Screening for mutations in human HPRT cDNA using the polymerase chain reaction (PCR) in combination with constant denaturant gel electrophoresis (CDGE).

Previously, we reported the modification of denaturing gradient gel electrophoresis called constant denaturant gel electrophoresis (CDGE). CDGE separates mutant fragments in specific melting domains. CDGE seems to be a useful tool in mutation detection. Since the hypoxanthine phosphoribosyltransferase (HPRT) gene is widely used as target locus for mutation studies in vitro and in vivo, we have examined the approach of analyzing human HPRT cDNA by polymerase chain reaction (PCR) and CDGE. All nine HPRT exons are included in a 716-bp cDNA fragment obtained by PCR using HPRT cDNA as template. When the full-length cDNA fragment was examined by CDGE, it was possible to detect mutations only in the last part of exon 8 and exon 9. However, digestion of the cDNA fragment with the restriction enzyme AvaI prior to CDGE enabled us to detect point mutations in most of exon 2, the beginning of exon 3, the last part of exon 8 and exon 9. With the use of two internal primer sets, including a GC-rich clamp on one of the primers in each pair, a region containing most of exon 3 through exon 6 was amplified and we were able to resolve fragments with point mutations in this region from wild-type DNA. The approach described here allows for rapid screening of point mutations in about two thirds of the human HPRT cDNA sequence. In a test of this approach, we were able to resolve 12 of 13 known mutants. The mutant panel included one single-base deletion, one two-base deletion and 11 single-base substitutions.


Allelic losses in mutations at the aprt locus of human lymphoblastoid cells.

We analyzed the nature of mutations at the autosomal locus coding for adenine phosphoribosyltransferase (aprt) in human cells to elucidate the process(es) governing mutagenesis at autosomal loci. A human lymphoblastoid cell line, WR10, was found to be heterozygous for mutated allele at the aprt locus, and was used for mutation analyses. By the use of a restriction fragment length polymorphism associated with the aprt locus in WR10 cells, the molecular characteristics of mutations arising spontaneously or induced by gamma-rays were investigated. Eighty-five percent (22/26) of the spontaneous mutant clones and 93% (64/69) of the gamma-ray-induced mutant clones resulted from loss of one of the two aprt alleles. Determination of the dosage of aprt genes in those mutants with allelic losses revealed that approximately half of them retained two copies of the mutated allele. These data suggest that the mutational events leading to APRT deficiency are analogous to those reported for tumor suppressor genes in malignancies.


Visible mutations induced by P-M hybrid dysgenesis in Drosophila melanogaster result predominantly from P element insertions.

This study supplied no evidence that P-M hybrid dysgenesis is a general release mechanisms for transposon movement. Newly induced mutations (23 singed, three yellow, and one white) were generated by hybrid dysgenesis and were molecularly analyzed for the presence or absence of P element insertions. Only one dysgenically-induced insertion mutation out of 27 analyzed was the result of a non-P insert; this frequency is not statistically above the non-dysgenic control level. Thus, it appears that individual transposable elements families are independently regulated.


Comparative toxicity and mutagenicity of N-hydroxy-2-acetylaminofluorene and 7-acetyl-N-hydroxy-2-acetylaminofluorene in human lymphoblasts.

Exponentially growing TK6 human lymphoblasts were exposed to either 0-50 microM N-hydroxy-2-acetylaminofluorene (N-OH-AAF) or 0-10 microM 7-acetyl-N-hydroxy-2-acetylaminofluorene (7-acetyl-N-OH-AAF) in both the absence and presence of a partially purified preparation of hamster-liver N-arylhydroxamic acid N,O-acyltransferase (AHAT). Neither N-arylhydroxamic acid was toxic to the lymphoblasts, nor mutagenic at the thymidine kinase (tk) locus, in the absence of AHAT over the concentration range examined. In the presence of AHAT, an enzyme that activates N-arylhydroxamic acids to electrophilic N-acetoxyarylamine intermediates, both compounds caused toxicity and mutagenicity in TK6 cells. The 7-acetyl-N-OH-AAF was approximately 10-fold more toxic and mutagenic than the unsubstituted N-OH-AAF. These data demonstrate that metabolism of these N-arylhydroxamic acids, presumably to N-acetoxyarylamine intermediates by AHAT, is a key event in the biological activity of these agents. In addition, the presence of electron-withdrawing 7-acetyl substituent that is thought to stabilize N-acetoxy intermediates, appears to enhance the biological activity of the unsubstituted N-OH-AAF.


Enhancing effects of heterocyclic amines and beta-carbolines on the induction of chromosome aberrations in cultured mammalian cells.

The effects of post-treatment with heterocyclic amines and beta-carbolines on the induction of chromosome aberrations were studied in Chinese hamster CHO K-1 cells and SV40-transformed excision repair-deficient human XP2OSSV cells. The number of chromosome aberrations induced by UV and MMC were increased by post-treatment with Trp-P-1 and Trp-P-2, in both the presence and the absence of S9 mix. A alpha C, MeA alpha C, Glu-P-1, Glu-P-2, IQ, MeIQ, harman and harmine increased chromosome aberrations only in the presence of S9 mix. Glu-P-2, IQ, MeIQ, harman, and harmine did not induce chromosome aberrations by themselves at the concentrations used in this study. Trp-P-1, Trp-P-2, A alpha C, MeA alpha C and Glu-P-1 were weak clastogens by themselves, but at much higher concentrations than those at which they increased the induction of chromosome aberrations in cells pretreated with UV or MMC. Therefore, the increases in chromosome aberrations were not considered to be additive.


Structure-activity relationships of aromatic diamines in the Ames Salmonella typhimurium assay. Part II.

Structure-activity relationships in the case of aromatic monoamines, diversely substituted on the ring, using the mutagenic activity in the Ames test were studied in part I. This part II is based on the same general principles but applied to phenylene diamines (ortho, para and meta) diversely substituted on the ring.


Toxicological significance of dog liver cytochrome P-450: examination with the enzyme expressed in Saccharomyces cerevisiae using recombinant expression plasmid.

A complementary DNA (cDNA) coding for a form of beagle dog cytochrome P-450 (Dah1), which is the orthologue to the CYP1A1 cDNA of rat, mouse and human, was inserted between the alcohol dehydrogenase (ADH) promoter and terminator regions of the yeast expression vector pAAH5. On introduction of the resulting recombinant plasmid pDC-1, Saccharomyces cerevisiae strain AH22 cells synthesized up to 1.5 x 10(5) molecules per cell of cytochrome P-450 protein (P-450(Dah1)). The carbon monoxide-bound reduced form of P-450(Dah1) showed an absorption peak at 447 nm and specific content of P-450(Dah1) was about 0.1 nmole P-450 per mg of microsomal protein. P-450(Dah1) cross-reacted with antibodies to rat P-448-H (CYP1A2) and dog P-450-D2 (CYP1A2). P-450(Dah1) activated 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) most efficiently in the umu test and exhibited a high activity of aryl hydrocarbon hydroxylase toward benzo[a]pyrene.


Monitoring and assessment of mercury pollution in the vicinity of a chloralkali plant. III. Concentration and genotoxicity of mercury in the industrial effluent and contaminated water of Rushikulya estuary, India.

Aquatic mercury pollution of the Rushikulya estuary in the vicinity of the chloralkali plant at Ganjam, India was monitored over a period from October 1987 to May 1989. The concentrations of aquatic mercury in the water samples taken from the effluent channel and from different sites along the course of the estuary covering a distance of 2 km were periodically recorded and ranged from 0 to 0.5 mg/l. The bioconcentration and genotoxicity of aquatic mercury in the samples were assessed by the Allium micronucleus (MNC) assay. The frequency of cells with MNC was highly correlated not only with bioconcentrated mercury (root mercury) but also with the levels of aquatic mercury. The threshold assessment values such as effective concentration fifty (EC50) for root growth, lowest effective concentration tested (LECT), and highest ineffective concentration tested (HICT) for induction of MNC in Allium MNC assay for the present aquatic industrial mercury were determined to be 0.14, 0.06 and 0.02 mg/l, respectively.


Effects of supercypermethrin, a synthetic developmental pyrethroid, on four biological test systems.

The genotoxic potential of the insecticide supercypermethrin, a second-generation pyrethroid, was studied on four different test systems. It was non-mutagenic to Salmonella typhimurium strains TA1535, TA100, TA1538, TA98 and TA97 in the presence and absence of S9 mixture. It induced gene conversion at the tryptophan locus and induced point mutations at the isoleucine locus in Saccharomyces cerevisiae cells. A slight increase in the frequency of aberrant anaphases and telophases in root tips of Hordeum vulgare and Vicia faba was observed, but no genotoxic effects were detected in Drosophila melanogaster.


Methyl parathion-induced sperm shape abnormalities in mouse.

Metacid 50, the commercial grade of methyl parathion (O,O-dimethyl-O-4-nitrophenyl phosphorothionate), a commonly used organophosphorus insecticide, was tested for its genotoxicity in Swiss albino mice using the sperm abnormality assay. Sperms of albino mice were examined at two time intervals, 1 week and 5 weeks after a single acute oral treatment with the pesticide at four dose levels, viz., 75.0, 37.5, 18.75 and 9.375 mg/kg body weight corresponding to 1/2 LD50, 1/4 LD50, 1/8 LD50 and 1/16 LD50 values respectively. A dose-related statistically significant increase in the percentage of abnormal sperm observed indicates the genotoxic potency of methyl parathion.


Genotoxicity of 2,4,6-trichlorophenol in V79 Chinese hamster cells.

The genotoxicity of the rodent carcinogen 2,4,6-trichlorophenol (TCP) was studied without exogenous metabolic activation in V79 Chinese hamster cells. TCP did not induce mutation at the hprt locus to 6-thioguanine resistance or structural chromosome aberrations. However, it produced statistically significant, dose-related increases in hyperdiploidy and micronuclei. From these results it appears that TCP causes chromosome malsegregation as its major mode of genotoxic action.


Detection of vincristine-induced hyperploidy in meiotic II metaphases of male Chinese hamsters.

Induction of hyperploidy in germ cells of male Chinese hamsters treated with vincristine at dose levels of 0.25, 0.50 or 0.75 mg/kg of body weight was investigated. Animals were killed at 6, 24, 48, 72 and 96 h after administration of the chemical by a single intraperitoneal injection. The testes were removed and processed for spermatogonial, meiotic I, and meiotic II metaphases. Significantly increased frequencies of hyperploidy were obtained in meiotic II cells harvested 6, 24 and 48 h but not 72 and 96 h after treatment, indicating the importance of multiple sampling times. Analysis of spermatogonial cells shows that the frequencies of hyperploidy in the treated samples were comparable to those of controls. Limited sampling times used in the present study as well as small sample size or possible loss of hyperploid cells may be responsible for the negative findings for spermatogonial cells. Examination of meiotic I cells from 53 animals reveals the presence of one animal with an elevated level of hyperploidy unrelated to the vincristine treatment.


