Effect of a beta 2-agonist (broxaterol) on respiratory muscle strength and endurance in patients with COPD with irreversible airway obstruction.

The effect of broxaterol, a new beta 2-agonist, on respiratory muscle endurance and strength was studied in a double-blind, placebo-controlled, randomized crossover clinical trial in 16 patients with chronic obstructive pulmonary disease (COPD) with irreversible airway obstruction (FEV1 = 57.1 percent of predicted). One patient withdrew from the study because of acute respiratory exacerbation. Inspiratory muscle strength was assessed by maximal inspiratory pressure (MIP) and endurance time was determined as the length of time a subject could breathe against inspiratory resistance (target mouth pressure = 70 percent of MIP, Ti/Ttot = 0.4). Broxaterol (B) or placebo (P) was given orally for seven days at the dose of 0.5 mg three times a day with a washout period of 72 h between study treatments. Measurements were performed before administration of B or P and 2 h (six patients) or 8 h (nine patients) after the end of each treatment. No significant changes in FEV1 or FRC were observed after B or P suggesting that diaphragmatic length was maintained constant with each treatment. The MIP did not significantly change, while endurance time increased after B in the patients tested at 2 h (from 234.8 +/- 48.1 s to 284.0 +/- 48.0 s, p less than 0.05) and at 8 h (from 187.2 +/- 31.1 s to 258.2 +/- 40.4 s, p less than 0.005). No changes were observed after P. Minute ventilation, airway occlusion pressure (P0.1), integrated electromyographic activities of the diaphragm (Edi), and intercostal parasternals (Eic) (normalized to the value obtained during MIP) showed no change during the endurance run with different treatments. We conclude that in a group of COPD patients with irreversible airway obstruction, B significantly improves respiratory muscle endurance, and that this does not arise as a result of an effect on neuromuscular drive or pulmonary mechanics, but may be mediated by peripheral factors.


Electrophysiologic study of the effects of aminophylline and metaproterenol on canine myocardium.

Aminophylline and beta-adrenergic agonists are widely used in the treatment of obstructive lung diseases. It has been suggested that combined aminophylline and beta-agonist therapy may promote the development of atrial and ventricular arrhythmias. The effects of these agents in combination on myocardial conduction and tissue refractoriness have not been documented. We evaluated the electrophysiologic effects of intravenous aminophylline and inhaled metaproterenol on canine myocardium. Aminophylline produced significant decreases from baseline in the AH interval (85 +/- 6.5 [SD] to 63 +/- 4.1 ms [p less than 0.02]), Wenckebach cycle length (WCL) (226 +/- 8.7 to 182 +/- 5.8 ms [p less than 0.02]), and ventricular effective refractory period (VERP) (166 +/- 6.0 to 148 +/- 4.9 ms [p less than 0.01]). Metaproterenol produced similar results, except metaproterenol significantly decreased the atrial effective refractory period (AERP) from 152 +/- 6.6 to 130 +/- 3.2 ms (p less than 0.02), an effect not seen with aminophylline alone. Metaproterenol also produced significantly greater reductions in AH interval and WCL, as well as a greater increase in heart rate than aminophylline did. When compared with aminophylline alone, combined metaproterenol and aminophylline therapy produced significantly greater reductions in the AH interval (63 +/- 4.1 versus 48 +/- 1.2 ms for combined therapy [p less than 0.01]), HV interval (32 +/- 1.2 versus 28 +/- 2.0 ms for combined therapy [p less than 0.02]), WCL (182 +/- 5.8 versus 150 +/- 7.1 ms for combined therapy [p less than 0.02]), and VERP (148 +/- 4.9 versus 132 +/- 2.0 ms for combined therapy [p less than 0.02]). We conclude that both aminophylline and metaproterenol significantly enhance AV nodal and His-Purkinje conduction. Metaproterenol produced significant changes in both atrial and ventricular tissue refractoriness. Metaproterenol produced significantly greater changes than aminophylline alone, and inhaled metaproterenol combined with intravenous aminophylline produced greater changes in AV nodal and His-Purkinje conduction and ventricular refractoriness than did aminophylline alone in a canine model.


Release of endosomal content induced by plasma membrane tension: video image intensification time lapse analysis.

Rhodamine-labeled vinculin microinjected into chicken embryo fibroblasts and rhodamine-labeled alpha 2-macroglobulin added to the fibroblast culture medium were sequestered in endocytotic vesicles and digested. When a sealed microcapillary coated with fibronectin or polylysine was attached to the fibroblasts and pulled at speeds of 100-200 microns/h, stretching the plasma membrane, a variable fraction of the endosomes released the rhodamine label. Release from individual vesicles was rapid, reaching completion in less than 30 to 120 s. The microfilament disrupting agent cytochalasin B prevented release, as did the microtubule stabilizing drug taxol. Colcemide, which disrupts microtubules, did not inhibit the release. Release was dependent on extracellular calcium, as it was prevented by 10 mM EGTA in the incubation medium. We postulate that opening of vesicular channels consequent to centripetal transmission of tension generated in the plasma membrane along microfilaments may be a mechanism of release of endosomal content.


Impairment of MHC class I transcription in a mutant bovine B cell line.

To better define the regulation and expression of bovine major histocompatibility complex (MHC) antigens, the bovine B lymphoblastoid cell line, BL3, was exposed to gamma-irradiation and surviving cells were immunoselected for MHC class I antigen loss. The resulting class I expression loss variant, BL3.1, was characterized at both the protein and genetic levels to ascertain the nature of the defect. Microfluorimetry analysis revealed a 3--5-fold surface density reduction of all class I products on BL3.1 cells relative to the parental BL3 cells. This decreased surface expression was specific for MHC class I and not for MHC class II or the non-MHC-linked gene product, immunoglobulin (Ig). Northern and quantitative slot blot analyses demonstrated a corresponding diminution of class I RNA in BL3.1 suggesting a transcriptional level defect. Nuclear run-off and transcription inhibition experiments confirmed no post-transcriptional changes while Southern blot analysis provided no evidence for alterations within or near the class I genes. To help elucidate the mechanism of altered class I expression, the parent, BL3, and variant, BL3.1, were cultured with factors known to enhance MHC class I transcription. Interferon (IFN)-gamma, lipopolysaccharide (LPS), and activated peripheral blood lymphocyte (PBL) supernatant cultured with both cell lines induced MHC class I transcription and surface expression 2--3-fold greater than the untreated controls. It is likely, therefore, that a genetic alteration outside of the class I genes has occurred within BL3.1 impairing expression of MHC class I.


Mucosal immune response to RDEC-1 infection: study of lamina propria antibody-producing cells and biliary antibody.

Infection of rabbits with Escherichia coli RDEC-1 is a useful model for diarrheal disease caused by mucosally attaching E. coli. Understanding of the protective immunity induced by RDEC-1 infection in rabbits should provide information useful in the design of vaccines for protection against this infection and other mucosally attaching organisms as well. Thus, to define the time course and location of specific immunoglobulin A secretion in relation to bacterial colonization during primary RDEC-1 infection, we infected rabbits with RDEC-1, which express AF/R1 adherence pili, and compared sites of anti-AF/R1 antibody-containing cells in the intestinal mucosa with the sites of luminal colonization and mucosal attachment of RDEC-1. Also, anti-AF/R1 antibodies in intestinal fluids and bile were measured by enzyme-linked immunosorbent assay, and attachment sites of RDEC-1 to the intestinal epithelium were determined by immunohistochemical examination. Anti-AF/R1 pilus antibody-containing cells were most numerous in the proximal intestine (duodenum and jejunum). In contrast, both luminal colonization and attachment of RDEC-1 to epithelial cells were densest in the distal intestine (cecum and colon). Anti-AF/R1 antibodies were present in approximately equal amounts in fluids collected from all levels of the gut after week 1 postinfection. Anti-AF/R1 antibody levels in undiluted bile exceeded those in gut flushes by at least 2 orders of magnitude. Loss of RDEC-1 attachment to epithelial cells preceded resolution of diarrheal illness despite the presence of large numbers of organisms in the intestinal lumen. Our studies indicate that during RDEC-1 infection (i) sites of greatest mucosal anti-AF/R1 antibody secretion are proximal to sites of maximal RDEC-1 luminal colonization and attachment, (ii) bile is a major source of specific antibodies in the intestinal lumen, and (iii) interference with RDEC-1 attachment to epithelial cells may permit resolution of disease.


R-plasmid-encoded adhesive factor in Klebsiella pneumoniae strains responsible for human nosocomial infections.

Klebsiella pneumoniae strains involved in hospital outbreaks of nosocomial infections, such as suppurative lesions, bacteremia, and septicemia, were resistant to multiple antibiotics including broad-spectrum cephalosporins. Epidemiologic investigations revealed that the reservoir for these K. pneumoniae strains was the gastrointestinal tracts of the patients. The study of the adherence ability of the strains reported here showed that these bacteria adhered to the microvilli of the Caco-2 cell line. This adhesion was mediated by a nonfimbrial protein with a molecular mass of 29,000 Da designated CF29K. Pretreatment of bacteria with antibodies raised against CF29K or Caco-2 cells with purified CF29K prevented the adhesion of K. pneumoniae strains to Caco-2 cells. CF29K immunologically cross-reacted with the CS31A surface protein of Escherichia coli strains involved in septicemia in calves. Genes encoding CF29K were located on a high-molecular-weight conjugative R plasmid, which transferred to E. coli K-12. Transconjugants expressed a large amount of CF29K protein and adhered to the brush border of Caco-2 cells. These findings show that K. pneumoniae strains were able to colonize the human intestinal tract through a plasmid-encoded 29,000-Da surface protein. Hybridization experiments indicated that the gene encoding resistance to broad-spectrum cephalosporins by the production of CAZ-1 enzyme and the gene encoding the adhesive property to intestinal cells were both located on a 20- to 22-kb EcoRI restriction DNA fragment. Genes encoding aerobactin and the ferric aerobactin receptor were also found on this R plasmid.


Role of nitrogen regulator I (NtrC), the transcriptional activator of glnA in enteric bacteria, in reducing expression of glnA during nitrogen-limited growth.

During nitrogen-limited growth, transcription of glnA, which codes for glutamine synthetase, requires sigma 54-RNA polymerase and the phosphorylated from the nitrogen regulator I (NRI; also called NtrC). In cells in which the lac promoter controlled expression of the gene coding for NRI, increasing the intracellular concentration of NRI lowered the level of glutamine synthetase. The reduction in glutamine synthetase does not appear to result from the NRI-dependent sequestering of any protein that affects transcription of glnA. Our results also suggest that the negative effect of a high concentration of NRI on glnA expression is a major determinant of the level of glutamine synthetase activity in nitrogen-limited cells of a wild-type strain. We propose that the inhibition results from an impairment of the interaction between NRI-phosphate and RNA polymerase that stimulates glnA transcription. We discuss a model that can account for this reduction in glutamine synthetase.


