quantitation of the structural changes; (3) staining for actin, epithelial membrane antigen, keratin, and vimentin by immunoperoxidase technique; (4) staining for complex glycoproteins by a panel of 13 lectins; and (5) phenotyping and quantitation of the interstitial inflammatory infiltrates by monoclonal antibodies, using immunoperoxidase technique. The results reveal that: (1) The ischemic kidney tissue displays marked tubulointerstitial damages including abundant interstitial chronic inflammatory infiltrates, with good preservation of glomerular structure, which is consistent with the standard criteria of chronic tubulointerstitial nephritis. (2) The antigenic profile of the ischemic tubular epithelium displayed marked alterations including a neo-expression of vimentin and keratin, as well as a loss of endogenous avidin binding activity, Ia antigen and several complex surface glycoproteins detectable by lectins. (3) Neither electron dense deposits nor immunoglobulins are detectable in the kidneys from experimental or control animals. (4) Tubulitis, defined as infiltration of tubular epithelium by inflammatory cells, was present in up to 42.2% of tubular cross sections of the ischemic kidneys. (5) The interstitial inflammatory infiltrates were composed of B lymphocytes, T helper lymphocytes, and macrophages whereas the T non-helper lymphocytes were scanty, a phenotypic pattern similar to that of several other experimental rat models of chronic tubulointerstitial nephritis. It is concluded that: (1) In the Sprague-Dawley rats, ischemia alone can cause a constellation of changes fulfilling the accepted features of chronic interstitial nephritis; (2) ischemia alters the antigenic profile of the tubular epithelium and thereby may initiate a cell mediated immune response, accounting for the observed tubulitis and interstitial inflammation; and (3) ischemia may well be the final common pathway for chronic tubulointerstitial nephritis of diverse etiologies.


Abatement of bleomycin-induced increases in vascular permeability, inflammatory cell infiltration, and fibrotic lesions in hamster lungs by combined treatment with taurine and niacin. The bleomycin (BL) hamster model of interstitial pulmonary fibrosis has been widely used to study the pathogenesis of interstitial pulmonary fibrosis and to screen potentially desirable antifibrotic agents. We have recently shown that taurine and niacin in combination, diminished BL-induced increases in lung lipid peroxidation and hydroxyproline content in hamsters. In the present study, we have evaluated the effects of taurine and niacin on the bronchoalveolar lavage (BAL) cells, and morphologic and morphometric features of the lung in the same model of pulmonary fibrosis. The hamsters were divided into 4 groups: saline; taurine + niacin + saline; BL; and taurine + niacin + BL. Treatment of taurine and niacin began 2 days before the first intratracheal instillation of saline or BL and thereafter daily throughout the study for taurine + niacin + saline and taurine + niacin + BL groups. Hamsters received BL or saline in three consecutive doses at weekly intervals by intratracheal route. Twenty days after the last intratracheal instillation, the hamsters were sacrificed for various studies. Combined treatment with taurine and niacin suppressed BL-induced inflammation and almost completely abrogated pulmonary fibrosis in hamsters. Two independent studies showed that taurine and niacin in combination significantly reduced BL-induced increases in bronchoalveolar inflammatory cell counts, protein content, and acid phosphatase activity. By both light and electron microscopy, the lungs of hamsters treated with BL and taurine and niacin had much fewer inflammatory cells, less epithelial necrosis and collagen deposition than hamsters treated with BL alone. The results of this investigation suggest that combined treatment with taurine and niacin is effective against the development of lung fibrosis in the BL-hamster model and offers a novel therapeutic modality in the prevention of the fibrotic processes.


Hemoglobin-acetaldehyde adducts in human alcohol abusers. Previous observations have indicated that acetaldehyde can bind irreversibly to proteins in vitro, yielding immunogenic determinants, which can stimulate production of antibodies against the acetaldehyde adducts. We have developed sensitive two-site enzyme-linked immunosorbent assays for measurement of hemoglobin-acetaldehyde adducts. These adducts were measured from the red blood cells of 169 alcohol abusers, 66 social drinkers, 18 abstainers, and 73 hospitalized control patients. While the immunoreactive acetaldehyde adducts were found to be increased in 50% of the alcohol abusers (p less than 0.01), 24% of the social drinkers (p less than 0.05) also exceeded the reference interval obtained from the abstaining controls. Adducts were also increased in 17 (23%) hospitalized controls, seven of whom could retrospectively be verified as heavy drinkers. Upon abstinence from ethanol, the adducts decreased during a period of 1-3 weeks. The studies indicate that acetaldehyde adducts are frequently elevated in the erythrocytes of human alcohol consumers. Measurements of such adducts may prove to be valuable in the early identification of excessive alcohol consumption and of hazardous social drinking and in the comprehensive assessment and treatment of patients with alcohol-related diseases.


A simplified method for the preparation of tetanus toxin binding fragment for neurobiology. The non-toxic binding fragment of tetanus toxin (fragment C) binds avidly to neural tissue and has a growing number of neurobiological uses. Its current utility is limited by both its high commercial cost and the complex procedure for its preparation requiring highly purified tetanus toxin. We have developed a short procedure which prepares fragments of tetanus toxin from crude C. tetani extracts. The resultant proteins are atoxic with molecular sizes and immunological properties closely resembling fragment C. These proteins undergo retrograde axonal and apparent transneuronal transport in a fashion similar to fragment C.