The effect of gamma-irradiation on the toxicity of malathion in V79 Chinese hamster cells and Molt-4 human lymphocytes.

There is growing interest in the irradiation of food and agricultural products for insect disinfestation, sprout inhibition, delayed ripening and the reduction of microbiological loads. Extensive research has been done on this process, and irradiation to a maximum dose of 10 kGy is recognized as safe by national and international regulatory agencies. The question has been raised, however, whether irradiation of pesticide residues might produce radiation products that were more toxic or less toxic than the original pesticide. To address this question, we observed the effects of 10 kGy of gamma-radiation on malathion as measured by sister-chromatid exchange (SCE), micronuclei formation, cell survival, growth rate and polyploid formation. We found no significant differences between the effects of irradiated and unirradiated malathion on any of these end-points. Polyploid formation was the most dramatic effect of both irradiated and control malathion on V79 Chinese hamster cells. Cell survival, polyploid formation and growth rate were slightly better in cells treated with irradiated malathion. In Molt-4 human lymphocyte cells, micronuclei formation was not affected by irradiated or unirradiated malathion. Compared to malathion alone, the lack of such biological effects indicates that none of the presumed radiation-induced breakdown products increased or decreased the endpoints studied. The number of SCE was consistently, but not significantly, higher in the cells treated with irradiated malathion. There were no significant differences in cell survival or micronucleus formation in the human lymphocyte cell line Molt-4 treated with irradiated or control malathion. Thus, the irradiation of the pesticide malathion to 10 kGy, a recommended upper dose for most food irradiations, does not significantly alter its toxicity in these in vitro systems.


The genotoxicity of organotin compounds in SOS chromotest and rec-assay.

In these days pollution by organotin compounds in the environment extends widely and effects on human health are feared. We studied the genotoxicity of various organotin compounds (butyltins, phenyltins, methyltins) and inorganic tin (SnCl4), which are present in the environment, with the SOS chromotest and the rec-assay. Mono-n-butyltin oxide, n-butyltin trichloride and di-n-butyltin dichloride showed high SOS-inducing potency in the SOS chromotest with Escherichia coli PQ37. Di-n-butyltin dichloride, tri-n-butyltin chloride, bis(tri-n-butyltin)oxide, dimethyltin dichloride and trimethyltin chloride were recognized as genotoxic chemicals by the rec-assay.


Genotoxicity assessment of pirmenol, a new antiarrhythmic drug.

The genotoxicity of pirmenol was tested in the E. coli and S. typhimurium mutagenesis assay, an in vitro mammalian cell chromosome-aberration assay and an in vivo mouse micronucleus assay. The E. coli tester strain WP2s was exposed to concentrations of pirmenol as high as 10,000 micrograms/plate both in the absence (S9-) and presence (S9+) of metabolic activation. Five strains of S. typhimurium (TA98, TA100, TA1535, TA1537, TA1538) were exposed to concentrations of pirmenol as high as 5000 micrograms/plate in the absence and presence of S9. Pirmenol was not mutagenic toward either E. coli or S. typhimurium. Chinese hamster lung V79 cell cultures were exposed to pirmenol at concentrations of 500-2500 micrograms/ml (S9-) and 500-3000 micrograms/ml (S9+). Pirmenol increased the frequency of structural chromosome aberrations (SCAs). The minimum clastogenic concentration was 1500 micrograms/ml (both S9- and S9+) with a peak clastogenic response of 6% (S9-) and 34% (S9+) cells with aberrations. Although there were statistically significant results in the S9- experiment, the percent cells with aberration values for treated groups were within the historical control range (0-6%) of this laboratory. The observed effects in both the absence and presence of S9 appear at high concentrations compared to human circulating plasma levels of 1-3 micrograms/ml and the clastogenicity was confined to chromosome gaps and breaks. Consequently, this in vitro effect would not be expected to be reflected by either in vivo clastogenic or carcinogenic activity. This was supported by findings in the mouse micronucleus study of pirmenol in which single oral doses administered to male CD-1 mice at 5, 55, or 115 mg/kg (80% LD50) produced no statistically significant increases in the frequency of micronucleated polychromatic erythrocytes in bone marrow at 24, 48 or 72 h postdosing. Additionally, no evidence of carcinogenicity was seen in a mouse or rat bioassay.


A survey of lymphocyte chromosomal damage in Slovenian workers exposed to occupational clastogens.

Assays for sister-chromatid exchanges (SCE), unstable chromosome and chromatid aberrations and micronuclei were performed on blood lymphocytes from persons exposed protractedly to radiation or chemical hazards in the workplace. There was a general tendency with all endpoints examined for the yields to increase with years of working in the industry. This was especially marked for SCE. By comparison with a control group of administrative workers the levels of damage were higher, usually significantly so, in the occupational groups. These comprised workers at a nuclear research reactor, a hospital diagnostic X-ray department, a coal mine and a mercury ore mine.


Sister-chromatid exchanges in human lymphocytes induced by dimethoate, omethoate, deltamethrin, benomyl and their mixture.

Dimethoate and omethoate, two common organophosphorus insecticides, induced a dose-related increase in the frequency of sister-chromatid exchanges (SCEs) in human lymphocytes in vitro (P of the regression lines less than 0.01). Two other common pesticides, the pyrethroid insecticide deltamethrin and the systemic fungicide benomyl, induced a modest increase in SCEs which bordered on statistical significance (P = 0.053 and 0.055, respectively). Mixtures of the four pesticides at total concentrations of 41.5 and 83 micrograms/ml (composed of 43% dimethoate, 43% omethoate, 12% deltamethrin and 1.2% benomyl) induced a dose-dependent increase in SCEs (P less than 0.01). The effects of these mixtures of pesticides were variable using lymphocytes from different individuals, although these differences did not attain statistical significance. Moreover, low concentrations of the four pesticides that did not increase SCEs significantly when tested alone, were positive for SCE induction when tested as a mixture. The experiments show that sub-threshold doses of pesticides may increase SCEs when present in a mixture.


Evaluation of the ligase chain reaction (LCR) for the detection of point mutations.

The ligase chain reaction (LCR) was evaluated as an amplification method for an in vivo mutation assay. Specifically, the ligase was tested for its ability to selectively amplify a DNA sequence mutated at a single base, in the presence of an excess of wild-type DNA. As a model template a 370-bp DNA fragment of the mouse Ha-ras protooncogene containing an A to T mutation at the second position of codon 61 was used. With the commercially available ligase Ampligase (Epicenter), 250 molecules of mutant fragments could be detected by an enzyme-linked immunoassay with digoxigenin marker (giving a theoretical detection limit of 1 target gene per 10(4) copies of genome). In the analysis of mixtures with corresponding wild-type DNA fragments, a 1:1 mixture resulted in a clearly stronger signal than control samples lacking wild-type and mutant DNA. However, the signal obtained from a 100-fold dilution of the mutant DNA with wild-type DNA could not be distinguished from the background noise. In this particular form, LCR lacks sufficient selectivity to be applied to an in vivo situation, where the ratio of mutant to wild-type DNA sequences might be expected to lie around 1:10(6).


Chinese hamster ovary cells deficient or proficient in O6-alkylguanine-DNA alkyltransferase activity are equally sensitive to X-rays.

In mammalian cells, under aerobic conditions, ionizing radiations and radiomimetic chemical agents can induce an enzymatic activity involved in DNA repair, O6-alkylguanine-DNA alkyltransferase (O6-AT). This catalytic protein is active against alkyl-radical-induced DNA damages. This induction was proposed to be linked to the formation of hydroxyl radicals. The possible involvement of O6-AT in the defense mechanism of the cell against aerobic radiation damage was investigated by comparing the X-ray sensitivity of two Chinese hamster ovary (CHO) cell lines, the first deficient (CHO mex-) and the second proficient by transfection of O6-AT (CHO mex+). The colony-forming ability after X-irradiation was appreciably reduced in CHO mex- in comparison to CHO mex+ cells. Nevertheless, pretreatment of proficient cells with O6-methylguanine, a specific inhibitor of O6-AT, reduced the DNA repair activity but did not modify the degree of sensitivity to X-rays of the CHO mex+ cells. Since the glutathione concentrations as well as the DNA damage amounts induced by X-irradiation were comparable in the variously treated cell lines, these results suggest that the observed induction of O6-AT by ionizing radiation in aerobic conditions could be a generalized rather than a specific response to damage by radicals.


Efficiency of DNA-histone crosslinking induced by saturated and unsaturated aldehydes in vitro.

Using a filter-binding assay based on precipitation of pUC13 plasmid DNA bound to calf-thymus histones, we have determined the efficiency of formation of DNA-protein crosslink formation induced by several aldehyde compounds in vitro. Formaldehyde, glutaraldehyde and acrolein were the most potent, causing 1 crosslink per 2.7 kbp of DNA at 1.5, 8 and 150 microM, respectively. All other compounds tested gave 1 crosslink per plasmid molecule in the mM concentration range as follows: acetaldehyde, 115 mM; propionaldehyde, 295 mM; butyraldehyde, 360 mM; crotonaldehyde, 8.5 mM; trans-2-pentenal, 6.3 mM. Significant decreases in the efficiency of DPXL formation were observed with monofunctional aldehydes of higher carbon chain length. For example, the concentration of formaldehyde needed to give 1 crosslink per molecule was almost 10(5) times less than that of acetaldehyde. Acetaldehyde differs from formaldehyde only by one saturated carbon. The presence of an unsaturated bond between the 2-3 carbons improved the potential for crosslink formation. For example, acrolein was over 500-fold more potent than propionaldehyde. Glutaraldehyde was almost as potent as formaldehyde, indicating that the bifunctional nature of this 5-carbon saturated aldehyde may be crucial to its high efficiency of DNA-protein crosslinking.


Lack of adaptive response to low doses of ionizing radiation in human lymphocytes from five different donors.

Various investigators reported a reduced yield of chromosome and chromatid aberrations in short-term cultures of human lymphocytes if a 'challenge' exposure to ionizing radiation was preceded by an 'adaptive' exposure. In order to examine the cell cycle dependence of the 'adaptive response', chromosome and chromatid aberration yields were estimated after challenge doses in the G1, S or G2 phase of lymphocytes which had been adapted in the early G1 phase. On testing two donors no protective adaptive response was found. Blood samples of four donors were tested for their capability to evoke the adaptive response in a standard experiment with the adaptive dose in the S phase and the challenge dose in the G2 phase. A synergistic response occurred in one out of two similar experiments performed with the same blood sample. The three other blood samples tested did not respond. Apparently these data indicate a high frequency of human lymphocyte cultures that do not display an adaptive response.


Mechanism of mutagenicity by 5-hydroperoxymethyl-2'-deoxyuridine, an intermediate product of ionizing radiation, in bacteria. HPMdU bacterial mutagenicity and oxidation of DNA bases.