Analysis of the autolysins of Bacillus subtilis 168 during vegetative growth and differentiation by using renaturing polyacrylamide gel electrophoresis.

The autolysins of Bacillus subtilis 168 were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels. Four bands of vegetative autolytic activity of 90, 50, 34, and 30 kDa (bands A1 to A4) were detected in SDS and LiCl extracts and in native cell walls by using B. subtilis 168 vegetative cell walls as the substrate incorporated in the gel. The four enzyme activities showed different substrate specificities and sensitivities to various chemical treatments. The autolysin profile was not medium dependent and remained constant during vegetative growth. During sporulation, band A4 greatly increased in activity just prior to mother-cell lysis. No germination-associated changes in the profile were observed, although a soluble 41-kDa endospore-associated cortex-lytic enzyme was found. By using insertionally inactivated mutants, bands A1 and A2 were positively identified as the previously characterized 90-kDa glucosaminidase and 50-kDa amidase, respectively. The common filamentous phenotype of various regulatory mutants could not be correlated to specific changes in the autolysin profile.


Translational control of pyrC expression mediated by nucleotide-sensitive selection of transcriptional start sites in Escherichia coli.

Expression of the pyrC gene, which encodes the pyrimidine biosynthetic enzyme dihydroorotase, is negatively regulated by pyrimidine availability in Escherichia coli. To define the mechanism of this regulation, an essential regulatory region between the pyrC promoter and the initial codons of the pyrC structural gene was identified. Mutational analysis of this regulatory region showed that the formation of a hairpin at the 5' end of the pyrC transcript, which overlaps the pyrC ribosome binding site, is required for repression of pyrC expression. Formation of the hairpin appears to be controlled by nucleotide-sensitive selection of the site of pyrC transcriptional initiation. When the CTP level is high, the major pyrC transcript is initiated with this nucleotide at a position seven bases downstream of the pyrC -10 region. This transcript is capable of forming a stable hairpin at its 5' end. When the CTP level is low and the GTP level is high, conditions found in cells limited for pyrimidines, the major pyrC transcript is initiated with GTP at a position two bases further downstream. This shorter transcript appears to be unable to form a stable hairpin at its 5' end. These results suggest a model for regulation in which the longer pyrC transcripts are synthesized predominantly under conditions of pyrimidine excess and form the regulatory hairpin, which blocks pyrC translational initiation. In contrast, the shorter pyrC transcripts are synthesized primarily under conditions of pyrimidine limitation, and they are readily translated, resulting in a high level of dihydroorotase synthesis. The data also indicate that a low level of pyrimidine-mediated regulation may occur at the level of transcriptional initiation.


Mutations at Glu-32 and His-39 in the epsilon subunit of the Escherichia coli F1F0 ATP synthase affect its inhibitory properties.

A collection of amino acid substitutions at residues Glu-32 and His-39 in the epsilon subunit of the Escherichia coli F1F0 ATP synthase has been constructed by cassette mutagenesis. Substitutions for residue Glu-32 appeared to cause abnormal inhibition of membrane-bound F1 ATPase activity, and replacement of His-39 by Arg, Val, and Pro affected F1F0 interactions.


Regulation of Anabaena sp. strain PCC 7120 glutamine synthetase activity in a Synechocystis sp. strain PCC 6803 derivative strain bearing the Anabaena glnA gene and a mutated host glnA gene.

The glnA gene from Synechocystis sp. strain PCC 6803 was cloned by hybridization with the glnA gene from Anabaena sp. strain PCC 7120, and a deletion-insertion mutation of the Synechocystis gene was generated in vitro. A strain derived from Synechocystis sp. strain PCC 6803 which contained integrated into the chromosome, in addition to its own glnA gene, the Anabaena glnA gene was constructed. From that strain, a Synechocystis sp. glnA mutant could be obtained by transformation with the inactivated Synechocystis glnA gene; this mutant grew by using Anabaena glutamine synthetase and was not a glutamine auxotroph. A Synechocystis sp. glnA mutant could not be obtained, however, from the wild-type Synechocystis sp. The Anabaena glutamine synthetase enzyme was subject to ammonium-promoted inactivation when expressed in the Synechocystis strain but not in the Anabaena strain itself.


Interactions of leukocyte integrins with intercellular adhesion molecule 1 in the production of inflammatory vascular injury in vivo. The Shwartzman reaction revisited.

We have investigated the role of leukocyte-endothelial cell interactions in a rabbit model of hemorrhagic vasculitis. Microvascular injury was produced in the skin by intradermal injection of Salmonella typhosa endotoxin followed 20 h later by intravenous zymosan, which activates complement. Hemorrhagic necrosis develops in the "prepared" skin sites which is characterized by microthrombi, neutrophil aggregation, platelet and fibrin deposition, and massive extravasation of erythrocytes. Hemorrhage in these Shwartzman-like lesions was quantitated by 99mTc-labeled autologous erythrocytes. Inhibition of the hemorrhagic response was obtained with mAb reactive with ICAM-1 as well as mAb against the leukocyte CD18 when either was administered intravenously just before intravenous zymosan challenge. This observation suggests that an intravascular event occurring in response to complement activation is required for the development of hemorrhagic vasculitis. We hypothesize that agents which successfully prepare the skin for the Shwartzman response after their intradermal injection do so by promoting increased intercellular adhesion molecule 1 (ICAM-1) expression on the vascular endothelium. Activation of complement then induces CD11/CD18 expression on circulating leukocytes thus producing an intravascular CD11/CD18-ICAM-1 (leukocyte-endothelium) adhesion event. Inhibition of intravascular leukocyte-leukocyte aggregation with mAb against CD11b (Mac-1) showed partial inhibition of hemorrhage, while mAb against CD11a (LFA-1) showed no inhibitory activity. This type of cytokine-primed, neutrophil-dependent vascular damage may be a model of human vasculitic processes where microvascular damage is produced in the absence of immune-complex deposition.


Genetic and pharmacological evidence for more than one human steroid 5 alpha-reductase.

The enzyme steroid 5 alpha-reductase catalyzes the conversion of testosterone into the more potent androgen, dihydrotestosterone, and impairment of this reaction causes a form of male pseudohermaphroditism in which genetic males differentiate predominantly as phenotypic females. We previously isolated cDNA clones that encode a human steroid 5 alpha-reductase enzyme. Here, we report molecular and genetic studies demonstrating that the gene encoding this cDNA is normal in subjects with the genetic disease steroid 5 alpha-reductase deficiency. We further show that in contrast to the major steroid 5 alpha-reductase in the prostate and cultured skin fibroblasts, the cDNA-encoded enzyme exhibits a neutral to basic pH optima and is much less sensitive to inhibition by the 4-aza steroid, finasteride (MK-906). The results provide genetic, biochemical, and pharmacological support for the existence of at least two steroid 5 alpha-reductase isozymes in man.


Dual antigenic recognition by cloned human gamma delta T cells.

The function of gamma delta T cells is still elusive. The nature of the antigens that they recognize and the mode of presentation of these antigens are largely unknown. The majority of human peripheral gamma delta T cells bear a V gamma 9/V delta 2 T cell receptor, and display nonclonal reactivity to mycobacteria, without restriction by MHC. It is unknown whether these cells have clonal antigenic specificity as well. Here we describe rheumatoid arthritis-derived V gamma 9/V delta 2 T cell clones, displaying dual antigenic recognition: a nonclonal, MHC-unrestricted recognition of mycobacteria, and a clonal recognition of a short tetanus toxin peptide presented by HLA-DRw53, a nonpolymorphic class II MHC molecule associated with susceptibility to rheumatoid arthritis. This is the first evidence that V gamma 9/V delta 2 T cells can recognize nominal antigenic peptides presented by class II MHC molecules. These results suggest that much like alpha beta T cells, V gamma 9/V delta 2 cells may contribute to the immune response against foreign antigens in an antigen-specific and MHC-restricted manner. The reactivity of these gamma delta T cells to mycobacteria may represent a superantigen-like phenomenon.


Long-term CD4+ memory T cells from the spleen lack MEL-14, the lymph node homing receptor.

We have characterized the surface phenotype and function of long-lived, Ag-specific memory CD4+ T cells generated in vivo by immunization with keyhole limpet hemocyanin (KLH). CD4+ T cells from the spleens of mice primed more than 2 mo previously with KLH, produced high levels of IL-2 and IL-3, and low levels of IL-4 and IFN-gamma in response to in vitro restimulation with specific Ag. The KLH-primed T cells mediated carrier-specific helper activity for the antibody production by NIP-primed B cells in secondary in vitro responses to NIP-KLH. Subsets of CD4+ T cells from KLH-primed mice were isolated on the basis of surface CD45RB (23G2) by magnetic separation and were examined for functional capacity in several assays of Ag-specific recall. Virtually all of the secretion of IL-2, IL-3, IL-4, and IFN-gamma in response to restimulation with Ag in vitro was associated with, and considerably enriched in, the CD45RB- subset of CD4+ T cells. Similarly, carrier-specific helper function and Ag-specific proliferation in vitro were also confined to the CD45RB-, CD4+ subset of T cells, confirming the previous association of this surface phenotype with memory Th cell activity. We also examined expression of the lymphocyte homing receptor, MEL-14 (gp90MEL), which is required for lymphocyte extravasation to peripheral lymph nodes and is present in high levels on naive T cells. MEL-14 positive and negative subsets of CD4+ T cells from long term KLH-primed mice were evaluated for Ag-specific memory function in terms of lymphokine production, Ag-induced proliferation, and helper activity. Each of these functions was associated exclusively with the MEL-14- subset of CD4+ T cells, which exhibited responses comparable to the CD45RB- subset. These data indicate that memory Th cell function in the spleen is contained within the MEL-14-, CD45RB- subset of CD4+ T cells and suggest that memory helper cells may have different patterns of recirculation from naive T cells.


Characterization of a novel rat thymocyte costimulating antigen by the monoclonal antibody 1.3.

