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PubMed2001

Role of Ca2+-sensitive protein kinase C in phenylephrine enhancement of Ca2+ sensitivity in rat tail artery.

We investigated the role of protein kinase C (PKC) isoforms on changes in sensitivity of contractile mechanisms to intracellular Ca(2+) (force /[Ca(2+)]i) by phenylephrine (0.1-100 microM) in rat tail arterial helical strips using simultaneous measurements of force and [Ca(2+)]i. Force/[Ca(2+)]Ii induced by phenylephrine was greater than that induced by 80 mM K+. Force/[Ca(2+)]i induced by phenylephrine in physiologic saline solution or low Ca(2+) solution was dependent on the agonist concentration. Removal of Ca(2+) completely abolished the phenylephrine-induced contraction. The PKC inhibitors staurosporine and calphostin C inhibited the increase in force/[Ca(2+)]i induced by phenylephrine to a much greater extent than that induced by 80 mM K+. LY379196, a specific PKCbeta inhibitor, did not inhibit the increase of calcium sensitivity due to phenylephrine. The classic PKC isoforms, alpha, betaI, and II not gamma were demonstrated in the artery by immunohistochemistry. These results suggest that in rat tail arterial smooth muscle, PKCalpha, and not beta or gamma, mediates the increase of changes in sensitivity of contractile mechanisms to intracellular Ca(2+) to high dose of alpha1 receptor stimulation (phenylephrine 100 microM) on nonphysiologic conditions.

Animals↗
PubMed2007

Rat chromosome 19 transfer from SHR ameliorates hypertension, salt-sensitivity, cardiovascular and renal organ damage in salt-sensitive Dahl rats.

OBJECTIVES: Unlike Dahl salt-sensitive (SS) rats, some strains of spontaneously hypertensive (SHR) rats develop only minor organ damage even when exposed to high-salt diet. In previous linkage studies, we identified quantitative trait loci on rat chromosome 19 (RNO19) linked to the SHR allele suggesting a protective effect against salt-induced hypertensive organ damage in SS. METHODS: To test the relevance of this finding, we generated and characterized a consomic strain SS-19SHR in which RNO19 from SHR was introgressed into the susceptible background of SS. We compared the effects of low-salt (0.2% NaCl) and high-salt (4% NaCl) diet exposure for 8 weeks on the development of hypertension and target organ damage in male consomic and SS animals (n=14-20, each). RESULTS: Systolic blood pressure, relative left ventricular weight and urinary protein excretion were significantly lower in SS-19SHR compared to SS under both low-salt and high-salt diet (P < 0.05, respectively). Left ventricular atrial natriuretic peptide mRNA expression showed a more pronounced 4.5-fold increase in SS compared to SS-19 (two-fold) after high-salt (P < 0.05). In comparison to low diet, high-salt exposure induced a significant increase in vascular aortic hypertrophy index, left ventricular interstitial fibrosis (+210%) and perivascular fibrosis (+195%) in SS but not in consomic SS-19SHR (P < 0.05, respectively). CONCLUSIONS: These results demonstrate a strong protective effect of RNO19 from SHR on the development of hypertension, salt-sensitivity, cardiovascular and renal organ damage in SS. In particular, we demonstrate a genetic effect protecting against the development of cardiac fibrosis in salt-sensitive hypertension.

Animals↗
PubMed1997

Draining lymph node cells of contact-sensitized mice induce suppression of contact sensitivity.

The application of hapten to the skin of mice can induce contact sensitivity (CS). It has also been well established that draining lymph node (DLN) cells can induce CS to the hapten used for skin painting when injected into naive mice. This is true for DLN cells recovered about 24 h after skin painting with hapten. It is unclear, however, whether DLN cells recovered shortly after hapten application have the same ability. By using an adoptive transfer assay system, we examined the ability of DLN cells recovered from mice at various times after skin painting with hapten to induce CS. DLN cells harvested 18-24 h after the application of fluorescein isothiocyanate (FITC) or 2,4-dinitrofluorobenzene (DNFB) induced strong CS when injected into naive mice. DLN cells harvested 3-6 h after the application of FITC or DNFB induced either only weak or no CS but induced suppression of the subsequent immunization to the two haptens. The suppression was hapten-specific, MHC restricted, and associated with the appearance of splenic suppressor T lymphocytes. Analyses with antibodies and ultraviolet (UV) B radiation demonstrated that suppression-inducing cells in DLNs were Ia+, Thy-1(-), and functionally UV-sensitive. These data suggest that epicutaneous sensitization with hapten first induces immunologically specific suppressor activity in the draining lymph nodes, whereas immunogenic activity becomes predominant later.

Adoptive Transfer↗
PubMed1991

A successful murine model for contact sensitization to a sesquiterpene-alpha-methylene-gamma-butyrolactone: sensitization to alantolactone in four strains of mice.

Induction of allergic contact hypersensitivity to a sesquiterpene lactone, alantolactone, was studied in four strains of mice: C3H/He, DBA/2, Balb/b, and Balb/c. The last three were successfully sensitized. A significant dose/response was demonstrated in these species, as well as an experimental "overload effect" in Balb/c and Balb/b strains. Histologic studies confirmed the allergic nature of the reaction. From the overall results, alantolactone can be considered a moderate sensitizer in mouse as well as in guinea pig. This study shows that the murine model can be used for experimental contact sensitization with moderate allergens, without the use of Freund's adjuvant for induction.

Animals↗
PubMed1975

Studies on the contact sensitization of man with simple chemicals. III. Quantitative relationships between specific lymphocyte transformation, skin sensitivity, and lymphokine activity in response to dinitrochlorobenzene.