The specific objective was to find what processes are responsible for the mutagenicity of 5-hydroperoxymethyl-2'-deoxyuridine (HPMdU), which is a product of ionizing radiation, and what role transition metal ions play in those processes. We found that HPMdU is a more potent mutagen than its decomposition products 5-hydroxymethyl-2'-deoxyuridine (HMdU) and 5-formyl-2'-deoxyuridine (FdU) in the Salmonella typhimurium strains tested, with the TA100 strain being the most sensitive. HMdU exerted intermediate mutagenicity and FdU was the weakest of the three compounds. At 50 nmoles/plate, HPMdU increased the number of revertants by 4-fold, whereas 1000 nmoles HMdU was required to enhance the number of revertants by 5-fold. Pretreatment of TA100 with o-phenanthroline, a membrane-permeable Fe and Cu chelator, caused an increase in mutagenicity of the low HPMdU doses but inhibited that of the 50 nmoles HPMdU/plate, while desferal, a membrane-impermeable Fe chelator, had virtually no effect. Azide (a catalase inhibitor) enhanced HPMdU mutagenicity, whereas 3-amino-1,2,4-triazole (a catalase and peroxidase inhibitor) and ammonium formate (a hydroxyl radical scavenger) were protective. Preincubation of TA100 cells with 20 and 40 nM HPMdU caused dose-dependent formation of the oxidized DNA base derivatives HMdU, thymidine glycol and 8-hydroxyl-2'-deoxyguanosine (8-OHdG), known hydroxyl radical-mediated oxidation products. Cumulatively, these results suggest that the genetic effects of HPMdU are due to its hydroperoxide moiety, which upon reacting with Fe generates hydroxyl radicals that in turn oxidize neighboring bases in cellular DNA. This also may be a mechanism by which ionizing radiation exerts its long-term effects.


Inorganic arsenic effects on human lymphocyte stimulation and proliferation.

Lymphocyte cultures from individuals exposed to high levels of hydroarsenicism showed a slower cell cycle kinetics than cultures from low-exposed individuals. Since this difference in proliferation could be due to chronic arsenic exposure, the in vitro effects of inorganic arsenic in human whole blood lymphocyte cultures were investigated. When lymphocytes were exposed to concentrations of arsenite and arsenate similar to those found in the blood of exposed subjects (10(-7), 10(-8) and 10(-9) M) during the last 24 h before harvesting, a dose-related inhibition of proliferation was observed. Cultures were also treated with 10(-9) M of arsenite and arsenate for 2, 6 and 24 h at the beginning of the cultures in the presence or absence of phytohemagglutinin (PHA). Inhibition of stimulation and proliferation was directly related to the length of treatment. The results show that, at the concentrations tested, arsenite and arsenate impair lymphocyte stimulation and proliferation and confirm the fact that chronic arsenic exposure can affect the proliferation of whole blood lymphocytes.


Selective reactivities of isocyanates towards DNA bases and genotoxicity of methylcarbamoylation of DNA.

The reactivities of methyl isocyanate (MIC) and phenyl isocyanate (PIC) with DNA, and the genotoxicity of MIC were investigated. MIC and PIC reacted with the exocyclic amino group of deoxycytidine, deoxyadenosine and deoxyguanosine to produce carbamoylated products. The reactions of both isocyanates with deoxycytidine were 2 and 4 orders of magnitude higher than with deoxyadenosine and deoxyguanosine, respectively. To explore the genotoxicity of MIC, M13mp9 RF DNA was modified with MIC and then introduced into E. coli. The plaque-forming efficiencies of DNA decreased with increasing dose levels, and the decreases were more pronounced in Uvr endonuclease-deficient strains (uvrA, uvrB and uvrC) than in the Uvr endonuclease-proficient strain, JM103. The differences in survival in JM103 and uvr- strains suggest that the methylcarbonyl adducts can be removed by the uvr excision-repair system. Modification of M13mp9 RF DNA with MIC induced MIC-dose-related, SOS-dependent mutations in the beta-galactosidase locus. These results demonstrate the genotoxic response of MIC-modified DNA in E. coli.


Trypanosoma brucei mitochondrial ribosomal RNA synthesis, processing and developmentally regulated expression.

The steady-state levels of the mitochondrial ribosomal RNAs of Trypanosoma brucei are repressed in the early bloodstream developmental stage of the parasite and accumulate approximately 30-fold during differentiation to the stage found in the midgut of the insect vector. In order to determine the mechanism regulating this developmental process, we have examined the transcription and processing of the 9S and 12S mitochondrial rRNAs of T. brucei. A short-lived RNA was detected in pulse labeling experiments which contains the mature 12S and 9S rRNAs and at least 1200 nucleotides of RNA transcribed from upstream of the 12S rRNA gene. This putative processing precursor RNA was identified in both intact cells and in run-on experiments using isolated mitochondria. The transcripts containing the upstream sequences are unstable and reach isotopic equilibrium within 15 min. Mature rRNAs in the insect developmental stage are stable and show no detectable turnover during a 36-h chase. Comparison of rRNA synthesis in bloodstream and insect life-stages indicates that mitochondrial rRNA levels are controlled not at the transcriptional level, but rather by a mechanism which likely modulates the stability of the mature rRNAs. These results suggest that a short-lived rRNA precursor is synthesized and processed at comparable rates in both bloodstream and insect stages of the parasite. Thus, it appears that differential stability of the mature 9S and 12S rRNAs plays a major role in modulating mitochondrial gene expression during the developmental cycle of T. brucei.


The effects of neuropeptides on mucous glycoprotein secretion from human nasal mucosa in vitro.

The role of neuropeptides in the regulation of macromolecule secretion from human nasal mucosa is incompletely understood. Previous in vitro explant culture studies have demonstrated the effects of neuropeptides on lactoferrin release from serous cells and 3H-glucosamine labeled respiratory glycoconjugate secretion from mucus-containing cells. The generation of a new monoclonal antibody, 7F10, has led to the development of an ELISA for high molecular weight respiratory mucous glycoproteins (MGP). This ELISA was used to measure the ability of sensory, parasympathetic and sympathetic neuropeptides to stimulate MGP release from human nasal mucosal fragments in short term explant culture in vitro. Significant MGP release was stimulated by the sensory neuropeptides gastrin releasing peptide (10 microM GRP: 10.6% +/- 2.4% increase, n = 8, P less than 0.01 vs. control), substance P (1 microM SP: 12.5% +/- 5.4%, n = 11, P less than 0.05), neurokinin A (1 microM NKA: 17.8 +/- 4.3%, n = 6, P less than 0.01), while calcitonin gene related peptide (CGRP) was without effect. Vasoactive intestinal peptide (VIP), a neurotransmitter from parasympathetic nerves, induced significant dose dependent MGP secretion, but had no additive or inhibitory interaction with methacholine-induced secretion. Neuropeptide Y (NPY), present in sympathetic nerves, had no effect on MGP secretion. These observations correlate with the effects of neuropeptides on serous cell lactoferrin secretion, and the presence of specific GRP, SP, and VIP binding sites on human nasal submucosal glands that have been detected by autoradiography. GRP and tachykinins (SP and NKA) from sensory nerves, and VIP released during parasympathetic reflexes may significantly stimulate mucous and serous cell secretion from human nasal mucosa in vivo.


Hydroxyl radical cleavage of tRNA in the ribosomal P site.

Hydroxyl radical is a useful probe of the accessibility of the sugar moiety of nucleic acids to solvent. Here we compare the accessibility of free and ribosome-bound yeast tRNA(Phe), Escherichia coli tRNA(Phe), and E. coli tRNA(Leu2) to attack by hydroxyl radicals generated from Fe(2+)-EDTA. When bound to the P site of 30S ribosomal subunits, a discrete region, corresponding almost precisely to the anticodon stem-loop, is strongly protected; weaker protection is observed in the 3' strand of the D stem and in the variable loop. The protected nucleotides constitute a well-defined substructure, corresponding to the lower half of the anticodon-D loop coaxial arm of the tRNA crystal structure. This result suggests that the 30S P site contains a pocket that becomes inaccessible to the Fe(2+)-EDTA complex when tRNA is bound, whose minimum dimensions can be inferred from the boundaries of the protected region of tRNA. When bound to the P site of 70S ribosomes, the entire tRNA backbone becomes inaccessible to hydroxyl radicals. Since previous studies have shown that virtually the entire footprint of a P-site tRNA on 16S and 23S rRNAs is mimicked by the extremities of the tRNA (the anticodon stem-loop plus the 3'-terminal aminoacyl-pentanucleotide), protection of the entire tRNA was unexpected. We conclude that protection of the elbow of tRNA is due either to interactions with ribosomal proteins or to enclosure in an inaccessible site formed by association of the two ribosomal subunits.


Induction of cytotoxic T lymphocytes with peptides in vitro: identification of candidate T-cell epitopes in human papilloma virus.

A set of overlapping peptides corresponding to the L1, E6, and E7 proteins of human papilloma virus 16 was tested for their ability to bind to major histocompatibility complex class I molecules and to stimulate cytotoxic T-lymphocyte (CTL) responses in vitro. A class I binding assay using intact RMA-S cells showed that 20 of the 99 human papilloma virus peptides bound to H-2Kb and/or Db molecules. Fifteen of the 20 class I-binding peptides stimulated primary CTL responses, whereas peptides that were negative in the binding assay failed to do so. Peptide-induced CTLs recognized the immunizing peptide very efficiently, requiring no more than 1-10 nM peptide for target cell lysis. However, two observations were made that have important implications for the design of peptide-based vaccines for inducing CTLs. (i) Not all major histocompatibility complex-binding peptides that contained known motifs characteristic of naturally processed peptides induced CTLs. (ii) The efficiency of CTL lysis was strongly decreased when the size of the target peptide differed by only one amino acid residue from that of the immunizing peptide. We conclude that peptides chosen for vaccination must correspond in length to naturally processed peptides.


Structure of the CD59-encoding gene: further evidence of a relationship to murine lymphocyte antigen Ly-6 protein.

The gene for CD59 [membrane inhibitor of reactive lysis (MIRL), protectin], a phosphatidylinositol-linked surface glycoprotein that regulates the formation of the polymeric C9 complex of complement and that is deficient on the abnormal hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria, consists of four exons spanning 20 kilobases. The untranslated first exon is preceded by a G+C-rich promoter region that lacks a consensus TATA or CAAT motif. The second exon encodes the hydrophobic leader sequence of the protein, and the third exon encodes the amino-terminal portion of the mature protein. The fourth exon encodes the remainder of the mature protein, including the hydrophobic sequence necessary for glycosyl-phosphatidylinositol anchor attachment. The structure of the CD59 gene is very similar to that encoding Ly-6, a murine glycoprotein with which CD59 has some structural similarity. The striking similarity in gene structure is further evidence that the two proteins belong to a superfamily of proteins that may also include the urokinase plasminogen-activator receptor and a squid glycoprotein of unknown function.


Catalysis of serine and tyrosine autophosphorylation by the human insulin receptor.