To define membrane-associated molecules that impart signals for the activation and expansion of double negative (DN) cells, mAb were raised against in vitro-cultured rat DN cells. One such mAb, 1.3, stimulated proliferation of DN cells along with submitogenic concentrations of PMA and IL-2 without affecting the mobilization of Ca2+. The 1.3 mAb precipitated a heterodimeric protein from DN cells and kidney (130/110 kDa). Although the tissue distribution and biochemical characteristics of the 1.3 determinant resemble the neutral aminopeptidase (AP-N) first described as the thymocyte activating molecule in the mouse, other data are contradictory; AP-N message was not detected in mRNA from 1.3 positive cells and the AP-N gene was absent in the genomic DNA from rat DN hybridomas expressing high levels of 1.3 Ag. In addition, the 1.3 mAb did not affect AP-N enzyme activity suggesting that 1.3 mAb does not function through this enzyme to transduce signals for proliferation. Thus, the 1.3 mAb defines a new and important thymocyte costimulating Ag.


CD31, a novel cell surface marker for CD4 cells of suppressor lineage, unaltered by state of activation.

In this study, we examined the role of CD31 as a cell surface marker for subsets of human CD4 cells. CD31, as defined by a newly developed mAb termed anti-1F11, can divide activated as well as resting CD4 cells into distinct functional subpopulations, based on its surface expression. Among CD4 cells freshly isolated from peripheral blood, anti-1F11 preferentially reacts with the CD45RA+ subset. The majority of helper activity for B cell IgG synthesis and memory function to recall Ag such as tetanus toxoid or mumps was found within the CD31- CD4 cell population, whereas CD31+ CD4 cells provided poor helper function for B cell IgG synthesis and were more responsive to Con A and autologous MHC (autologous MLR). The expression of CD31 on CD45RA+ CD4 cells did not change after activation, despite the loss of CD45RA from the cell surface. Conversely, CD31 was not acquired after activation of CD45RO+ CD45RA- CD4 cells. Furthermore, activated CD4 cells expressing CD31 can induce suppressor function for B cell IgG synthesis, whereas the reciprocal population of activated CD4 cells (CD31-) provide strong helper function for B cell IgG production. Finally, IL-4 production could only be induced by stimulation with PMA and ionomycin in either resting or activated CD31- CD4 cells. Thus, CD31 may prove useful in defining CD4 populations with reciprocal functional programs. Moreover, unlike other markers used for this purpose, the expression of CD31 does not change after activation and may serve as a more useful marker for identification of cells of suppressor or helper lineage.


Sustained engraftment of mice transplanted with IL-1-primed blood-derived stem cells.

IL-1 is considered the primary mediator of the acute phase response. One of the characteristic manifestations of this response is early neutrophilia that is probably caused by release of mature neutrophils from the bone marrow into the peripheral blood. In the present study, we assessed whether IL-1 had a similar releasing effect on the number of circulating progenitor cells and stem cells. Female BALB/c mice were injected i.p. with increasing (0.1-1.0 micrograms/mouse) concentrations of rhu-IL-1 alpha. IL-1 injection resulted in a marked dose-dependent increase in the number of polymorphonuclear neutrophils, granulocyte-macrophage colony-forming units (CFU-GM), and cells forming spleen colonies (CFU-S day 8 and day 12). The maximal increase was found at 4 to 8 h after injection of 1 micrograms IL-1 per mouse, yielding a mean fivefold elevation in neutrophil count, and a mean 30-fold and 10-fold increase in the number of circulating CFU-GM and CFU-S, respectively. In a subsequent series of experiments, lethally irradiated (8.5 Gy) female recipient animals were transplanted with 5 x 10(5) blood mononuclear cells derived from male IL-1-treated animals. Long-term survival was obtained in 68% of mice transplanted with peripheral blood cells derived from donor animals at 6 h after a single injection of 1 micrograms IL-1. The mean number of circulating CFU-GM in these donor animals was 557/ml blood. At 6 mo after transplantation, greater than 95% of the bone marrow cells were of male origin, as determined using in situ hybridization with a Y-chromosome specific probe. In contrast, long-term survival was reached in less than 10% of mice transplanted with an equal number of blood cells derived from saline-treated controls or donor animals treated with a dose of 0.1 micrograms IL-1. These results indicate that a single injection of IL-1 induces a shift of hematopoietic progenitor cells and marrow repopulating cells into peripheral blood and that these cells can be used to rescue and permanently repopulate the bone marrow of lethally irradiated recipients.


Tumor-bearing mice exhibit a progressive increase in tumor antigen-presenting cell function and a reciprocal decrease in tumor antigen-responsive CD4+ T cell activity.

Splenic CD4+ T cells from BALB/c mice bearing a syngeneic tumor (CSA1M) 2 to 3 wk after the inoculation with CSA1M cells produced IL-2 and macrophage-activating factor upon in vitro cultures. This lymphokine production was achieved without stimulation of these T cells with exogenous stimulating tumor Ag. However, elimination of APC from spleen cells resulted in almost complete abrogation of the capacity of CD4+ T cells to produce IL-2/macrophage-activating factor. The lymphokine production was regained when APC from CSA1M-bearing mice were added back to cultures. APC from normal or another syngeneic tumor (Meth A)-bearing mice failed to regain the lymphokine production. These observations demonstrated that the lymphokines were produced by CD4+ T cells from CSA1M-bearing hosts through their collaboration with APC binding CSA1M tumor Ag in the tumor-bearing state. The lymphokine-producing capacity of whole spleen cells from tumor-bearing mice reached the maximal level around 2 to 3 wk after tumor implantation but gradually decreased with the progress of tumor-bearing stages. Importantly, tumor-bearing stage-related changes were observed in a different fashion in the capacities of anti-CSA1M CD4+ T cells vs CSA1M tumor Ag-binding APC. The capacity of APC increased with the progress of tumor-bearing stages as demonstrated by the stimulation of CSA1M-immunized T cells with APC from different CSA1M-bearing stages. In contrast, the reactivity of anti-CSA1M T cells to APC from a given CSA1M-bearing stage decreased with the tumor-bearing stage. These results demonstrate a stage-related increase tumor Ag-binding APC function, as well as a reciprocal reduction in tumor Ag-responsive CD4+ T cell activity.


Long-term consequences of 239PuO2 exposure in dogs: persistent T lymphocyte dysfunction.

Young Beagle dogs were exposed by inhalation to aerosols of 239PuO2 and observed for their lifespans as part of a large, ongoing study of the biological effects of inhaled radionuclides. The purpose of our study was to compare certain immune responses of the 239PuO2-exposed dogs at middle age (7-10 years old) and old age (12-14 years old), with those of unexposed, age-matched or young (3-4 years old) animals. Some of the aged, exposed dogs had developed lung tumours. Lymphocyte proliferative responses to phytohaemagglutinin (PHA) were lower in aged control dogs than in either young or middle-aged control dogs. Both aged and middle-aged, radiation-exposed dogs had decreased responses to PHA when compared to age-matched controls. Responses to concanavalin A (Con A) were not affected by age in control dogs, but tended to decrease in the oldest group of radiation-exposed dogs. Responses to both PHA and Con A were severely depressed in tumour-bearing dogs. The cytolytic activity of natural killer cells was not affected by age, radiation exposure, or tumour presence. We concluded that inhalation of 239PuO2 by young Beagle dogs resulted in an earlier-than-normal decrease in the ability of T cells to respond to mitogenic stimulation. In other words the depressed responses to PHA that were observed might represent radiation-induced, accelerated ageing of the T cell response.


Iron(II) sulphate (Fricke solution) oxidation yields for 8.9 and 13.6 keV X-rays from synchrotron radiation.

The oxidation yields (G) for 8.86 and 13.55 keV X-rays produced by synchrotron radiation were measured using an iron(II) sulphate (Fricke) solution. Monoenergetic X-rays were produced using a silicon crystal monochromator. The X-rays were absorbed in 0.4 M sulphuric acid-iron(II) sulphate solution and FeIII ion yields were measured and corrected for escape fractions resulting from scattering using Monte Carlo calculations. Doses in the solution were determined using a thin window, parallel plate chamber calibrated against a primary standard free-air chamber at the Electrotechnical Laboratory (Osaka, Japan). Yields (G) of 1.50 +/- 0.06 and 1.43 +/- 0.06 mumol J-1 were obtained for 8.86 and 13.55 keV X-rays respectively.


Oxygen effect for DNA double-strand break induction determined by pulsed-field gel electrophoresis.

The induction of DNA double-strand breaks (dsb) following irradiation under oxygenated and hypoxic conditions with and without misonidazole was measured by pulsed-field gel electrophoresis (PFGE) in a human bladder carcinoma cell line. The dose-response curve for DNA dsb detection by PFGE was biphasic with an apparent reduction in rate of dsb induced with dose. Oxygen enhancement ratios (OER) for cell survival (at a surviving fraction of 0.1) and for DNA damage assessed by PFGE (at 80% retained) were 2.0 and 3.0 respectively. Dose-modifying factors for misonidazole (15 mM), of 1.9 (survival) and 2.4 (DNA damage) were found. Although the magnitude of the inter-experiment variations limit the precision with which cell survival and DNA electrophoresis can be compared, the data do support a simple correlation between these two measures of response. When DNA dsb induction frequency was assessed from the number average molecular weight, values of 2.7 (+/- 0.3), 0.7 (+/- 0.1) and 2.6 (+/- 0.5) x 10(-9) dsb/bp/Gy were found for irradiation under oxic, hypoxic alone and hypoxic + misonidazole conditions respectively. This gives an OER of 3.9 and a DMF of 3.7.


Radon: current challenges in cellular radiobiology.

Radon is by far the most important contributor to the collective dose equivalent. Most of what is known about the hazards of radon daughters comes from epidemiological studies of miners. There are a few well defined areas in which in vitro research can complement such studies: First, more data on the relative effects of differing energy (LET) alpha-particles would help: (1) understand the significance of the depth of sensitive cells in the bronchial epithelium--which varies between individuals, as well as between smokers and non-smokers, and between miners and non-miners; (2) understand the relative hazards of radon and thoron daughters. Second, reliable methods for predicting high LET responses from low LET response, would enable Japanese A-bomb survivor data to be applied with confidence. Third, understanding the effects of single-particle traversals of cells relative to multiple traversals could allow reliable extrapolation of epidemiological miner data to low exposures. Fourth, a better understanding of the nature of the interaction between tobacco and radiation damage would help predict the effect of radon on non-smokers.


Repair of DNA strand breaks at hyperthermic temperatures in Chinese hamster ovary cells.