Calcitonin gene-related peptide potentiates substance P-induced plasma extravasation in the rat trachea. Antidromic stimulation of vagal sensory nerves is known to produce plasma extravasation in the rat trachea. This neurogenic inflammation is thought to be mediated by substance P or other tachykinins released from sensory nerve endings. We sought to determine whether calcitonin gene-related peptide (CGRP), which is also released from sensory nerve endings, can potentiate substance P-induced plasma extravasation in the rat trachea. To accomplish this, we measured the amounts of Evans blue dye extravasated into the trachea after intravenous injections of substance P alone and combined with CGRP. We found that when substance P and CGRP were injected together, the amount of plasma extravasation produced in the trachea was substantially greater than the amount produced when substance P was injected alone. This potentiation was critically dependent on the dosage of CGRP and was not observed when relatively high dosages were used. We also found that CGRP had a potent hypotensive effect and speculate that reduced blood pressure may account for the lack of potentiation observed at the higher CGRP dosages. Based on these findings, we conclude that CGRP can potentiate substance P-induced plasma extravasation in the rat trachea and may therefore play a role in modulating neurogenic inflammation of the airways.


Detection of proliferating hepatocytes by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) in patients with acute hepatic failure. This study evaluated whether liver regeneration could take place after massive or submassive necrosis of liver cells in 25 patients with several kinds of hepatic failure by immunohistochemical staining for proliferating cell nuclear antigen (PCNA). PCNA positivity was significantly higher (P less than 0.01) in the patients who survived than in the patients who died. Furthermore, PCNA-positive hepatocytes were recognized diffusely in the lobule of the liver in survivors. There was positive correlation between PCNA positivity and plasma concentration of AFP (alpha-fetoprotein), (r = 0.77, P less than 0.01). These results show that liver regeneration could take place after massive necrosis of liver cells in survivors from acute hepatic failure and that immunohistochemical staining for PCNA is useful for prognostic evaluation.


Phenotypic variation of surface antigenic determinants in Trichomonas vaginalis detected by monoclonal antibodies. We produced a large panel of murine monoclonal antibodies against surface determinants of Trichomonas vaginalis using the hybridoma technique. An immunoenzymatic technique (E.L.I.S.A.) was used to screen positive hybrid cells producing specific antibodies against the protozoan surface. Eleven monoclonal antibodies (Mabs) out of seventy-seven positives were further characterized. We tested antibody reactivity in order to investigate the antigenic variance among 13 different strains of Trichomonas vaginalis of different geographic origin. To elucidate the complexity of antigenic expression in Trichomonas vaginalis, further characterization of the antigenic pattern in our 13 clinical isolates was carried out by immunoblotting techniques. We demonstrate that some monoclonal antibodies react with antigens varying in molecular weight in the different strains tested. We also demonstrate the pivotal role of protozoan proteases in antigenic rearrangement.


A new bacterial staining method involving Gram stain with theoretical considerations of the staining mechanism. In order to investigate the mechanism of Gram staining of bacteria, tests with anionic dyes followed by treatment with cationic octyltrimethylammonium (OTMA) were carried out. The study revealed that tetrabromophenolphthalein ethylester (TBPE) gave the most reliable staining of Gram-negative bacteria with negative staining of Gram-positive bacteria. Tests on many species of bacteria showed that TBPE positive bacteria were Gram-negative and vice versa, without exception.


Propylene glycol as a vehicle for percutaneous absorption of therapeutic agents. Propylene glycol (PG) was evaluated as a vehicle for the in vivo percutaneous absorption of the hydrochloride salts of desipramine, nortriptyline, procainamide, and N-acetyl-procainamide. Each drug was administered topically to hairless (hr-1/hr-1) mice in water and in aqueous 10% and 50% PG. Mean drug concentrations in blood, brain, heart, liver, and lung were measured by high-pressure liquid chromatography after either two or three hours of topical absorption. The presence of PG generally enhanced the absorption of each drug, and the degree of enhancement appeared to be related to the percentage of PG in the dosing solution.


Development of a flow-cytometric HLA-A locus mutation assay for human peripheral blood lymphocytes. A flow-cytometric technique was developed to measure the frequency of variant lymphocytes lacking expression of HLA-A2 or A24 allele products among donors heterozygous for HLA-A2 or A24. It was found that the variant frequency of lymphocytes in peripheral blood was of the order of 10(-4) and increased with donor age. Molecular analyses of mutant clones revealed that about one-third were derived from somatic recombinations and that the remaining two-thirds did not show any alterations after Southern blotting analysis. In contrast, mutants obtained after in vitro X-ray mutagenesis study were found to be mostly derived from large chromosomal deletions. A small-scale study on atomic bomb survivors did not show a significant dose effect.


Time-saving in biological dosimetry by using the automatic metaphase finder Metafer2. The amount of time-saving by using the Metafer2 metaphase finder for routine analysis of radiation-induced chromosome aberrations (biological dosimetry) was determined. Metaphases were prepared by standard methods from cultures of human peripheral blood lymphocytes and stained either with Giemsa or with the FPG method. The metaphase finder was used for detecting metaphases on the microscope slides and for automatically processing the evaluation data. In our laboratory, standardized analysis of 1000 metaphases requires at least 3 working days for cell culturing and slide preparation and 51.5 working hours for cytogenetic analysis. When using the metaphase finder the time required for cytogenetic analysis is reduced to 17.3 working hours (time-saving factor: 51.5/17.3 h = 3.0). In our prolonged method, including more than one scoring of each slide and karyotyping of metaphases with chromosome aberrations, the analysis times for 1000 cells are 132 and 70 working hours, respectively (time saving factor: 132/70 h = 1.9).