The protein kinase activity of human insulin receptors purified from Sf9 insect cells after infection with a recombinant baculovirus was evaluated. The following experimental observations led to the unexpected conclusion that this receptor protein catalyzes both serine and tyrosine autophosphorylation at significant stoichiometries. (i) Phosphorylation of lectin-purified insulin receptors with [gamma-32P]ATP resulted in rapid receptor tyrosine phosphorylation (7 mol of P per high-affinity binding site) and the delayed onset of insulin-stimulated receptor serine phosphorylation (about 7% of total phosphorylation). The tyrosine kinase inhibitor (hydroxy-2-naphthalenylmethyl)phosphonic acid (HNMPA), which has no effect on protein kinase C or cyclic AMP-dependent protein kinase activities, inhibited both the receptor serine and tyrosine phosphorylation. (ii) Phosphorylation of a synthetic peptide substrate composed of insulin receptor residues 1290-1319 on serines-1305/1306 by partially purified insulin receptors was also inhibited by HNMPA. (iii) Insulin receptors sequentially affinity-purified on immobilized wheat germ agglutinin and immobilized insulin showed no apparent contaminant proteins on silver-stained SDS/polyacrylamide gels yet catalyzed autophosphorylation on receptor serine and tyrosine residues when incubated with [gamma-32P]ATP. These results suggest that the catalytic site of the insulin receptor tyrosine kinase also recognizes receptor serine residues as substrates for the phosphotransfer reaction. Furthermore, insulin-stimulated receptor serine phosphorylation in intact cells may occur in part by an autophosphorylation mechanism subsequent to tyrosine phosphorylation of the insulin receptor.


Targeted cleavage of mRNA by human RNase P.

Ribonuclease P from Escherichia coli can cleave RNAs in simple, hydrogen-bonded complexes of two oligoribonucleotides that resemble the aminoacyl stem and 5' leader sequence of tRNA precursors. RNase P from human (HeLa) cells cannot catalyze the cleavage in vitro of the 5'-proximal oligoribonucleotide that contains the leader sequence in such simple complexes but can do so when the 3'-proximal oligoribonucleotide (external guide sequence) is altered to resemble three-quarters of a tRNA molecule. In such a complex, the efficiency of cleavage of the mRNA for chloramphenicol acetyltransferase, as the 5'-proximal oligoribonucleotide, depends on the structural details of the external guide sequence and on the choice of target site within the mRNA. The presence of the appropriately designed external guide sequence in cells in tissue culture reduces chloramphenicol acetyltransferase activity and the level of the corresponding intact mRNA in the cells. Thus, it appears that the use of such external guide sequences may provide a general technique for gene inactivation.


Extracellular matrix regulates smooth muscle responses to substance P.

Little is known about the extracellular factors that determine a cell's responsiveness to neurotransmitters. This is a particularly important issue for pharmacologically diverse cell types such as neurons and smooth muscle. This report demonstrates that the contractile responses of amniotic smooth muscle to a specific neuropeptide, substance P, is controlled by a molecule(s) intimately associated with the extracellular basement membrane. This molecule(s) normally represses the expression of substance P responsiveness in this tissue. When the amniotic smooth muscle is separated from the basement membrane by dissociation, normally unresponsive cells exhibit a progressive increase in responsiveness to substance P, beginning within the first 24 hr in culture. The induction of substance P responses was completely inhibited when the cells were plated onto isolated amniotic basement membrane rather than onto polyornithine or collagen I. Similar changes in the responsiveness to another agonist, histamine, did not occur. The data demonstrate that extracellular matrix exerts a major instructive influence in determining the responsiveness of avian amniotic smooth muscle to specific ligands. We suggest that similar regulatory mechanisms may operate in other tissues.


Purification of a murine protein-tyrosine/threonine kinase that phosphorylates and activates the Erk-1 gene product: relationship to the fission yeast byr1 gene product.

We report the purification to near homogeneity of a 45-kDa phorbol ester-stimulated protein kinase that phosphorylates and activates the Erk-1 gene product. This kinase, which we provisionally denote MEK for MAPK/Erk kinase, phosphorylated kinase-inactive Erk-1 protein primarily on a tyrosine residue and, to a lesser extent, on a threonine. We extend our previous results and show that two forms of purified MEK activated the myelin basic protein kinase encoded by Erk-1. MEK was inactivated by the serine/threonine phosphatase 2A but not by the protein-tyrosine phosphatase 1B. Sequence analysis of peptides generated by trypsin digestion of MEK revealed similarity to the proteins encoded by the Schizosaccharomyces pombe byr1 and Saccharomyces cerevisiae STE7 genes. These data are discussed with regard to a possible signal transduction mechanism.


Increased tyrosine kinase activity of c-Src during calcium-induced keratinocyte differentiation.

In cultured human epidermal keratinocytes, induction of differentiation by Ca2+ and ionophore treatment was found to result in rapid elevation of c-Src tyrosine kinase activity and inactivation of the c-Yes tyrosine kinase. Activation of c-Src kinase was accompanied by tyrosine dephosphorylation, which might be explained by a rapid increase in intracellular protein-tyrosine phosphatase activity. Ca(2+)-induced differentiation was also associated with altered tyrosine phosphorylation of several cellular proteins and correlated with a marked redistribution of intracellular phosphotyrosine from membrane and adhesion sites to the nucleus. Some of the c-Src protein was also found in the nucleus after Ca2+ treatment, and Ca(2+)-activated c-Src bound to three cellular proteins (120 kDa, 65 kDa, and 34 kDa). In agreement with these results, immunohistochemistry on human epidermis revealed an increase in c-Src expression and tyrosine phosphorylation in cells undergoing differentiation, which strongly suggests a possible role of non-receptor tyrosine kinases in epithelial cell maturation.


Inhibition of human immunodeficiency virus and growth of infected T cells by the immunosuppressive drugs cyclosporin A and FK 506.

The effects of the immunosuppressive drugs cyclosporin A and FK 506 were studied on cells chronically infected with human immunodeficiency virus type 1 (HIV-1) as well as on uninfected and newly infected cells. When cells chronically infected with HIV-1 or with HIV-2 were cocultivated with uninfected cells in the presence of cyclosporin A or FK 506 there was a delay in the formation of syncytia and of cytopathic effects. This inhibitory effect was not due to decreased membrane expression of CD4. In addition, there was an approximately 100-fold reduction in the yield of infectious HIV-1 when the infected cells were grown in the presence of these drugs, a finding consistent with other evidence of decreased HIV expression. Both drugs were found to inhibit the growth of chronically infected cells at concentrations that did not inhibit the growth of the uninfected cells. These results, demonstrating that cyclosporin A and FK 506 interfere with HIV production and selectively inhibit the growth of infected cells, suggest that they may be useful in the treatment of this infection and indicate further cellular targets for antiviral agents.


[Peptide inhibitors of the renin-angiotensin system. 2. Polymer-bound tri-, penta- and nonapeptide inhibitors of angiotensin converting enzyme].

Inhibitors of the angiotensin converting enzyme (ACE, EC 3.4.15.1) are important in the treatment of the high blood pressure. The therapeutically used drugs captopril, enalapril and ramipril are enzymatic stable short pseudo-peptides. They are stabilized against enzymatic degradation and therefore usefully for oral application. But for some indications e.g. post operative treatment and shock therapy well dosed infusions are needed. For this purpose we attached nona-, penta- and tripeptide inhibitors of the ACE to immunologically inert dextran polymers. The inhibitors are derived as well from the bradykinin potentiating nonapeptide BPP9 alpha (Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro) and the bradykinin potentiating pentapeptide BPP5 alpha (Pyr-Lys-Trp-Ala-Pro), both originally isolated from snake venoms, as from acylated tripeptides with the structure Acyl-AA1-Arg-Pro. We estimated the influence on the biological activity of two different linkers to the dextran polymers. The coupling to the polymer was achieved on the one hand via the aldehyd moiety (DAD-AK) and on the other hand by the carboxyl residue (KMD). In the case of DAD-AK-polymers the condensation of the peptides was performed by the N-hydroxysuccinimide ester of the polymer. Because of the instability of the KMD-OSU in this case water soluble carbodiimides are used. The polymer bound peptides inhibit the isolated ACE, but in the most cases with a reduced activity. Only the tripeptide DPhe-Arg-Pro has a enhanced activity in the polymer bound state. The polymer bound inhibitors show a prolongated action on normotensive rats by intravenous application.(ABSTRACT TRUNCATED AT 250 WORDS)


[The application of the problem-solving method in a situation of surgical intervention; a case report].

This study used the problems solving method for directing nursing assistance to the hospitalized surgical patient. We present the elaborate instrument founded in the Bailey e Claus model which is analyzed and exemplified. The authors consider the available resources concluding that the proposed is possible to be established. They realize how necessary is in nursing all the taking decision process to reach the proposed objectives, toward the resolution of the patient problems.


Comparison of L-5-hydroxytryptophan and L-5-hydroxytryptophan inosinate as agents for increasing brain serotonin formation in rats.

The effects of L-5-hydroxytryptophan (L-5HTP) and L-5HTP inosinate injection on brain 5-hydroxyindoles in rats were compared. L-5HTP and L-5HTP inosinate caused indistinguishable dose-dependent increases in 5HTP and 5HIAA (5-hydroxyindoleacetic acid) but not serotonin concentrations in whole brain at 1 hr in rats. Our results do not substantiate a previous claim that L-5HTP inosinate is superior to L-5HTP itself in increasing brain serotonin formation.


[A case of medically treated type A acute aortic dissection: trial of antifibrinolytic therapy while monitoring the thrombotic and fibrinolytic parameters].

A 56-year-old male was admitted with severe chest and abdominal pain. Enhanced CT revealed type-A acute aortic dissection. Enhancement of false lumen was complete in the aortic arch, but it was incomplete in the ascending and descending aorta. This finding suggested a thrombosing tendency of the false lumen. Marked elevation of Thrombin-Antithrombin III complex (TAT) and Plasmin-alpha 2 Plasmin inhibitor complex (PIC) indicated the activation of both coagulation and fibrinolysis in the false lumen. Aprotinin and Tranexamic acid were used in order to suppress the fibrinolysis. Both TAT and PIC were completely normalized at the 20th hospital day. Enhanced CT on the 40th hospital day showed that the false lumen had disappeared, and the adventitia had thickened. Care should be taken to notice activated coagulation and fibrinolysis in the false lumen. This report appears to be the first statement about the effectiveness of antifibrinolytic therapy to facilitate thrombosing and healing of the false lumen in the treatment of acute aortic dissection.


A monoclonal antibody-based immunodiagnostic assay for onchocerciasis.