The repair of DNA double-strand breaks was measured by pH 7.2 filter elution in cells incubated at 25-45 degrees C either before or after X-irradiation. Exposure to 45 degrees C for 15 minutes immediately prior to X-irradiation significantly increased both the half-time for DNA double-strand break closure and the number of DNA double-strand breaks remaining in nuclear DNA 180 minutes after irradiation. Exposure to temperatures between 41 and 45 degrees C immediately after X-irradiation accelerated DNA double-strand break closure and resulted in no increase in the number of DNA double-strand breaks remaining in the cell's genome 180 minutes after irradiation. The results indicate either that the radiosensitization produced by the administration of hyperthermic temperatures before and after irradiation result from two characteristically different molecular mechanisms, or that neither the rate of DNA strand break closure nor the number of DNA strand breaks remaining in nuclear DNA after irradiation accurately predict hyperthermic radiosensitization. These conclusions assume that no DNA strand breaks are below the resolution of this DNA damage assay and that a comparison between cytotoxicity and DNA repair after exposure to high radiation doses is valid.


Cellular recovery in two sub-lines of the L5178Y murine leukaemic lymphoblast cell line differing in their sensitivity to ionizing radiation.

Cellular recovery was assessed in two sublines of L5178Y murine lymphoma cells of differing radiosensitivity (LY-S and LY-A4) using low dose-rate irradiation and split-dose experiments. No increase in cell survival was observed in the LY-S cell line until the dose-rate was reduced to 2 cGy/min, whereas in the LY-A4 cell line 20 cGy/min was low enough to detect changes in survival. The extent of this change, as assessed by dose reduction factors at 2 logs of cell kill, was greater in the LY-A4 cell line. Fitting these data with the incomplete repair model of Thames led to anomalous values for the half-time of repair. In split-dose experiments the maximum observed recovery ratio increased as a function of dose in a manner that is consistent with the linear-quadratic equation. As was found previously with radiosensitive human tumour cells, the LY-S cell line showed more split-dose recovery at any given dose than the LY-A4 cell line.


The transition from late G1 to early S phase is most vulnerable to the coclastogenic effect of ultraviolet radiation plus arsenite.

It has previously been reported that chromosome aberrations induced by ultraviolet (UV) radiation can be enhanced by treatment with sodium arsenite for 24 h post-irradiation. Using synchronized CHO-K1 cells, it has now been established that cells in the transitional stage from late G1 to early S phase are most vulnerable to the coclastogenic effect of treatment with UV radiation and arsenite. This result cannot be explained by the special vulnerability of cells in the late G1 to early S transition to UV clastogenicity, as the coclastogenic effects of UV and caffeine or UV and arabinofuranosylcytosine were detected when treating the mid-S but not late-G1 or G2 phase cells.


Radiomimetic cell cycle delay induced by tetranodecanoyl phorbol acetate is enhanced by caffeine and by the protein kinase inhibitor 2-aminopurine.

The tumour promoter and protein kinase C agonist, 12-O-tetranodecanoyl-phorbol-13-acetate (TPA), has been reported to show a radiomimetic action because it transiently delays the passage of HeLa cells through the G2 phase, as do ionizing radiation and other DNA damaging agents. Caffeine is known to override the G2 delay imposed by DNA damage; it is shown here that caffeine does not override the radiomimetic delay imposed by TPA in HeLa, but instead enhances it, without affecting G2 progression in control cells. Most of the other agents which more specifically affect some of the diverse range of caffeine targets either do not affect G2 progression after TPA, or delay G2 progression in control cells and exert a further delay in the presence of TPA. The exception is 2-aminopurine, a protein kinase inhibitor which has been shown to have an action similar to that of caffeine is allowing progression of the cell cycle to mitosis after the inhibition of DNA synthesis, without affecting normal cycle progression through G2. This agent, like caffeine, also has the contrary action of retarding cycle progression after TPA. It is concluded that the G2 delays induced by ionizing radiation and by TPA operate by different mechanisms, which are modulated in opposite senses by mechanisms involving protein kinase inhibition.


In vitro and in vivo studies using BW12C: toxicity, haemoglobin modification and effects on the radiosensitivity of normal marrow and RIF-1 tumours in mice.

BW12C binds to haemoglobin, shifting the oxygen saturation curve to the left, and is under investigation as an inducer of tumour hypoxia. The intrinsic cellular toxicity of the drug to RIF-1 and EMT6 cells in monolayer culture was studied, and IC50 values of 100 micrograms ml-1 for 24 h exposure and 10 micrograms ml-1 for 4-day exposure were measured. The LD50 (95% CL) in C3H mice was shown to be 124 (118-130) mg kg-1 for normal, rapid i.v. injection of the drug, and 173 (164-181) mg kg-1 for slow injection. The well-tolerated dose of 70 mg kg-1, used for all subsequent studies, was shown to produce a maximum haemoglobin modification of 70% 5 min after i.v. administration. This effect decayed with a half-life (+/- 2 se) of 76 +/- 8 min, giving 50% modification at 30 min and 22-25% modification at 2 h after administration. A dose of 70 mg kg-1 BW12C administered 30 min before irradiation protected animals against lethality, and increased the radiation LD50 (95% CL) from 7.16 (7.05-7.27) to 7.86 (7.70-8.02) Gy, representing a DMF of 1.1. In contrast the same drug dose and schedule did not alter normal marrow CFUs radiosensitivity at doses up to 6 Gy. The dose of 70 mg kg-1 did, however, cause marked radioprotection in RIF-1 intramuscular leg tumours. Four- to seven-fold increases in survival were measured by clonogenic cell survival immediately or 24 h after treatment. Protection was maximal 15 to 30 min after administration, and absent by 2 h. The drug did not protect RIF-1 cells in culture against radiation damage, indicating that the in vivo effect is indirect. BW12C is therefore an effective tumour radioprotector in this tumour model, in a manner consistent with an increase in tumour hypoxic fraction, although factors other than changes in blood chemistry may also be involved.


Single-strand break disappearance in quiescent and phytohaemagglutinin-stimulated human peripheral blood lymphocytes exposed to a single low dose of gamma-radiation.

Quiescent and phytohaemagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBL) were irradiated with 4 Gy of gamma-rays and assayed using the alkaline filter elution technique to determine (1) the rate of removal of single-strand breaks (ssb) and (2) the occurrence of excision repair events as indicated by the accumulation of ssb in the presence of the excision repair inhibitor 1-beta-D-arabinofuranosylcytosine (araC). The percentage of ssb disappearance, in the absence of araC, at 5 min after irradiation was significantly higher in PHA-stimulated PBL than in quiescent PBL [40.4 +/- 8.4% (mean +/- SD) and 71.3 +/- 6.8% in quiescent and PHA-stimulated PBL, respectively]. In the presence of araC, both quiescent and PHA-stimulated PBL rapidly accumulated araC-associated ssb, indicating the inhibition of early (base excision) repair processes acting on alkali-stable base damages. Results with PBL from two different donors indicated a significantly higher rate of accumulation of araC-associated ssb in PHA-stimulated PBL than in quiescent cells. In PBL from a third donor no such difference in the rate of accumulation of araC sites was observed. After 1 h repair incubation, the same number of araC-associated ssb was found in the two different cell populations from all three donors.


Depression by sodium ions of calcium uptake mediated by non-N-methyl-D-aspartate receptors in cultured cerebellar neurons and correlation with evoked D-[3H]aspartate release.

In a previous study we noted that the release of D-[3H]aspartate evoked by non-N-methyl-D-aspartate (non-NMDA) receptor agonists in cultured rat cerebellar granule cells was enhanced in the absence of extracellular Na+. To explain this apparent paradox, we tried in the present investigation to correlate the effect of Na+ removal on the kainate (KA)- and quisqualate (QA)-induced D-[3H]aspartate release with that on KA- and QA-induced 45Ca2+ accumulation. The releasing activity of KA, which was only partially Ca2+ dependent in the presence of Na+, became totally Ca2+ dependent in its absence. Moreover, the releasing activity of QA, which was Ca2+ independent in the presence of Na+, became 50% Ca2+ dependent in the absence of the monovalent cation. The releasing action of both agonists was in all cases antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and that induced by KA was also sensitive to kynurenic acid. When glutamate was tested as an agonist in the presence of Na+, it was found that its D-[3H]aspartate releasing action was Ca2+ independent and was largely due to heteroexchange. The evoked release was Ca2+ independent, scarcely sensitive to CNQX, and insensitive to NMDA antagonists. In Na(+)-free medium, the glutamate-evoked D-[3H]aspartate release was lower (due to the abolishment of heteroexchange), but was totally Ca2+ dependent and antagonized by CNQX and kynurenate. KA (30 microM-1 mM) stimulated the accumulation of 45Ca2+ in a dose-dependent and CNQX-sensitive way, the effect being progressively higher as the Na+ concentration in the medium was decreased. Li+ affected KA-induced 45Ca2+ accumulation in a way similar to Na+, although 45Ca2+ uptake was somewhat lower in Li(+)-containing medium. The voltage-activated calcium channel antagonists La3+ and (-)-202-791 caused only a limited inhibition of the KA-induced 45Ca2+ influx both in the presence and in the absence of Na+. Under all the conditions tested [presence and absence of Na+ and of (-)-202-791], the kainate-induced 45Ca2+ uptake was scarcely sensitive to the NMDA antagonist 2-amino-5-phosphonovalerate. QA and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid also stimulated 45Ca2+ influx in a CNQX-sensitive way, the effect being enhanced in Na(+)-free media. These agonists were, however, less effective than KA.(ABSTRACT TRUNCATED AT 400 WORDS)


Expression of the neuronal surface glycoprotein Thy-1 does not follow appearance of its mRNA in developing mouse Purkinje cells.

In developing rodent nervous system, although the appearance of Thy-1 mRNA, as seen by in situ hybridisation, is in general quickly followed by the appearance of immunohistochemically detectable protein, there are certain sites where a delay of several days occurs between expression of detectable message and protein. Mouse Purkinje cells exemplify this behaviour and are the dominant Thy-1-expressing cell in early postnatal cerebellum, so allowing quantitative, homogenate-based methods to be used to test whether such a lag in protein expression does occur. Measurement of Thy-1 mRNA (by slot blot) and protein (by radioimmunoassay) shows a substantial excess of Thy-1 message, compared to protein accumulating in the tissue, during the first postnatal week, which is not found in tissues (rat cerebellum, and rat or mouse cerebrum) where no lag is apparent in appearance of Thy-1 protein from the section-based methods. The species of Thy-1 mRNA produced by Purkinje cells does not appear to change during development, as assessed either in terms of its size (by northern blotting) or in the heterogeneous pattern of transcription initiation sites used (assessed by S1 nuclease protection analysis). Appearance of Thy-1 protein in these cells, therefore, seems to be regulated posttranscriptionally.


Coordinate regulation of the cyclic AMP system with firing rate and expression of tyrosine hydroxylase in the rat locus coeruleus: effects of chronic stress and drug treatments.