Five murine monoclonal antibodies raised against Onchocerca volvulus cuticular extracts and termed MOVs (1-4 and 6) were selected based on reactivity with O. volvulus cryosections, and non-reactivity with cryosections of human skin and/or nodular tissue. Two others MOVs 5 and 7 reacted with both. Using the peroxidase-anti- peroxidase (PAP) histochemical method, the target epitopes of MOV 1 were located in the cuticle's basal and cortical layers, those of MOV 2 in the cortical layer; whilst MOV 3-7 stained the basal layer. A sandwich ELISA was then developed. The trapping polyclonal antibody was raised in rabbits utilising the same antigens as for preparation of the MOVs. Once captured on microtiter plates, target antigens were identified by the sequential binding of a MOV, followed by a goat anti-mouse globulin/peroxidase or alkaline phosphatase conjugate that catalysed a colorimetric reaction in the presence of appropriate substrates. In this system, MOV 1 emerged as the most specific and potent reagent capable of recognizing antigens of Onchocerca sp. with a minimal detection limit of 78 ng per test. MOV 1, failed to react with extracts of Loa Loa, Ascaris lumbricoides and Ascaris suum in the test. The developed assay relied on the use of MOV 1, required only 1 ml of urine or 0.05 ml serum. About 97.8% of the 47 urines and 50% of the 20 sera from patients studied gave positive results. Only 1 (3%) of 32 control urines and up to 80% of the 10 control sera studied tested positive, suggesting urine as a better specimen source.(ABSTRACT TRUNCATED AT 250 WORDS)


The T-cell repertoire in myasthenia gravis involves multiple cholinergic receptor epitopes.

Antibodies against the alpha-subunit of the acetylcholine receptor (AChR) are found in most patients with myasthenia gravis and are considered to contribute to the receptor damage which leads to the characteristic signs and symptoms of the disease. This B-cell response is T-cell driven. Elevated T-cell reactivities to AChR and its alpha-subunit have been described in myasthenia gravis, and AChR alpha-subunit peptide reactive T-cell lines and clones preferentially recognizing certain defined sequence segments have been reported, thereby disclosing the possibility of specific immunotherapy. We have defined the T-cell repertoire to AChR, its alpha-subunit and the synthetic peptide sequences 100-117, 113-130, 143-163, 161-179, 207-225, 221-240, and 235-255 of the alpha-subunit in an immunospot assay which is based on secretion of interferon-gamma (IFN-gamma) by individual memory T cells upon stimulation with specific antigen in short-term cultures. Most patients with myasthenia gravis displayed T-cell reactivities to 1 to 6 different peptides. The mean numbers of T cells recognizing individual peptides varied in the myasthenia gravis patients between 1 per 77,000 and 1 per 167,000 peripheral blood mononuclear cells. None of the seven peptides evaluated could be identified as an immunodominant T-cell epitope, and any of them was found to dominate in individual patients. The numbers of T cells reacting with AChR and recombinant human AChR alpha-subunit were slightly higher (mean numbers 1 per 26,000 and 1 per 50,000 mononuclear cells, respectively). Such cells, as well as AChR alpha-subunit peptide reactive T cells, were also found in patients with other neurological diseases and in healthy subjects, but at lower frequencies and numbers. In myasthenia gravis, the elevated numbers of memory T cells recognizing multiple AChR alpha-subunit peptides may be crucial for the development of the disease, and the IFN-gamma released by such T cells might be important for its perpetuation.


T-cell epitopes on the human acetylcholine receptor alpha-subunit residues 10-84 in myasthenia gravis.

In myasthenia gravis the production of anti-acetylcholine receptor antibodies is modulated by acetylcholine receptor-specific T cells. Most B- and T-cell epitopes are located on the alpha-subunit of the receptor. In order to map the fine specificity of the antigen-specific T cells in myasthenia gravis, T-cell stimulation in response to 70 hexapeptides was studied in 24 patients and 24 healthy individuals. The hexapeptides overlapped with one amino acid and represented residues 10-84 of the NH2-terminal part of the alpha-subunit of the receptor. The IFN-gamma secretion from single T cells was used to detect T-cell stimulation. A significant difference in the T-cell response to several of the peptides was found between patients and healthy controls. The majority of the hexapeptides induced T-cell stimulation in at least one of the patients. Peptide-induced T-cell stimulation was evident in all but one of the patients. The results indicate that different epitopes and multiple T-cell clones are involved in the T-cell recognition of the acetylcholine receptor.


Detection of potyviruses with antisera to synthetic peptides.

Eight peptides corresponding to conserved regions of the coat protein of potyviruses were synthesized. All the peptides were recognized by anti-virus or anti-core-virus. Antisera raised to the synthetic peptides were tested with purified viruses and viral antigens present in plant sap. In many cases, the extent of cross-reactivity between different potyviruses was not correlated with the degree of sequence homology between the peptide used for immunization and the corresponding region in the coat protein of the potyvirus tested. An antiserum raised to a peptide of 18 residues containing a highly conserved region was found to react with all seven potyviruses tested.


Human and murine monoclonal antibodies directed against a conserved sequence from gp41 (aa583-599) of human immunodeficiency virus type 1.

Human spleen cells from an HIV-seropositive donor were immunized in vitro with the aa583-599 peptide conjugated to an heptalysyl core. This sequence was derived from the putatively HIV-immunosuppressive region of HIV1 gp41. The same conjugated peptide was used to immunize mice. One human and one mouse IgM monoclonal antibody (mAb) directed against the aa583-599 peptide were obtained. The two mAb had distinct patterns of reactivity against a panel of 42 peptides with modified sequences. Neither of the mAb inhibited the immunosuppressive effect of aa583-599 octopus-lys-conjugated peptide on anti-CD3 Ab-induced lymphoproliferation. In addition, both mAb did not neutralize cell-free virus transmission or enhance HIV infection. However, HmAb inhibited formation of syncytia between HIV1-infected (but not HIV2-infected cells) and non-infected target cells at concentrations above 20 micrograms/ml, whereas MmAb did not have any effect. The degree of conservation of the aa583-599 region makes HmAb a candidate for use as a group-specific reagent in future HIV1 passive immunotherapy protocols.


The effect of calcium mobilization on LPS-induced IL-1 beta production depends on the differentiation stage of the monocytes/macrophages.

The role of elevated intracellular calcium concentration [Ca2+]i in the LPS-induced activation of interleukin-1 beta (IL-1 beta) production was examined in cells representing different stages of myeloid differentiation (undifferentiated monocytic leukaemia cell line THP-1, THP-1 cells induced to adherent, macrophage-like cells by phorbol ester treatment and normal peripheral blood-derived adherent monocytes). LPS did not elevate the [Ca2+]i as measured by the Fura-2 fluorescence technique. When these cells were stimulated with LPS in the presence of the calcium ionophore A23187, a clear increase in the IL-1 beta protein production was observed in the undifferentiated THP-1 cells but not in the more differentiated cell types. This ionophore-induced increase was also seen in the IL-1 beta mRNA levels. Thus these data confirm the previous findings demonstrating that elevation of [Ca2+]i is not involved in the LPS-dependent signal transmission. However, the LPS-induced signals are greatly potentiated by the elevated [Ca2+]i, but only in undifferentiated monocytic cells.


Hyperalgesia mediated by spinal glutamate or substance P receptor blocked by spinal cyclooxygenase inhibition.

Inhibition of cyclooxygenase by nonsteroidal anti-inflammatory drugs (NSAIDs) in the periphery is commonly accepted as the primary mechanism by which these agents produce a selective attenuation of pain (analgesia). NSAIDs are now shown to exert a direct spinal action by blocking the excessive sensitivity to pain (hyperalgesia) induced by the activation of spinal glutamate and substance P receptors. These findings demonstrate that the analgesic effects of NSAIDs can be dissociated from their anti-inflammatory actions. Spinal prostanoids are thus critical for the augmented processing of pain information at the spinal level.


Women and access to health care.

This paper is concerned with access to health care for women in developing countries, with specific reference to Latin American and Caribbean countries. It reviews the available literature on the concept of access as it relates to other variables such as accountability, affordability and acceptability of health services, taking into consideration the effects of the generalized socio-economic crisis that has affected most countries during the last decade, as well as equity objectives. Various approaches to defining variables affecting access to health care appear in the literature reviewed. While some of them indicate that ability to pay for services act as a major determinant of access to health care, others point to behavioral issues related to motivation, health seeking behavior or perception of illness as a deterrent to women in the low socioeconomic strata, while others indicate that sociocultural issues, such as values, education, religion or demographic variables related to age, influence access to health care. The paper concludes with some comments on policies and strategies for securing access to health and healthcare, indicating the need to move away from traditional solutions including framing gender-based health differences in status and access adequately, promoting and strengthening social participation of women in policy making.


Aprotinin increases release of von Willebrand factor in cultured human umbilical vein endothelial cells.

Through the perioperative administration of the proteinase inhibitor aprotinin, hemostasis can be improved and postoperative bleeding reduced after cardiac operations. The mechanism of action has been only partially clarified. The goal of our study was to investigate the influence of aprotinin on the synthesis of von Willebrand factor (vWF) in human endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultivated in vitro and incubated with different aprotinin concentrations (55, 100, and 215 mol/L). With all investigated aprotinin concentrations, there was an increase in vWF synthesis compared with basal secretion (p less than 0.001). When the HUVEC were preincubated with aprotinin and stimulated with thrombin, there was a further significant increase in vWF synthesis. HUVEC that, were first incubated with aprotinin and then stimulated with thrombin demonstrated a significant increase in vWF synthesis compared with basal secretion in nonincubated cells (p less than 0.0001). Also, compared with the cells that had received thrombin stimulation alone, the combination of aprotinin incubation and thrombin stimulation led to a significantly higher vWF concentration (p less than 0.05). Because vWF is necessary for the interaction with platelet factor glycoprotein Ib and platelet adhesion, the demonstrated increase in vWF synthesis could be one of the mechanisms of action of aprotinin leading to its blood-sparing effect.


Acute radiation enteritis in rats: bile salts and trypsin.

Surgically exteriorized rat intestine divided into segments containing saline solution, a bile salt binder, or a trypsin inhibitor were radiated (1100 cGy) and subsequently evaluated for histologic damage. Five days after radiation, crypt cell numbers, mucosal height, and mucous cell numbers were significantly greater in segments protected by the binding of bile salts or the absence of trypsin activity compared with saline-bathed mucosa. Thus both proteolytic enzymes and bile salts in the lumen contribute to the acute enteritis that follows a single dose of x-radiation.


[The mechanism of tachycardic arrhythmias].

The mechanisms of cardiac tachyarrhythmias are discussed on the basis of experimental animal studies. Two major mechanisms are discussed in detail: membrane oscillations arising form electrically unstable cells and acting as ectopic foci of a tachycardia, and disturbances of impulse propagation which cause vortex-like circulating excitation with re-entry. These vorteces from the origin of the re-entrant tachycardias and of ventricular fibrillation.


Alterations of the levels of glycoproteins Ib-IX and IIb-IIIa in platelets stored at 22 degrees C.

Platelets stored in CLX blood bags, under normal blood banking conditions, were studied for up to 7 days to determine if changes occurred in the levels of membrane glycoproteins (GP) Ib-IX and IIb-IIIa. Radiolabeled monoclonal antibodies (MAB) were used to estimate the number of glycoprotein molecules on the surface membrane of intact platelets. GP IX and GP IIb-IIIa levels remained essentially unaltered during storage. In contrast, the content of GP Ib at day 7 decreased by 45% of the total when fresh. The aggregation response to ristocetin, which requires GP Ib, was also diminished after 7 days. Addition of protease inhibitors, leupeptin and/or aprotinin did not appear to influence the degradation of this glycoprotein. We conclude that storage at 22 degrees C had deleterious effects on the GP Ib content of platelets.