Recent studies have demonstrated that chronic stress increases the firing rate and expression of tyrosine hydroxylase (TH) in neurons of the locus coeruleus (LC), the major noradrenergic nucleus in brain. The present study was undertaken to examine the influence of chronic stress and other treatments known to influence the activity of LC neurons on the cyclic AMP (cAMP) second messenger system in these neurons. Chronic (5 days) cold exposure significantly increased levels of TH immunoreactivity in the LC, as previously reported, but not in substantia nigra (SN) or ventral tegmentum (VT), two dopaminergic nuclei studied for comparison. Chronic cold exposure increased levels of cAMP-dependent protein kinase activity in soluble, but not particulate, fractions of the LC, and increased basal and GTP- and forskolin-stimulated adenylate cyclase activity in this brain region. In contrast, levels of the protein kinase and adenylate cyclase in VT, SN, and frontal cortex were not significantly influenced by cold exposure. To study further the relationship between regulation of LC firing rate, TH expression, and the cAMP system in the LC, other treatments known to influence TH were examined. Reserpine treatment, shown previously to increase levels of TH, was found to increase both LC firing rate and levels of soluble cAMP-dependent protein kinase activity in the LC. 6-Hydroxydopamine, shown previously to increase levels of TH and firing rate of LC neurons, also increased soluble levels of protein kinase activity. Other treatments known to either increase (adrenalectomy) or decrease (chronic imipramine) levels of TH in the LC were also found to increase or decrease, respectively, levels of cAMP-dependent protein kinase activity in this brain region.(ABSTRACT TRUNCATED AT 250 WORDS)


Blood-brain barrier pericytes are the main source of gamma-glutamyltranspeptidase activity in brain capillaries.

Cerebral endothelial cells form the selective permeability barrier between brain and blood by virtue of their impermeable tight junctions and the presence of specific carrier systems. These specialized properties of brain capillaries are reflected in the presence of proteins that are not found in other capillaries of the body. gamma-Glutamyltranspeptidase (GGT) has been widely used as a marker for brain capillaries and differentiated properties of brain endothelial cells. By using histochemical and biochemical methods we have investigated the expression of GGT in isolated capillaries, cultured brain endothelial cells and pericytes, and cocultures of astrocytes and brain endothelial cells. It was surprising that the majority of GGT activity was associated with pericytes, but not endothelial cells, suggesting that GGT is a specific marker for brain pericytes. The remaining GGT activity that was associated with endothelial cells rapidly disappeared from cultured cells but was reinduced in cocultures with astrocytes. Our results emphasize the need for pure endothelial cells for the investigation of blood-brain barrier characteristics.


Role of the central adrenergic system in the regulation of prostaglandin biosynthesis in rat brain.

The role of endogenous catecholamines in the regulation of brain prostaglandin (PG) synthesis was studied in the rat. Male rats were injected in the brain lateral ventricle or in the ventral noradrenergic bundle with either the catecholaminergic neurotoxin 6-hydroxydopamine or vehicle. Other groups of rats were injected intraperitoneally with the tyrosine hydroxylase inhibitor, alpha-methyl-p-tyrosine, or with the inhibitor of dopamine-beta-hydroxylase, FLA-63. All these drugs produced a significant depletion of norepinephrine (NE) content in the cortex and hypothalamus. The rats that had lower levels of NE exhibited reduced capacity to synthesize PGE2 but not thromboxane B2 and 6-keto-PGE1 alpha in the cortex and hypothalamus. However, induced production of PG, stimulated by the bacterial endotoxin lipopolysaccharide (LPS), remained unchanged, namely, a similar (2- to 2.5-fold) increase of PG synthesis was noted in control and in NE-depleted rats. We suggest that the regulation of PG synthesis under basal condition requires intact adrenergic input, whereas LPS-induced production of PG is independent of the adrenergic innervation.


Synapsin I regulates glutamate release from rat brain synaptosomes.

Introduction of the dephosphorylated from of synapsin I into rat brain synaptosomes using freeze-thaw (transient) permeabilization significantly decreased the K(+)-induced release of glutamate. In contrast, introduction of synapsin I that had been phosphorylated by Ca2+/calmodulin-dependent protein kinase II was without effect on glutamate release. Addition of dephosphosynapsin I after freeze-thaw treatment also had no effect. Thus, the action of synapsin I was dependent on the phosphorylation state of synapsin I and on its entry into the synaptosomes. Our results implicate synapsin I as an important component in the regulation of neurotransmitter release in the mammalian nervous system.


Kappa-opioids decrease excitatory transmission in the dentate gyrus of the guinea pig hippocampus.

In the guinea pig hippocampus, kappa 1-opioid binding sites were primarily localized in the molecular layer of the dentate gyrus as shown by autoradiography using either the kappa 1-selective radioligand 3H-U69,593 or the nonselective radioligand 3H-diprenorphine in the presence of unlabeled mu- and delta-blocking ligands. In this region, the electrophysiological effects of kappa 1-receptor activation were identified using extracellular and intracellular recordings of dentate granule cell responses. The amplitude of the extracellularly recorded population spike was reduced by U69,593 with an EC50 of 26 nM; this effect was reversible and blocked by the opioid antagonist naloxone. The kappa 1-selective antagonist norbinaltorphimine also blocked the effect of U69,593 with an apparent equilibrium dissociation constant (Ki) of 0.26 nM determined by Schild analysis in the physiologic assay. This value agreed well with the Ki for norbinaltorphimine at kappa 1-binding sites measured by radioligand binding displacement (0.24 nM). These results indicate that the electrophysiologic response observed was likely mediated by kappa 1-receptors. As seen with U69,593, dynorphin B, an endogenous opioid peptide that is present in the dentate gyrus, also inhibited the population spike response. mu- and delta-selective opioid agonists had no effect on the amplitude of the maximally evoked response. Intracellular recordings of dentate granule cells showed no direct effects of U69,593 on the granule cells themselves. However, analysis of synaptic potentials revealed that U69,593 significantly reduced the amplitude of glutaminergic EPSPs evoked by afferent stimulation without affecting IPSP amplitudes. The specific effect of U69,593 application on granule cell EPSPs indicates that presynaptic kappa 1-receptor activation inhibits glutamate release from perforant path terminals in the molecular layer of the dentate gyrus. These results suggest that endogenous dynorphins present in the granule cells may act as feedback inhibitors of the major excitatory input to the dentate gyrus.


Differential effects of neurotrophic factors on neurotransmitter development in the IMR-32 human neuroblastoma cell line.

The human neuroblastoma cell line IMR-32 exhibits both cholinergic and adrenergic properties. We have used IMR-32 cells to study the effects of CDF (CAT development factor) and bFGF (basic fibroblast growth factor) on the development of neurotransmitter properties. CDF treatment increases CAT activity in a dose-dependent manner, independent of cell density. Time course studies show that there is a threefold increase in the specific CAT activity in IMR-32 cells treated with CDF for 6 d. CDF does not, however, affect the level of tyrosine hydroxylase (TH) activity, or the rate of cell proliferation. bFGF, on the other hand, induces TH activity and decreases CAT activity in a dose-dependent manner. bFGF's effect on TH is enhanced by increasing cell density, while its reduction of specific CAT activity is independent of cell density. Time course studies show a 30-fold increase in TH activity per cell and a threefold decrease in CAT activity per cell, after treatment with bFGF for 6 d. In contrast to the effects of CDF, bFGF enhances cell proliferation in IMR-32 cells. Double-labeled immunofluorescence studies showed that 95% of the cells stain for CAT and 65% stain for TH following treatment with CDF and bFGF, respectively. When these factors are combined, approximately 75% of the cells express both CAT and TH, demonstrating that IMR-32 cells are bipotential with regard to neurotransmitter-associated enzyme expression. We also show that insulin-like growth factor I and NGF selectively induce CAT activity and cell proliferation, respectively, whereas epidermal growth factor has no effect.(ABSTRACT TRUNCATED AT 250 WORDS)


The NMDA receptor antagonist D-2-amino-5-phosphonopentanoate (D-AP5) impairs spatial learning and LTP in vivo at intracerebral concentrations comparable to those that block LTP in vitro.

This series of experiments investigated whether the NMDA receptor antagonist D-2-amino-5-phosphonopentanoate (D-AP5) could induce impairments of spatial learning across a dose range comparable to its impairment of hippocampal long-term potentiation (LTP) in vivo. Estimations of the extracellular concentration of D-AP5 in hippocampus using microdialysis were also made to compare whether these impairments occur at concentrations similar to those required to impair LTP in the in vitro hippocampal slice. Rats were chronically infused with D-AP5 into the lateral ventricle at a range of concentrations (0-50 mM) via osmotic minipumps. They were first trained to find and escape onto a hidden platform in an open-field water maze task. After the behavioral learning, they were anesthetized with urethane and an attempt was made to evoke and monitor hippocampal LTP. Extracellular samples of D-AP5 in hippocampus were then taken using microdialysis, and finally, the animals were killed and tissue samples dissected. The microdialysis and tissue samples were analyzed for D-AP5 content using HPLC with fluorescence detection. The results established, first, that D-AP5 impairs spatial learning in a linear dose-dependent manner, highly correlated with its corresponding impairment of hippocampal LTP in vivo. No concentration of D-AP5 was observed to block LTP without affecting learning. Second, the microdialysis estimates indicated that, subject to certain assumptions, D-AP5 causes these impairments at extracellular concentrations comparable to those that impair LTP in vitro. Third, comparison of the whole tissue and microdialysis samples revealed a concentration ratio of approximately 30:1, indicating that 97% of the intracerebral D-AP5 is inaccessible to the dialysis probes. Infusion of 20 mM EGTA was found to cause a sevenfold increase in D-AP5 in the dialysis perfusates, suggesting that at least part of the inaccessible D-AP5 is trapped by a calcium-dependent mechanism. Two further behavioral control studies indicated that the D-AP5-induced impairment of spatial learning is unlikely to be secondary to a drug-induced motor disturbance, and that the performance of the D-AP5 group whose concentration was just sufficient to block hippocampal LTP completely was statistically indistinguishable from that of a group of rats with bilateral hippocampal lesions induced by ibotenic acid. Taken together, these findings offer support for the hypothesis that activation of NMDA receptors is necessary for certain kinds of learning.


Glutamate uptake disguises neurotoxic potency of glutamate agonists in cerebral cortex in dissociated cell culture.