Multimeric analysis of von Willebrand factor by vertical sodium dodecyl sulphate agarose gel electrophoresis, vacuum blotting technology and sensitive visualization by alkaline phosphatase anti-alkaline phosphatase complex.

To detect von Willebrand factor multimers in plasma samples and factor VIII concentrates, a vertical discontinuous SDS electrophoresis was developed. A vacuum blotting system allowed to improve the transfer to the nitrocellulose membrane. The visualization of the separated multimers was sensitized by applying an alkaline phosphatase anti-alkaline phosphatase staining technique. The reported method clearly shows structural abnormalities of von Willebrand factor and deficiency of high multimers, the vacuum transfer is efficient and the sensitivity of the staining system is very high.


Reperfusion increases neutrophils and leukotriene B4 receptor binding in rat focal ischemia.

Neutrophils are critically involved with ischemia and reperfusion injury in many tissues but have not been studied under conditions of reperfusion after focal cerebral ischemia. The present studies were conducted to confirm our previous observations quantifying neutrophils in rat permanent focal stroke using a myeloperoxidase activity assay and to extend them to transient ischemia with reperfusion. In addition, leukotriene B4 receptor binding in ischemic tissue was evaluated as a potential marker for inflammatory cell infiltration. Histological, enzymatic, and receptor binding techniques were used to evaluate neutrophil infiltration and receptor binding in infarcted cortical tissue 24 hours after permanent middle cerebral artery occlusion (n = 25) or temporary occlusion for 80 (n = 12) or 160 (n = 22) minutes followed by reperfusion for 24 hours in spontaneously hypertensive rats. Sham surgery (n = 26) produced no changes in any parameter measured. After permanent middle cerebral artery occlusion, neutrophil accumulation was observed histologically, but the infiltration was moderate and typically within and adjacent to blood vessels bordering the infarcted cortex. After temporary middle cerebral artery occlusion with reperfusion, marked neutrophil infiltration was observed throughout the infarcted cortex. Myeloperoxidase activity was increased (p less than 0.05) after permanent occlusion and to a greater extent after temporary occlusion with reperfusion. Myeloperoxidase activity (units per gram wet weight) in ischemic cortex was increased over that in nonischemic (control) cortex 32.2-fold, 54.6-fold, and 92.1-fold for permanent occlusion and 80 and 160 minutes of temporary occlusion with reperfusion, respectively (p less than 0.05). Sham surgery produced no changes in myeloperoxidase activity. Leukotriene B4 receptor binding also was increased (p less than 0.05) after focal ischemia and paralleled the increases in myeloperoxidase activity. Ischemic cortex-specific receptor binding (femtomoles per milligram protein) was 3.87 +/- 0.63 in sham-operated rats and 4.57 +/- 0.98, 8.98 +/- 1.11, and 11.12 +/- 1.63 for rats subjected to permanent occlusion and 80 and 160 minutes of temporary occlusion with reperfusion, respectively (all p less than 0.05 different from sham-operated). Cortical myeloperoxidase activity was significantly correlated with the degree of cortical leukotriene B4 receptor binding (r = 0.66 and r = 0.79 in two different studies, p less than 0.01). These data indicate that neutrophils are involved in focal ischemia and that there is a dramatic accumulation of neutrophils in infarcted tissue during reperfusion that can be quantified using the myeloperoxidase activity assay. Leukotriene B4 receptor binding increases in infarcted tissue in a parallel manner, which suggests that the increased leukotriene B4 binding is to receptors located on the accumulating neutrophils.


Enzyme-linked immunosorbent assay for the detection of substances that carry blood group A specificity.

An enzyme-linked immunosorbent assay (ELISA) was developed to estimate the amount of material carrying blood group A activity in biologic samples. A soluble synthetic form of the A antigenic determinant (A trisaccharide, ATS) conjugated to peroxidase competes with the blood group A substance present in a biologic sample for anti-A attached to a solid phase by a second antibody coating the plastic micro-wells. A reference curve is constructed by using known quantities of ATS to compete with a fixed amount of ATS-peroxidase conjugate. The A substance activity in a sample is obtained by extrapolating the degree of inhibition of the binding of the ATS-peroxidase conjugate to an equivalent amount of ATS in the reference curve. The assay is reproducible, specific, and sensitive. It has been used in pharmacologic studies to estimate the concentration of ATS in the blood and urine of rats, rabbits, and baboons and in a study with human samples, testing the potential clinical use of ATS to neutralize anti-A when therapeutically indicated. It is also useful for the detection of ABO natural products in secretions, thus allowing the accurate classification of secretor and nonsecretor individuals.


Prestorage removal of Yersinia enterocolitica from red cells with white cell-reduction filters.

Prestorage removal of phagocytic white cells (WBCs) may increase the survivability of contaminating bacteria in units of stored red cells. Fourteen units of whole blood were inoculated with 65 colony-forming units per mL of Yersinia enterocolitica (serotype O:3) and processed into AS-3-preserved red cells. Five red cell units were filtered with a prototype third-generation filter and five red cell units with a second generation filter. WBC reduction was performed on the day of collection. Four red cell units were not filtered. Three noninoculated whole blood units served as negative controls; two were filtered (one with each type of WBC-reduction filter) and one remained unfiltered. All red cell units were then stored at 4 degrees C for 42 days. One of the five filtered red cell units (20%) in each filter group supported growth of Y. enterocolitica. In contrast, 4 (100%) of 4 unfiltered inoculated red cell units had growth (p = 0.04). Overall, 2 (20%) of 10 units of WBC-reduced red cells supported the growth of Y. enterocolitica, as compared to 100 percent of unfiltered red cell units after inoculation (p = 0.015). Bacterial contamination was not detected in any of the three noninoculated units. It can be concluded that prestorage WBC filtration significantly reduces the potential for growth of Y. enterocolitica in red cells stored at 4 degrees C for 42 days.


The replication of cymbidium ringspot tombusvirus defective interfering-satellite RNA hybrid molecules.

A DNA copy of DI RNA of cymbidium ringspot tombusvirus was cloned downstream of a phage T7 promoter. In vitro-transcribed RNA replicated in Nicotiana clevelandii when co-inoculated with full-length viral genomic RNA transcripts and protected plants from apical necrosis. Artificial deletion mutants derived from the DI RNA clone showed that most of the central sequence block is necessary for replication. Hybrid DI RNA-satRNA clones were prepared and in vitro-synthesized RNA was inoculated to plants in the presence of helper viral RNA. There was replication only of in vitro transcripts derived from hybrid clones where satRNA sequences were inserted upstream or downstream from the central block, but not of those derived from clones where satRNA sequence replaced the central block. Progeny RNA of biologically active clones was either full-length or showed deletions depending on the insertion of satRNA sequences in DI RNA. DI RNA-satRNA constructs having part of the 5' region exchanged were not replicated.


Processing of linear longer-than-unit-length potato spindle tuber viroid RNAs into infectious monomeric circular molecules by a G-specific endoribonuclease.

Different cDNA constructs were used for the in vitro synthesis of RNA transcripts that contain a complete monomeric unit of the potato spindle tuber viroid (PSTVd) plus an additional repeat of a part of the circular RNA genome. These permutated linear longer-than-unit-length PSTVd RNAs were incubated with the G-specific endoribonuclease RNase T1 which generated monomeric circular PSTVd RNA molecules that were infectious when mechanically inoculated to tomato plants. Besides the correct monomeric PSTVd RNA, smaller and larger circular RNAs were also formed during the reaction. The comparison of different transcripts revealed that correct in vitro processing of PSTVd RNA can proceed at alternative sites indicating that circularization is driven by RNA structure and not governed by a particular sequence. Based on these data, we propose a novel model for the processing of multimeric replicative viroid RNA intermediates through RNA cleavage and ligation catalyzed by a host endoribonuclease.


Epitope mapping of the gp53 envelope protein of bovine viral diarrhea virus.

Epitopes recognized by nine monoclonal antibodies (mAbs) on the envelope protein, gp53, of two strains of bovine viral diarrhoea virus (NADL and Oregon C24V) were mapped by competitive binding assays and by the characterization and sequence analyses of mAb neutralization escape mutants. This defined an antigenic domain on gp53 that was shared by many BVDV strains, while other less conserved epitopes were possibly distinct. Sequencing of escape mutant viruses revealed that a cluster of three amino acids in the N-terminal half of gp53 were involved in the main antigenic domain shared by both NADL and Oregon C24V viruses, while an amino acid 31 residues further toward the N-terminus was involved in a second site present only on the NADL strain. Since other amino acids defining these epitopes were located at distant positions within the gp53 protein, it is likely that a major domain [corrected] on gp53 consist of composite, conformation-dependent epitopes.


Presentation of hepatitis B virus preS2 epitope on bluetongue virus core-like particles.

A chimeric protein containing most of the hepatitis B virus preS2 region (amino acid residues 1-48) upstream to, and colinear with the amino-terminus of bluetongue virus VP7 protein (preS2-VP7) was expressed by a recombinant Autographa californica nuclear polyhedrosis virus (AcNPV). The chimeric protein formed BTV core-like particles (CLPs) in Spodoptera frugiperda cells only when the cells were coinfected with this recombinant virus and a recombinant baculovirus that expresses unmodified VP7 and VP3 of BTV. The ratio of preS2-VP7 incorporated into CLPs was influenced by the relative multiplicities of infection of the two viruses. Immunoelectron microscopy of the chimeric particles indicated that the preS2 epitope was exposed on the surface of the CLPs. When insect cells were coinfected with the preS2-VP7 recombinant virus and a baculovirus vector that synthesized only the VP3 protein, no CLPs were identified.


[Determination of melittin antigenic determinants in a model membrane].

Monospecific polyclonal antibodies labelled with colloid gold were obtained against melittin in different conformations (monomer, tetramer and complex with BSA). They were used for electron analysis of the position of antigenic determinants of melittin in DMPC model membrane. The antibodies reacted in cross-immunoassay in different titres with all antigenes and this evidences for the presence of common antigenic determinants in their structures. It was found that melittin antigenic determinants in the model membrane are exposed and accessible to reaction with antibodies and, therefore, they serve the agent for stimulation of immune response and production of antibodies.


Biologically selected recombinants between feline leukemia virus (FeLV) subgroup A and an endogenous FeLV element.