The pharmacological properties of glutamate agonists were compared in astrocyte-rich and astrocyte-poor cultures derived from embryonic rat cerebral cortex. The object of this investigation was to determine the extent to which glutamate uptake might influence the receptor-mediated neurotoxic actions of these compounds. In astrocyte-rich cultures, using 30 min exposures, we observed that the potencies of the poorly transported agonists NMDA (35 microM) and D-glutamate (89 microM) were higher than that of L-glutamate (205 microM). In astrocyte-poor cultures, L-glutamate was much more potent, with an EC50 of 5 +/- 4 microM (3-12 microM), for a 30 min exposure, whereas the potencies of NMDA and D-glutamate were essentially unchanged. L- and D-aspartate were also more effective in astrocyte-poor cultures, again with EC50 values of approximately 6-10 microM, as compared with 130 and 108 microM, respectively, in astrocyte-rich cultures. In other experiments, blocking sodium-dependent glutamate uptake in astrocyte-rich cultures, by using a sodium-free medium, made glutamate as potent an agonist as in astrocyte-poor cultures. Finally, we directly assessed the glutamate uptake system in astrocyte-rich and astrocyte-poor cultures and found that uptake was reduced approximately 25-fold in the astrocyte-poor cultures. These results show that in the presence of abundant astrocytes the neurotoxic potencies of L-glutamate, L-aspartate, and D-aspartate are substantially under-estimated.


Regulation of hepatic lipogenic enzyme gene expression by diet quantity in rats fed a fat-free, high carbohydrate diet.

This investigation concerns the effects of the level of intake of a high carbohydrate diet on transcriptional rate, mRNA concentration and enzyme induction for lipogenic enzymes in rat liver. Six hours after refeeding fasted rats, the transcriptional rates in livers reached low maximum levels with small quantities of diet, but the mRNA concentrations continued to increase as diet intake increased. Greater diet intake primarily increased transcriptional rates and mRNA concentrations of lipogenic enzymes. After refeeding for 16 h, the mRNA concentrations were sigmoidly increased relative to the diet quantity and reached maximum levels of 20-, 110-, 22- and 16-fold above each fasted level for acetyl-CoA carboxylase, fatty acid synthase, malic enzyme and glucose-6-phosphate dehydrogenase, respectively. After 3 d of refeeding (in a steady state of lipogenic enzyme activities), however, the transcriptional rates, mRNA concentrations and activity inductions of all the enzymes were sigmoidly increased relative to diet quantity, but were not different among the enzymes. Consequently, fatty acid synthesis and triglyceride levels in the liver were not increased by feeding less than 70% of ad libitum intake but were greatly increased by feeding greater than 70% of ad libitum intake.


A novel method of induced renal hypothermia.

When the kidney is removed from cold storage for implantation into the recipient it gradually rewarms (second warm ischaemic time) and a prolonged second warm ischaemic time is a risk factor for delayed graft function. A cooling jacket has been designed to prevent this rewarming during transplantation. This study evaluates the efficacy of this device. Surface and core temperatures of less than 15 degrees Centigrade were maintained for 120 minutes. Renal function was significantly better in cooled than in uncooled kidneys in a single kidney canine model. Induced renal hypothermia, using a device such as this, should be a routine manoeuvre in renal transplantation.


The effects of an LHRH agonist on testicular function in the cryptorchid rat.

To assess the effects of an LHRH analog (LHRH-ethylamide des gly10 D-Leu6) on the function of the cryptorchid testis, an animal model was created, using prepubertal male Sprague-Dawley rats (21 days old). The animals were divided into six groups: 1) sham operated; 2) sham + LHRH treated; 3) cryptorchid; 4) cryptorchid + LHRH treated; 5) cryptorchid and orchidopexed; 6) cryptorchid, LHRH treated and orchidopexed. Two weeks post cryptorchidism, the animals were treated with either saline or LHRH-analog (one microgram./rat/day) subcutaneously for a total of twenty five days. Orchidopexy was performed following hormonal treatment. The effects of treatment were assessed in terms of body and reproductive organ weights, serum testosterone and LH levels, sperm counts and motility and fertility. The results demonstrated that experimental cryptorchidism impaired reproductive function, which was restored by orchidopexy, alone. Treatment of cryptorchid rats (groups 4 and 6) with LHRH did not produce any lasting improvement in spermatogenesis or fertility.


Systemic treatment of early breast cancer by hormonal, cytotoxic, or immune therapy. 133 randomised trials involving 31,000 recurrences and 24,000 deaths among 75,000 women. Early Breast Cancer Trialists' Collaborative Group.

In a worldwide collaboration, information was sought and centrally checked on mortality and recurrence for each woman in any randomised trial that began before 1985 of any aspect of systemic adjuvant therapy for early breast cancer. Checked data were available for 75,000 women (about 90% of those ever randomised), of whom 32% had died and another 10% had experienced recurrence. The parts now reviewed include 30,000 women in tamoxifen trials, 3000 in ovarian ablation trials, 11,000 in polychemotherapy trials, 15,000 in other chemotherapy comparisons, and 6000 in immunotherapy trials. Highly significant reductions in the annual rates both of recurrence and of death are produced by tamoxifen (25% SD 2 recurrence and 17% SD 2 mortality: 2p less than 0.00001), by ablation below age 50 (26% SD 6 recurrence and 25% SD 7 mortality: 2p = 0.0004), and by polychemotherapy (28% SD 3 recurrence and 16% SD 3 mortality: 2p less than 0.00001), but not by ablation at older ages or by immunotherapy. (Tamoxifen also reduced the risk of development of contralateral breast cancer by 39% SD 9: 1p less than 0.00001). For tamoxifen and for polychemotherapy the avoidance of recurrence is chiefly during years 0-4 (this difference being maintained but not increased afterwards), but the avoidance of mortality is highly significant both during and after years 0-4, so the cumulative differences in survival produced by these relatively brief treatments (median: 2 years tamoxifen, 1 year polychemotherapy) are larger at 10 than at 5 years. There is little information beyond year 10 (except for ovarian ablation, which produces separately significant mortality reductions both during and after years 0-9). Both direct and indirect randomised comparisons show long-term polychemotherapy (eg, 12 months) to be no better than shorter (eg, 6 months) regimens, but do show polychemotherapy to be significantly better than single-agent chemotherapy. Indirect randomised comparisons do not reveal significant differences between different forms of polychemotherapy, or differences between different tamoxifen doses, but do show that long-term tamoxifen (eg, 2 years, or even 5 years) is significantly more effective than shorter tamoxifen regimens. In old age (70+) tamoxifen is of demonstrated efficacy, but chemotherapy has not been evaluated. Between ages 50 and 69 direct comparisons show that chemotherapy plus tamoxifen is better (1p less than 0.00001) than chemotherapy alone both for recurrence and for mortality, and better (1p less than 0.00001) than tamoxifen alone for recurrence.(ABSTRACT TRUNCATED AT 400 WORDS)


HIV-1 sensitivity to zidovudine and clinical outcome in children.

In adults with the acquired immunodeficiency syndrome, long-term monotherapy with zidovudine selects for human immunodeficiency virus type 1 (HIV-1) strains with substantially reduced in-vitro susceptibility to the drug. We have assessed the relation between in-vitro resistance to zidovudine and clinical outcome in children, in whom disease progression is more rapid than in adults. We studied 23 children with symptoms of HIV-1 disease during extended monotherapy with zidovudine. An in-vitro assay was used to determine the concentration of zidovudine required to inhibit by 50% the replication of viral isolates (IC50) obtained after 9 to 39 months of treatment. Viral stocks of high enough titre to yield reproducible results were obtained from 19 of the children. During the following 6 months of therapy, 9 children were stable, 7 deteriorated, and 3 died. There was a highly significant relation between decreased zidovudine susceptibility and poor clinical outcome (p less than 0.001) but no relation between IC50 and age at start of therapy or length of time on treatment. Age-adjusted CD4 lymphocyte counts were lower at the start of treatment (p = 0.02) and at the time of sampling (p = 0.01) in children whose viral isolates had an increased zidovudine IC50. Initial serum p24 antigen levels were not predictive of subsequent emergence of resistant virus, but at the time of sampling for viral sensitivity higher p24 antigen levels were associated with raised IC50 (p = 0.004). The findings suggest that most children who become unresponsive to monotherapy with zidovudine, as judged by clinical criteria, will have changes in in-vitro sensitivity to the drug. In these children, an alternative antiretroviral therapy should be considered.


Coagulation-factor deficiencies and abnormal bleeding in Noonan's syndrome.

Noonan's syndrome is characterised by a dysmorphic facies, congenital heart disease, and short stature, and is inherited as an autosomal dominant trait. Because abnormal bleeding has also been reported, we investigated a group of patients for coagulation-factor deficits. Of the 72 individuals studied (37 male, 35 female, mean age 11.4 years), 47 (65%) had a history of abnormal bruising or bleeding. 29 patients (40%) had a prolonged activated partial thromboplastin time. Specific abnormalities in the intrinsic pathway of coagulation (partial factor XI:C, XII:C, and VIII:C deficiencies) were found in 36 patients (50%). Multiple abnormalities among these 36 patients included combined factor XI:C and XII:C deficiencies (4 patients) and factor XI:C and VIII:C deficiencies (4), and 1 patient had combined factor VIII:C, XI:C, and XII:C deficiency. There was poor correlation between a history of abnormal bleeding and coagulation-factor deficit. In five families, similar coagulation-factor deficiencies were present in first-degree relatives with the syndrome. The pattern of inherited bleeding abnormalities seen in Noonan's syndrome suggests autosomal regulation of the intrinsic coagulation pathway.


Platelet-derived growth factor BB for the treatment of chronic pressure ulcers.

A randomised, phase I/II, double-blind, placebo-controlled study was designed to assess the effect of topically applied recombinant human BB homodimeric platelet-derived growth factor (rPDGF-BB) on healing of chronic pressure ulcers. Twenty patients were randomly allocated daily treatment for 28 days with 1, 10, or 100 micrograms/ml rPDGF-BB (0.01, 0.1, or 1.0 micrograms per cm2 ulcer area) or placebo. Patients treated with 100 micrograms/ml rPDGF-BB showed a greater healing response than the placebo group, but the lower doses had little effect. After 28 days, ulcers treated with 100 micrograms/ml rPDGF-BB were smaller than those treated with placebo (mean [SE] volume 6.4 [4.0] vs 21.8 [5.6]% of day 0 volume). There were no toxic effects. These preliminary findings suggest that rPDGF-BB is a potent wound-healing agent in soft tissue.


Abnormal sulphur oxidation in systemic lupus erythematosus.