In efforts to elucidate the proximal leukemogens that might be produced during a feline leukemia virus (FeLV) infection of cats, homologous recombinations between molecularly cloned exogenous and endogenous FeLV proviruses of known sequences were examined in cell cultures in vitro. A plasmid containing an infectious member of the most commonly occurring FeLV subgroup (FeLV subgroup A or FeLV-A) was coexpressed with noninfectious constructs containing the envelope (env) gene of an endogenously inherited FeLV-like feline genomic element in transfected feline fibroblasts. The viruses generated were selected for their ability to propagate in human cells which are resistant to infection by the parental ecotropic FeLV-A or the noninfectious endogenous constructs. An analysis of the recombinants thus derived identified a limited number of sites in the env gene which were preferentially utilized in the generation of recombinant FeLVs under the selection conditions used. These sites were clustered in the surface glycoprotein (SU) moiety of the env gene, and it appeared that most, but not all, of the SU gene product of FeLV-A, beginning from the N-terminus, can be replaced by sequences from an endogenous element, still allowing the virus to be biologically viable. In fact, these substitutions in the env gene expanded infectivity of the parental FeLV-A from ecotropic to polytropic cell tropism. Additionally, substitutions in the SU region yielded many recombinants in which a primary neutralizing pentapeptide epitope of FeLV-A was altered because of its variance in the endogenous element. In several of the recombinants, this sequence was also found to be frequently mutated. Consistent with the changes identified in this antibody-binding domain, the recombinant viruses were only weakly inhibited by a monoclonal antibody directed against this epitope, while FeLV-A was highly sensitive to neutralization.


Interferon-inducible gene expression in chimpanzee liver infected with hepatitis C virus.

The molecular host response to hepatitis C virus (HCV) infection was examined by isolation of HCV-induced genes from a cDNA library constructed from chimpanzee liver during the acute phase of hepatitis C. Two cDNA clones, 130-7 and 130-51, were obtained by differential hybridization with cDNA probes prepared from poly(A)+ RNAs of infected and uninfected livers. Northern blot analysis revealed that the 130-7 and 130-51 cDNAs were expressed as 1.5- and 1.0-kb products, respectively, in chimpanzee liver and that their induction rates of the two were 20 and 4, respectively. Nucleotide sequence analyses of these cDNA inserts showed that the sequence of cDNA 130-7 was that of a class I major histocompatibility antigen and that the sequence of cDNA 130-51 was 98% homologous with a human interferon-inducible mRNA. These results suggest that HCV infection may actively induce interferon, which in turn induces the expressions of these interferon-inducible genes. Furthermore, the high expression of HLA class I antigen in the acute phase of hepatitis C suggests that liver cell injury in HCV infection may be mediated by cytotoxic T cells that recognize viral antigen in association with HLA class I antigen.


The influence of N-linked glycosylation on the antigenicity and immunogenicity of rubella virus E1 glycoprotein.

Rubella virus E1 glycoprotein contains three functional N-linked glycosylation sites. The role of N-linked glycosylation on the antigenicity and immunogenicity of E1 glycoprotein was studied using vaccinia recombinants expressing E1 glycosylation mutants. Expressed E1 glycosylation mutant proteins were recognized by a panel of E1-specific monoclonal antibodies in radioimmunoprecipitation, immunofluorescence, and immunoblotting, indicating that carbohydrate side chains on E1 are not involved in the constitution of epitopes recognized by these monoclonal antibodies. This observation was further supported by the fact that removal of oligosaccharides on E1 by glycosidase digestion did not significantly change the antigenicity of E1. All the glycosylation mutants were capable of eliciting anti-RV E1 antibodies. The single glycosylation mutants (G1, G2, and G3), but not the double mutant (G23) or the triple mutant (G123), were found to be capable of inducing virus neutralizing antibodies. Among the single glycosylation mutants, only G2 and G3 were active in producing hemagglutination inhibition antibodies in mice. Our findings suggest that although carbohydrate on E1 is not directly involved in the antigenic structures of E1, it is important in maintaining proper protein folding and stable conformation for expression of immunological epitopes on E1.


[Multi-visceral upper abdominal resection and orthotopic liver transplantation--a surgical treatment concept for regionally metastatic tumors of the endocrine pancreas].

Islet cell carcinoma is rare. Even in advanced metastasizing tumors, relatively long survival times were reported. Aim of surgical treatment is complete removal of the tumor mass, if possible. When not, palliative procedures were described to be efficient in some of the cases. In the literature, among the first patients with regional metastasizing intraabdominal malignancies and upper abdominal exenteration, some cases with neuroendocrine tumors were reported. Aim of the following study is to describe the results in 4 patients with metastasizing islet cell carcinomas, which were treated by multi-visceral upper abdominal exenteration and combined orthotopic liver transplantation.


Enhanced effect of mutant granulocyte-colony-stimulating factor (KW-2228) on the growth of normal and leukemic hemopoietic progenitor cells in comparison with recombinant human granulocyte-colony-stimulating factor (G-CSF).

We tested the in vitro effect of a novel granulocyte colony-stimulating factor (G-CSF) derivative (KW-2228) on the growth of G-CSF-dependent hemopoietic progenitor cells: granulocyte precursor cells (CFU-G), leukemic blast progenitors freshly obtained from 9 patients with acute myeloblastic leukemia (AML) and cells of a G-CSF-dependent human AML cell line (OCI/AML 1a). KW-2228 showed a higher stimulating effect than recombinant human G-CSF (rhCSF) on CFU-G; 3 out of 9 leukemic blast progenitors and OCI/AML 1a cells. The difference in biochemical stability between rhG-CSF and KW-2228 was considered to explain the superior colony-stimulating activity of KW-2228. The results show that KW-2228 will be a new granulopoietic factor.


Thyroid hormone regulation of human pituitary alpha subunit gene expression.

The thyroid hormone T3 exerts an inhibitory effect upon pre-translational expression of the alpha subunit gene in the anterior pituitary. In the intact animal oestrogen alone is without effect upon alpha gene expression but oestrogen modulates the influence of T3 deficiency upon the alpha gene. T3 effects upon alpha transcription are mediated via sequences within 98 bp of the transcriptional start site of the gene and similar 5' flanking sequences of the alpha gene mediate both T3 regulation and oestrogen modulation of T3 inhibition. This oestrogen effect may be mediated directly via interaction of the oestrogen receptor with the alpha gene thyroid hormone response element to which the oestrogen receptor is able to bind.


Increased release of tissue plasminogen activator and its inhibitor in human parotid saliva upon stimulation.

Parotid saliva from 12 healthy volunteers was collected prior to and after 5 and 25 min of stimulation at a constant flow rate of 0.25 or 1.0 ml min-1. In the salivary samples the concentrations of tPA (tissue-type plasminogen activator), PAI-1 (plasminogen activator inhibitor type-1), albumin and total protein were determined and the activity of amylase, tPA and PAI assessed. Presence of both tPA and PAI-1 antigen was demonstrated in all samples, and in unstimulated saliva the ratio between the activator and its inhibitor was 1:7. Upon stimulation we found a significantly increased concentration of PAI-1, a less pronounced increase in tPA concentration, unchanged amylase and total protein levels and significantly decreased albumin concentration. tPA activity was significantly reduced after prolonged stimulation which had no effect on PAI activity. In stimulated saliva a significant positive correlation between concentration of tPA and PAI-1 was demonstrated. Stimulation with citric acid had no effect on output of albumin which is passively filtered from blood, whereas the increase in flow rate corresponded to the significantly increased secretion rate of total protein and amylase which is secreted by gland cells. The secretion pattern of tPA and PAI-1 differed significantly from that of albumin in showing markedly increased output rate during the stimulation period, and the relative increase in output of PAI-1 was significantly higher than that of amylase and total protein. Thus, the results from this study suggest an active release of both tPA and its main inhibitor PAI-1 into saliva.


[An experimental model of intravitreal neovascularization in rabbits].

The author developed a consistent model of intravitreal neovascularization in the eyes of pigmented rabbits by incomplete posterior vitreous detachment after air injection followed by implantation of basic fibroblast growth factor (b-FGF; 250 ng or 1 microgram) in the vitreous and simultaneous intravitreal injection of DL-alpha-aminoadipic acid in physical saline solution (1 mg/kg). Newly formed vessels were observed in the proliferative fibrous membrane that surrounded the b-FGF pellet. In fluorescein angiography, we observed fluorescein leakage from the newly formed vessels. Histologically, the newly formed vessels showed fenestration of endothelial cells. This method provides an easy and consistent model to study neovascularization.


Long-term prognostic significance of ambulatory electrocardiographic findings in apparently healthy subjects greater than or equal to 60 years of age.

To determine the long-term prognostic significance of frequent or complex ectopic beats and ST-segment changes on 24-hour ambulatory electrocardiogram (ECG) in apparently healthy older subjects, 98 volunteers were followed up from the Baltimore Longitudinal Study of Aging who were 60 to 85 years old and free of cardiac disease by history, physical examination and maximal treadmill testing at the time of ambulatory ECG between 1978 and 1980. Over a mean follow-up period of 10 years, coronary events developed in 14 subjects: angina pectoris in 7, nonfatal myocardial infarction in 3 and sudden cardiac death in 4. The incidence of coronary events did not differ significantly between subjects who developed the following arrhythmias and those who did not, respectively: greater than or equal to 30 supraventricular ectopic beats in any hour, 18 vs 13%; greater than or equal to 100 supraventricular ectopic beats in 24 hours, 20 vs 12%; paroxysmal atrial tachycardia, 15 vs 14%; greater than or equal to 30 ventricular ectopic complexes (VECs) in any hour, 17 vs 14%; greater than or equal to 100 VECs in 24 hours, 18 vs 14%; or repetitive VECs, 20 vs 13%. The mean 24-hour heart rate (75 +/- 8 vs 72 +/- 9 beats/min) as well as the maximal (116 +/- 20 vs 111 +/- 18 beats/min) and minimal (51 +/- 6 vs 53 +/- 7 beats/min) heart rate also did not differ between the coronary event and non-event groups.(ABSTRACT TRUNCATED AT 250 WORDS)


Expression of galanin in rat primary sensory afferents after moxibustion to the skin.

We examined the effects of moxibustion on primary sensory neurons in the skin of rats using immunocytochemistry combined with a fluorescent retrograde tracer dye, fluoro gold (FG). Galanin-like immunoreactive (IR) fibers were often observed in the dermis of treated skin at 18 hours after moxibustion, while such fibers were rarely detected in untreated (control) skin. Moreover, most of galanin-IR fibers also displayed substance P (SP)-like immunoreactivity. About 20-30% of the dorsal root ganglion (DRG) neurons labeled when FG was injected intradermally into the moxibustion-treated skin showed galanin-like immunoreactivity, while the proportion of FG-labeled neurons with such immunoreactivity was less than 10% in control DRGs. These results show that moxibustion induced galanin expression by primary sensory neurons containing SP. The possible functions of this peptide are discussed in relation to the effects of moxibustion.


A sacred symbol of an organ associated with birth resembling omega.

As a result of analogous thinking Birth = Creation, then the organs associated with birth have been projected as partaking in creation. Birth of a new life-form begins with reproduction of union of a pair as male-female. They depend upon their reproductive organs. Their external sex-organs are the first to initiate reproduction and both are considered auspicious. Reproduction leads to pregnancy of the female whose internal organ becomes the organ where the fetus grows until an independent life-form issues into the world. As this organ resembles the letter Omega of the Greek alphabet those who believe in prudery speak of it as the omega-shaped organ. Nevertheless, it is the place where a new life form develops and it is considered auspicious. Everything promoting the formation of a life-form is considered auspicious.