S-carboxy-L-methylcysteine was used to assess the activity of the S-oxidation pathway of sulphur metabolism in 35 patients with systemic lupus erythematosus (SLE); 25 (71%) showed impaired sulphoxidation and 21 (60%) produced virtually no sulphoxides, compared with 17 (36%) and 2 (4%), respectively, of 47 healthy controls. The substrate/product ratio of cysteine oxygenase (plasma cysteine/sulphate) was significantly higher in SLE patients than in controls (median [interquartile range] 362 [224-588] vs 65 [44-111]; p less than 0.00001). The alternative pathway of sulphur metabolism, S-methylation, catalysed by thiolmethyltransferase, was not impaired in the SLE patients. There is a biochemical difference in sulphur metabolism between SLE and rheumatoid arthritis, since both pathways are impaired in the latter disorder.


Assessment of bullet disruption in armed conflicts.

Under international humanitarian law, the Hague Declaration of 1899 forbids the use of small arms ammunition that disrupts in the body after impact. However, assessment of whether bullets have undergone disruption is subjective and accusations that one or both sides of a conflict have used such ammunition cannot be substantiated. We describe a method by which disruption of bullets after impact can be assessed objectively. We reviewed 1287 wound radiographs of 1201 patients in four International Committee of the Red Cross hospitals. Radiographs were scored according to the F (fracture) and M (presence of metallic bodies) variables of the Red Cross wound classification; bullet disruption is evidenced by metallic fragments on the radiograph. One hospital had a significantly higher proportion of patients wounded by bullets that disrupted after impact; these wounds were associated with comminuted fractures. The findings show that the presence of metallic fragments on a radiograph is a reliable indicator of wound severity and bullet disruption. The study has important implications for the upholding of international humanitarian law, because with our method the use and manufacture of bullets that contravene the Hague Declaration can be identified.


Allogeneic bone-marrow transplantation without protective isolation in adults with malignant disease.

Bone-marrow transplant (BMT) patients are severely immunocompromised immediately after the procedure and they are commonly nursed in strict protective isolation to reduce the risk of both infection and graft-versus-host disease (GvHD). We have studied a consecutive series of patients to see whether protective isolation is of benefit as prophylaxis against infectious complications of BMT. 50 consecutive patients who had malignant disease and received their first BMT from siblings or unrelated donors were nursed in standard single rooms with visitors instructed to wash their hands. A subset of 20 patients living locally spent a median of 25 days in hospital after BMT; they also spent some time at home on a median of 8 days before engraftment and 3 patients went home on more than 90% of their hospital days. 16 patients (32%) had positive bacterial cultures and/or focal infection. Gram-positive bacteraemia was found in 12 subjects (24%) but there were no gram-negative or deep fungal infections. Grade II or III acute GvHD developed in 17 patients (34%). There were no deaths from infection or acute GvHD. Transplant-related mortality was 6% in the first 100 days and 18% overall with a median follow-up of 22 months. Our mortality data compare favourably with those from institutions with strict isolation procedures. We conclude that BMT may be safely completed in some institutions without either protective isolation or the need to confine patients continuously in hospital.


Physicians, elderly drivers, and dementia.

In western countries, the proportion of drivers who are elderly is increasing rapidly; over a quarter of the driving population will be 55 years of age or older by the year 2000. Although elderly drivers tend to drive less and may avoid night driving, the number of car accidents and the severity of injuries sustained in such accidents by distance driven increases strikingly after the age of 65. But how can doctors identify those elderly drivers who are a danger to themselves and others? And, once they are identified, how should clinicians balance the effects of removing a driving licence, which may greatly affect a patient's lifestyle, against public safety and breach of confidentiality if the patient refuses to give up his or her licence voluntarily? Medical decisions about illnesses that predispose to loss of consciousness are fairly clear-cut, but normal ageing processes and dementia can be much more difficult to identify and to assess; moreover, there seems little correlation between tests of mental performance and driving ability. Comparisons between practices in different countries may provide some answers, but the introduction of modified driving tests for elderly drivers with some evidence of mild impairment but who wish to retain their driving licence should be considered.


Efficacy of antibiotic prophylaxis for prevention of native-valve endocarditis.

Whether antibiotic prophylaxis can prevent bacterial endocarditis is hotly debated. In an attempt to settle this issue, we have assessed the efficacy of prophylaxis for bacterial endocarditis on native valves in a nationwide, case-control study in the Netherlands. Cases were patients with known cardiac disease in whom endocarditis developed within 180 days of a medical or dental procedure for which prophylaxis was indicated. Of a total of 438 patients with endocarditis diagnosed during 2 years, 48 were eligible for the study. Controls were patients with the same cardiac status in whom endocarditis did not develop within 180 days of a similar procedure; of a total of 889 controls from five hospitals, 200 were eligible. Overall, about 1 in 6 patients in both groups had received prophylaxis. The best estimate of protective efficacy was 49% for first-ever endocarditis occurring within 30 days of a procedure. Endocarditis developed within 30 days of a procedure in only 13% of patients with a previously diagnosed heart lesion which predisposed to the disease. The findings suggest that strict adherence to generally accepted recommendations for prophylaxis might do little to decrease the total number of patients with endocarditis in the community.


Loss of heterozygosity on chromosome 7q and aggressive primary breast cancer.

Genetic alterations are believed to be important in the origin and dissemination of breast cancer. Cytogenetic rearrangements on chromosome 7 are common in breast tumours. We used the c-met proto-oncogene probe, which detects sequences on chromosome 7q31, to analyse tumour and blood leucocyte DNA samples from 245 patients with primary breast cancers. The pmetH polymorphic probe detected a high frequency of allele loss (40.5%) among the 121 informative (heterozygous) patients. This genetic alteration was not significantly associated with standard prognostic features including tumour size, histopathological grade, and lymph-node or steroid receptor status. However, patients with loss of heterozygosity on chromosome 7q31 in primary tumour DNA had significantly shorter metastasis-free survival (p = 0.00022) and overall survival (p = 0.0036) after surgery than patients without this alteration. These findings indicate that this region of chromosome 7 might be the site of a breast tumour or metastasis suppressor gene.


Colour Doppler ultrasound assessment of arteriovenous haemodialysis fistulas.

Satisfactory function of the arteriovenous fistula (AVF) is essential for adequate haemodialysis in patients with chronic renal failure. Existing methods to assess AVF function are imprecise (eg, clinical examination) or invasive (eg, angiography). We assessed the value of colour flow-doppler ultrasound (CFDU) in the investigation of clinically suspected AVF dysfunction. 51 patients with suspected impairment of AVF function were studied by CFDU, and 28 also underwent angiography. The findings were compared with the reference standard of the findings at reoperation. CFDU showed AVF stenoses in 18 patients, which were all confirmed at reoperation; the results of angiography in 13 of these 18 patients showed complete agreement with the findings of CFDU and surgery. CFDU showed partial or complete AVF thrombosis in 33 patients, confirmed in all patients at reoperation; 15 patients also underwent angiography, and thrombi were not found in 6 (in 4 because of technical failure). 7 patients had aneurysms on CFDU that were confirmed by surgery in all patients and by angiography in 2 of the 3 patients studied. CFDU enables reliable non-invasive assessment of AVF morphology and function and may become the procedure of choice for AVF assessment in patients with suspected AVF abnormalities.


Erythrocyte choline uptake after renal transplantation.

Erythrocyte membrane choline transport is abnormally high in chronic renal failure. The aim of this study was to find out whether, and how quickly, this abnormality is reversed by renal transplantation. Ten adults with chronic renal failure were studied before and for up to 6 months after renal transplantation. Plasma creatinine concentration and erythrocyte uptake of radiolabelled choline were measured periodically for up to 6 months. The maximum rate of choline transport (Vmax) was abnormally high before transplantation and fell to normal values during the first week after transplantation. This fall matched that in plasma creatinine. A temporary rise in plasma creatinine in one patient, reversed by methylprednisolone treatment, was accompanied by a rise in choline flux, and graft failure in another patient was accompanied by a return to pretreatment rates of choline transport. Thus, the high rates of choline uptake in uraemia are rapidly reduced on recovery of renal function. The speed of the changes suggests the involvement of a plasma factor.


Ciprofloxacin for treatment of malakoplakia.

The tumour-like lesions of the rare disease malakoplakia, which consist of macrophages containing undigested coliform bacteria, are often misdiagnosed as a carcinoma. Although an infectious aetiology is likely, no antimicrobial therapy has been successful in the long-term. Since ciprofloxacin penetrates well into macrophages, this drug was given to two patients with advanced malakoplakia (500 mg twice daily). After long-term treatment all granulomatous lesions disappeared. Thus, malakoplakia can be cured by antibiotic treatment.


Idiopathic hemiparetic parkinsonism, a syndrome distinct from idiopathic parkinsonism.

Two women had a syndrome of progressive parkinsonism with ipsilateral rigidity, mild resting tremor, paresis, hyperreflexia, and an extensor plantar response. Symptoms had started 24 and 3 months after a surgical procedure in the affected limb. Neuroimaging studies were unhelpful. Both the parkinsonian features and the pyramidal tract signs responded well to dopaminergic drug treatment. We propose that the syndrome be called "idiopathic hemiparetic parkinsonism".


Role of assays for parathyroid-hormone-related protein in investigation of hypercalcaemia.

Parathyroid-hormone-related protein (PTHrP) has been implicated as a humoral mediator of hypercalcaemia in malignant disease. We have investigated the contributions of PTHrP and parathyroid hormone (PTH) to the hypercalcaemia seen in routine clinical practice by means of highly sensitive immunoradiometric assays. PTHrP concentrations in plasma and PTH concentrations in serum were measured in 121 consecutive patients with hypercalcaemia (corrected serum calcium above 2.65 mmol/l) identified from routine biochemical profiles in a district general hospital. Hypercalcaemia was due to primary hyperparathyroidism in 63 (52%) patients and to malignant disease in 40 (49%). Plasma PTHrP was detectable in 35 (88%) of 40 patients with solid tumours and 3 of 9 patients with haematological malignant disease; it was undetectable in 92% of patients with primary hyperparathyroidism. 7 patients with malignant disease had PTH concentrations above 4.0 pmol/l, consistent with coexisting primary hyperparathyroidism. Measurement of both PTH and PTHrP in all patients led to a change in the diagnosis in 7% of patients. This study provides direct evidence for a humoral role of tumour-derived PTHrP in hypercalcaemia, and shows how PTHrP assays can be used appropriately, in conjunction with PTH assays, to investigate hypercalcaemia in routine clinical practice.


Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex.

A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.


Susceptibility to cell death is a dominant phenotype: triggering of activation-driven T-cell death independent of the T-cell antigen receptor complex.

The failure of Thy-1 and Ly-6 to trigger interleukin-2 production in the absence of surface T-cell antigen receptor complex (TCR) expression has been interpreted to suggest that functional signalling via these phosphatidylinositol-linked alternative activation molecules is dependent on the TCR. We find, in contrast, that stimulation of T cells via Thy-1 or Ly-6 in the absence of TCR expression does trigger a biological response, the cell suicide process of activation-driven cell death. Activation-driven cell death is a process of physiological cell death that likely represents the mechanism of negative selection of T cells. The absence of the TCR further reveals that signalling leading to activation-driven cell death and to lymphokine production are distinct and dissociable. In turn, the ability of alternative activation molecules to function in the absence of the TCR raises another issue: why immature T cells, thymomas, and hybrids fail to undergo activation-driven cell death in response to stimulation via Thy-1 and Ly-6. One possibility is that these activation molecules on immature T cells are defective. Alternatively, susceptibility to activation-driven cell death may be developmentally regulated by TCR-independent factors. We have explored these possibilities with somatic cell hybrids between mature and immature T cells, in which Thy-1 and Ly-6 are contributed exclusively by the immature partner. The hybrid cells exhibit sensitivity to activation-driven cell death triggered via Thy-1 and Ly-6. Thus, the Thy-1 and Ly-6 molecules of the immature T cells can function in a permissive environment. Moreover, with regard to susceptibility to Thy-1 and Ly-6 molecules of the immature T cells can function in a permissive environment. Moreover, with regard to susceptibility to Thy-1 and Ly-6 triggering, the mature phenotype of sensitivity to cell death is genetically dominant.


Extrathymic positive selection of alpha beta T-cell precursors in nude mice.

T lymphocytes expressing alpha beta T-cell receptors with sufficient affinity to major histocompatibility complex (MHC) molecules expressed on thymus epithelial cells are positively selected and mature to functional T cells. But several studies have demonstrated that athymic nude mice grafted with MHC-incompatible thymuses developed T cells specific for nude host rather than thymic MHC. We examined this paradox by analysing the specificity of T lymphocytes derived from nude mice. We report here that nude T lymphocyte precursors transferred to allogeneic SCID (severe combined immunodeficiency) mice with a functioning thymus (but lacking T or B cells) generated host MHC-restricted effector T cells but also contained T cells restricted to donor MHC. If nude T cells were depleted from nude lymphohaemopoietic donor cells before or after transfer, only host MHC-specific T cells matured. The results may explain the unusual MHC specificities of nude T lymphocytes described in earlier studies and demonstrate two separate differentiation steps: in nude mice, T cells may be positively selected for self-MHC restriction specificity extrathymically; then a functional thymus is required for efficient T cell maturation.


Molecular characterization and silk gland expression of Bombyx engrailed and invected genes.

Genetic analysis in Drosophila has shown that engrailed (en) plays an important role in segmentation and neurogenesis. A closely related gene, invected (in), is coexpressed with en in the posterior developmental compartments where en is known to specify cell state. We report here the isolation of two en-like cDNAs from the middle silk glands of Bombyx mori larvae. Sequence analysis revealed that they are the counterparts of Drosophila en and in. Four highly conserved domains, including the homeodomain, were identified in these En and In proteins from Bombyx and Drosophila. In addition, two en-specific and one in-specific domains could also be found. These structurally homologous genes might share a similar role in Bombyx development. They were found to be coexpressed in the middle silk gland but not in the posterior silk gland during the fourth molt/fifth intermolt period. We speculate that these Bombyx en-like genes might be involved in the compartmentalization of the silk gland.


Selective killing of CD4+ cells harboring a human immunodeficiency virus-inducible suicide gene prevents viral spread in an infected cell population.

We have stably expressed in CD4+ lymphoid cells the herpes simplex virus type 1 thymidine kinase (HSV1-TK) gene under the control of the human immunodeficiency virus (HIV) promoter and transactivation response element sequences. Upon HIV infection these regulatory sequences were transactivated, switching on high-level expression of HSV1-TK. This in turn caused the death of HIV-infected cells when they were cultured in the presence of acyclovir, a nucleoside analog that becomes toxic after phosphorylation by HSV1-TK. The elimination of HIV-infected cells resulted in the arrest of HIV spreading in the culture. Complete protection of HSV1-TK-expressing cells was obtained using acyclovir concentrations that are commonly detected in the plasma of patients treated for HSV1 infection. Thus, expression of this DNA construct generates a pool of CD4+ booby-trapped cells that, as a population, are resistant to HIV infection. Our data provide a rationale for the use of suicide genes in the design of gene therapy of HIV infection.


Long-distance restriction mapping of the proximal long arm of human chromosome 21 with Not I linking clones.

Human chromosome 21 is the smallest of the 22 autosomes and 2 sex chromosomes. Hybridization of the human repetitive sequence Alu to pulsed-field gel-fractionated Not I-digested genomic DNA from a human-mouse hybrid cell line containing chromosome 21 as the sole human component identified chromosome 21 Not I restriction fragments. A Not I restriction map of regions of the chromosome was constructed, by identifying neighboring Alu bands with Not I linking clones. This approach simplifies the task of physical mapping and avoids ambiguities in Not I fragment assignments that arise from gel-to-gel mobility variations. A contiguous map was constructed with six Not I linking clones that covers at least the proximal one-third of the long arm of chromosome 21 and spans 20 megabases. A more detailed restriction map revealed 11 likely CpG islands in this region and localized 11 additional DNA markers.


Cloning and functional characterization of a family of human and mouse somatostatin receptors expressed in brain, gastrointestinal tract, and kidney.

Somatostatin is a tetradecapeptide that is widely distributed in the body. It acts on multiple organs including brain, pituitary, gut, exocrine and endocrine pancreas, adrenals, thyroid, and kidneys to inhibit release of many hormones and other secretory proteins. In addition, it functions as a neuropeptide affecting the electrical activity of neurons. Somatostatin exerts its biological effects by binding to specific high-affinity receptors, which appear in many cases to be coupled to GTP-binding proteins. Here we report the cloning, functional expression, and tissue distribution of two different somatostatin receptors (SSTRs). SSTR1 and SSTR2 contain 391 and 369 amino acids, respectively, and are members of the superfamily of receptors having seven transmembrane segments. There is 46% identity and 70% similarity between the amino acid sequences of SSTR1 and SSTR2. Stably transfected Chinese hamster ovary cells expressing SSTR1 or SSTR2 exhibit specific somatostatin binding, with an apparently higher affinity for somatostatin-14 than somatostatin-28, and NH2-terminally extended form of somatostatin-14. RNA blotting studies show that SSTR1 and SSTR2 are expressed at highest levels in jejunum and stomach and in cerebrum and kidney, respectively. A SSTR1 probe hybridized to multiple DNA fragments in EcoRI digests of human and mouse DNA, indicating that SSTR1 and SSTR2 are members of a larger family of somatostatin receptors. Thus, the biological effects of somatostatin are mediated by a family of receptors that are expressed in a tissue-specific manner.


Human immunodeficiency virus (HIV) type 1 can superinfect HIV-2-infected cells: pseudotype virions produced with expanded cellular host range.

In studies on viral interference, cloned T-cell lines chronically infected with human immunodeficiency virus (HIV) type 1 or HIV-2 were inoculated with several strains of these two AIDS retrovirus subtypes. HIV-2UC1-infected cells, which still express the CD4 receptor, could be superinfected with a variety of HIV-1 and HIV-2 strains. This event was accompanied by cytopathic effects in the cells and production of pseudotype virions with an expanded cellular host range. HIV-1- or HIV-2-infected clonal cell lines, which did not express CD4, could not be superinfected by any HIV strains but were coinfected after transfection of molecular clones into the persistently infected cells. These observations indicate that viral interference with HIV occurs at the cell surface and involves a down-modulation of the CD4 molecule. If the CD4 protein is expressed, superinfection can take place, and phenotypically mixed virus particles are produced. Since HIV-1 and HIV-2 dually infected individuals have been detected, these in vitro observations may have relevance to the in vivo state.


S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds.

Endothelium-derived relaxing factor (EDRF) activity has been attributed to the highly labile nitric oxide radical (NO). In view of the fact that the plasma and cellular milieux contain reactive species that can rapidly inactivate NO, it has been postulated that NO is stabilized by a carrier molecule that preserves its biological activity. Reduced thiol species are candidates for this role, reacting readily in the presence of NO to yield biologically active S-nitrosothiols that are more stable than NO itself. Because sulfhydryl groups in proteins represent an abundant source of reduced thiol in biologic systems, we examined the reaction of several sulfhydryl-containing proteins of diverse nature and function upon exposure to authentic NO and EDRF. We demonstrate that S-nitroso proteins form readily under physiologic conditions and possess EDRF-like effects of vasodilation and platelet inhibition. These observations suggest that S-nitrosothiol groups in proteins may serve as intermediates in the cellular metabolism of NO and raise the possibility of an additional type of cellular regulatory mechanism.


Depression of the Ca(2+)-ATPase activity of the rat liver endoplasmic reticulum by the liver tumour promoters, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane and phenobarbital.

The effects of a short-term in vivo administration of two liver tumour promoters (phenobarbital and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane on rat liver endoplasmic reticulum Ca(2+)-ATPase were investigated. The specific activity values of this membrane-bound enzyme significantly decreased (P less than 0.01) by 51% for phenobarbital-treated rats and by 48% for 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane-treated rats compared with control animals. The depression of liver endoplasmic reticulum Ca(2+)-ATPase appears to be a manifestation of the toxicological effect of tumour promoters.


Cytosolic long-chain acyl-CoA hydrolase, a suitable parameter to measure hepatic response to peroxisome proliferators.

The possibility of using cytosolic long-chain acyl-CoA as a parameter to measure the response of liver to peroxisome proliferators was studied. A subcutaneous (s.c.) injection of perfluorooctanoic acid (PFOA) to male Wistar rats caused an increase in activity of cytosolic long-chain acyl-CoA hydrolase. This increase in activity seems to be due to enzyme induction, since it was prevented by simultaneous administration of cycloheximide or actinomycin D with PFOA. The activity of cytosolic long-chain acyl-CoA hydrolase was increased in a dose-dependent manner by the administration of three peroxisome proliferators with diverse chemical structures: alpha-(p-chlorophenoxy)isobutyric acid (clofibric acid), 2,2'-(decamethylenedithio)diethanol (tiadenol) and PFOA. The increased activity produced by clofibric acid lasted throughout a 22-week treatment. A good correlation was found between the activities of cytosolic long-chain acyl-CoA hydrolase and peroxisomal beta-oxidation induced by the administration of the peroxisome proliferators. These results indicate that cytosolic long-chain acyl-CoA hydrolase is a suitable parameter for measuring the response of rat liver to challenges by peroxisome proliferators.