Glycosulfatase activity of H. pylori toward human gastric mucin: effect of sucralfate.

Colonization of gastric mucosa by Helicobacter pylori, a bacterium implicated in the etiology of gastric disease, involves the cell surface sulfated glycosphingolipid receptors for the attachment. Evidence has also been obtained recently that sulfated mucus glycoproteins have the ability to interfere with this process. Here, we show that H. pylori displays glycosulfatase activity, and report the specificity of this enzyme toward gastric mucosal sulfated glycoproteins and glycolipids. With 35S-labeled human gastric sulfated mucin as substrate, the enzyme activity was identified in the extracellular material elaborated by the bacterium. The glycosulfatase exhibited maximum activity at pH 5.7 in the presence of Triton X-100 and CaCl2, and gave on SDS-PAGE a protein band of 30 kDa. Specificity studies revealed that the enzyme effectively caused desulfation of N-acetylglucosamine-6-sulfate and galactose-6-sulfate present in carbohydrate chains of gastric mucins, as well as that of glucose-6-sulfate, a constituent of mucus glyceroglucolipids. However, the H. pylori glycosulfatase was ineffective toward galactosyl- and lactosylceramide sulfates which serve as receptors for this bacterium attachment and contain the sulfate ester group at C-3 of galactose. The glycosulfatase activity toward human sulfated gastric mucin was inhibited by sucralfate. The inhibitory effect was proportional to the concentration of sucralfate up to 120 micrograms/ml, at which a 78% decrease in mucin desulfation occurred. The results demonstrate that H. pylori, through its glycosulfatase activity, affects the sulfated mucin and glyceroglucolipid content of the protective mucus layer, and that antiucler drug sucralfate is able to counteract the detrimental action of this enzyme.


Serum trypsin in chronic renal failure and transplant patients.

The frequency and degree of elevated serum levels of trypsin (T) and correlation with other pancreatic enzymes were determined in several groups of patients with renal disease, i.e., patients with chronic renal failure (CRF), hemodialysis patients (HD), renal transplant recipients (RT), and in a control (C) group. Mean values of T were significantly higher in all other groups than in the C group (p less than 0.0001). A statistically significant correlation between T and creatininemia levels was found only for the RT group (p less than 0.0001). Correlations between T versus pancreatic amylase and T versus lipase activity were found to be statistically significant in the CRF and RT groups (p less than 0.01), but not in the HD group. Most patients in all groups had T values higher than the maximum value observed in the controls and, of them, most had very elevated values. The results suggest that in chronic renal pathology there are frequent and significant increases in serum T levels, circulating in parallel with the other pancreatic enzymes. It is possible that, together with the renal excretion impairment, there could also be subclinical pancreatic damage or a dysfunction of the other means of elimination of T that can be responsible for, or contribute to, the serum increase in the enzyme.


A health survey of toll booth workers.

The prevalence of respiratory and other health problems in a cohort of highway toll booth workers was surveyed by mailed questionnaire. In a low proportion of respondents (43.2%), a high prevalence of central nervous system complaints (headaches, irritability, or anxiety, and unusual tiredness), mucous membrane irritation (eye irritation, nasal congestion, and dry throat), and musculoskeletal problems (joint and back pains) was found. We believe these symptoms are reflective of the acute irritant and central nervous system effects of exposure to motor vehicle exhaust. The musculoskeletal complaints are likely the result of bending, reaching, and leaning out of the toll booth. The need for in-depth evaluation of the ventilation systems and the ergonomic and job stressors of work at toll booths is suggested by these results.


Detection of bone marrow metastases in small cell lung cancer patients. Comparison of immunologic and morphologic methods.

An immunocytochemical method, involving four monoclonal antibodies (MAbs) previously selected for their specific binding to small cell lung cancer (SCLC) cells in human bone marrow, was used for detection of bone marrow metastases in 81 patients with diagnosed SCLC. This procedure was compared with two routine morphologic methods with regard to diagnostic efficiency and sensitivity. Bone marrow involvement was found in 26 patients (32%), one of which had limited disease according to conventional clinical criteria. Eight of the positive cases were exclusively diagnosed by immunocytochemistry, whereas the histologic and cytologic methods separately identified two patients each. Immunocytochemistry had a detection level of tumor cells in the mononuclear cell fraction of approximately 1-2%, whereas no patients with less than 10% immunocytologically detectable tumor cells were diagnosed by cytomorphologic examination of bone marrow aspirates. Evidence was obtained that the diagnostic efficiency of any method increased with the number of samples examined. Of the four MAbs used, the anti-NCAM antibody, MOC-1, labeled tumor cells in all immunologically positive patients, and in all but one of these patients all cytologically confirmed tumor cells were stained. The antibodies MOC-31, which recognize a cluster-2 antigen, and NrLu10 bound nearly all tumor cells in most cases, whereas MLuC1 only diagnosed tumor cells in a fraction of the patients. The results show that the immunocytochemical application of these antibodies is superior to morphologic techniques in detecting SCLC bone marrow metastases. Further use of the method might provide prognostically and therapeutically useful information.


Proliferating cell nuclear antigen immunohistochemistry in rat aorta after balloon denudation. Comparison with thymidine and bromodeoxyuridine labeling.

The authors compared detection of proliferating vascular smooth muscle cells (SMC) in vitro and in vivo with proliferating cell nuclear antigen (PCNA) immunohistochemistry and two established methods: [3H]thymidine autoradiography and bromodeoxyuridine immunohistochemistry. Labeling with [3H]thymidine and bromodeoxyuridine of rat vascular SMC in culture stained 11% +/- 2% and 11% +/- 1% of cells, and PCNA immunohistochemistry 22% +/- 2% of cells. Proliferation in the media of the denuded rat aortae was highest 3 days after denudation: 4.2%, 3.8%, and 4.7% of labeled cells with [3H]thymidine, bromodeoxyuridine, and PCNA, respectively. In the intima, proliferation was highest 7 days after denudation with 42% [3H]thymidine, 40% bromodeoxyuridine, and 46% PCNA-positive cells. With double labeling, all [3H]thymidine-positive cells were PCNA positive, whereas some cells were only positive for PCNA. The authors conclude that PCNA immunohistochemistry compares favorably with [3H]thymidine autoradiography, and bromodeoxyuridine immunohistochemistry.


Distribution of albumin and alpha-fetoprotein mRNAs in normal, hyperplastic, and preneoplastic rat liver.

The nature of bile duct-like (oval) cells proliferating during chemical hepatocarcinogenesis has been controversial. To investigate this issue further, the authors compared the hepatic distribution of albumin (ALB) and alpha-fetoprotein (AFP) mRNAs in rats in which oval cell proliferation was induced by feeding a choline-devoid diet containing 0.1% ethionine (CDE, a hepatocarcinogenic diet) with that in normal rats and in rats in which biliary epithelial cell hyperplasia was induced by either bile duct ligation or feeding alpha-naphthylisothiocyanate (ANIT). Northern blot analysis in parenchymal and nonparenchymal liver cells isolated from these animals demonstrated that ALB mRNA was present in the hepatocytes of both control and experimental animals, whereas this transcript was detected in nonparenchymal epithelial cells only in CDE-fed rats. Alpha-fetoprotein mRNA was not seen in either parenchymal or nonparenchymal cells isolated from normal or hyperplastic livers induced by bile duct ligation or ANIT feeding. In CDE-fed rats, however, both parenchymal and nonparenchymal cell populations displayed AFP message. In situ hybridization directly demonstrated nonparenchymal cell expression of both ALB and AFP transcripts in CDE-fed rats. Most surprisingly, ALB and AFP mRNAs were also detected by in situ hybridization in occasional nonparenchymal cells located in portal tracts near the limiting plate in normal liver, as well as under conditions associated with bile duct hyperplasia. Immunohistochemical studies of intermediate filament proteins, cytokeratin 19 (a marker of glandular epithelia), vimentin (a marker of mesenchymal lineage), and desmin (a marker of muscle cell differentiation) demonstrated that oval cells, as well as normal and hyperplastic bile duct cells, were positive for cytokeratin 19 and negative for both vimentin and desmin. Cytokeratin-positive oval cells formed duct profiles and were connected to preexisting ductules and ducts. These results are construed to suggest that oval cells proliferating during CDE hepatocarcinogenesis are derived from epithelial cells within the biliary tree. The presence of cells with similar morphologic appearance, periportal location, and AFP and ALB expression in normal liver suggests that these cells may be the progenitors of oval cells induced by some carcinogenic regimens.


T lymphocytes expressing HECA-452 epitope are present in cutaneous acute graft-versus-host disease and erythema multiforme, but not in acute graft-versus-host disease in gut organs.

Lymphocytes in formalin-fixed skin biopsies from patients with cutaneous acute graft-versus-host disease (aGVHD) were studied with HECA-452 (an antibody recognizing lymphocytes with skin-homing properties) and a panel of antibodies recognizing pan-B (L26 [CD20]), pan-T (L60 [CD43] and A6 [CD45RA]), and T-helper subset (OPD4) antigens in paraffin sections. Biopsies from patients with erythema multiforme (EM) were similarly studied for comparison. In both conditions, T lymphocytes stained by OPD4 were predominantly confined to the dermis, whereas those stained by HECA-452 were concentrated in the epidermis; however, there was considerable variation between cases, and overlap between findings in the dermis and epidermis. Lymphocytes similarly studied in paraffin sections of liver, salivary gland, and gut affected by aGVHD were essentially unreactive with HECA-452, although they were largely stained by pan-T markers and showed some comparable reactivity with OPD4. The findings suggest that aGVHD of the skin is mediated by a different set of lymphocytes than in gut organs, and may have a similar immunologic mechanism to EM.


Assessment of proliferative activity in ovarian neoplasms by flow and static cytometry. Correlation with prognostic features.

We undertook a prospective flow and static cytometric study of proliferative activity in 74 malignant, borderline and benign ovarian neoplasms. Proliferative activity as assessed by S phase fraction (SPF), and immunostaining of tissue sections with Ki-67 antibody was compared with prognostically important clinicopathologic features. Malignant neoplasms had higher median percentage Ki-67 staining (27.6%) than borderline (12.3%) and benign (2.7%) tumors (P less than 0.05). Percentage Ki-67 staining correlated with SPF, DNA index, architectural grade, nucleolar grade, and mitotic count in malignant tumors; with nucleolar grade in benign tumors and with none of these variables in borderline tumors. Similarly, malignant neoplasms had a higher median SPF (11.5%) than borderline (3.4%) and benign (2.9%) neoplasms (P less than 0.05). In malignant neoplasms, SPF correlated with percentage Ki-67 staining, DNA index, age, and stage, but with none of these in borderline or benign neoplasms.