Dinitrochlorobenzene (DNCB) coupled to peripheral blood erythrocytes or leukocytes forms a particulate complex, DNCB-antigen. The addition of DNCB-antigen induced blastogenesis and DNA synthesis in leukocyte cultures from DNCB-sensitized human subjects and not in leukocyte cultures from nonsensitized controls. In general, sensitized subjects who displayed a higher degree of cutaneous reactivity to DNCB, as manifested by duration and intensity of dermatitis, also showed a greater blastogenic response to DNCB-antigen in vitro. This quantitative correlation, however, was not invariant. Certain soluble factor(s), or lymphokines are released following the addition of DNCB-antigen to leukocyte cultures prepared from some sensitive subjects who were rechallenged one or more times with DNCB. These lymphokines induce blastogenesis in secondary target leukocyte populations from nonsensitized subjects. Extended studies are presented which slow little or no lymphokine activity in peripheral blood leukocyte cultures during a primary immune response, despite high degrees of blastogenic activity in response to DNCB-antigen. Significant lymphokine activity was observed only following additional rechallenge with DNCB. Blastogenesis and skin reactivity specific for DNCB have been shown to develop at about the same time during a primary immune response. This, along with the quantitative correlation shown in this communication, suggests that both processes probably reflect thymic-dependent cellular immunity. The appearance of lymphokine activity following rechallenge with DNCB suggests that DNCB-induced lymphokines may represent an amplifying mechanism of the cellular immune response that involves recruitment of previously uncommitted lymphocytes.

Antigens↗
PubMed1990

The discrimination between nickel-sensitive and non-nickel-sensitive subjects by an in vitro lymphocyte transformation test.

A lymphocyte transformation test (LTT) was able to distinguish between nickel-sensitive subjects and non-nickel-sensitive controls. Sixty-one out of 66 (92%) nickel-sensitive subjects had positive stimulation indices in 6- and/or 7-day assays using 5 micrograms/ml of nickel sulphate, whereas none of the 43 controls gave positive results. Stimulation indices were not enhanced by the patch testing of subjects to nickel before performing the LTT. A weak correlation was seen between the results of the LTT and the macroscopic degree of patch-test reactivity. The concentration of nickel sulphate used (5 micrograms/ml) did not have a significant non-specific mitogenic effect.

Adult↗
PubMed1991

Prostacyclin activates tachykinin release from capsaicin-sensitive afferents in guinea-pig bronchi through a ruthenium red-sensitive pathway.

1. We have investigated the ability of prostacyclin (PGI2) to contract guinea-pig isolated bronchi and the possible involvement of capsaicin-sensitive primary afferents in the response to PGI2. 2. PGI2 (0.1-100 microM) produced concentration-dependent contractions of the guinea-pig isolated bronchi. In vitro capsaicin desensitization (10 microM for 30 min followed by washing) significantly reduced the PGI2-induced contraction at all concentrations tested. A capsaicin-resistant component of contraction (40-60% of the overall response) was also evident. 3. Ruthenium red (3 microM), an inorganic dye which acts as a selective functional antagonist of capsaicin, significantly decreased PGI2-induced contractions, without affecting the response to substance P, neurokinin A or acetylcholine. 4. MEN 10, 207, (Tyr5, D-Trp6,8,9, Arg10)-neurokinin A (4-10) (3 microM), a selective antagonist of NK2-tachykinin receptors, significantly decreased PGI2-induced contractions and neurokinin A-induced contractions, without affecting the response to acetylcholine. 5. The effect of ruthenium red and MEN 10,207 on the one hand, and that of ruthenium red and capsaicin on the other was non additive. 6. These results indicate that PGI2-induced contraction of the guinea-pig isolated bronchi involves two distinct mechanisms, one of which involves transmitter (tachykinins) release from peripheral endings of capsaicin-sensitive primary afferents. In as much as PGI2-activation of primary afferents is sensitive to ruthenium red, we suggest that PGI2 shares a common mechanism of tachykinin release with that activated by capsaicin.

Animals↗
PubMed1996

Multiple azo disperse dye sensitization mainly due to group sensitizations to azo dyes.

A female patient, with a previous episode of contact dermatitis caused by a blue dress, developed similar dermatitis due to a navy-blue dress. Patch tests revealed multiple allergic positive reactions to paraphenylenediamine (PPD), the navy-blue dress, its extracts, 6 azo disperse dyes in a textile series, as well as 3 dye components, including Disperse (DP) Red 153, which were present in the dress; these were composed of 9 azo disperse dyes, all dyes being of a different chemical structure. On the basis of chemical similarities between these 16 azo dyes including PPD, these are classified into the following 4 groups: thiazol-azoyl-PPD group (including DP Blue 106, DP Blue 124 and 5 used dyes), aminoazobenzene group (DP Red 1, DP Red 17, DP Brown 1 and 2 used dyes), PPD group (PPD and DP Orange 3) and benzothiazol-azoyl-PPD group (2 dyes in DP Red 153). With few exceptions, cross-sensitizations between dyes in the same group have been reported by other authors, or are suggested by us, in the former 3 groups. Multiple azo disperse dye sensitization is therefore considered to be attributable mainly to group sensitizations to azo dyes.

Aniline Compounds↗
PubMed1997

National rates and regional differences in sensitization to allergens of the standard series. Population-adjusted frequencies of sensitization (PAFS) in 40,000 patients from a multicenter study (IVDK).

Sensitization rates to contact allergens vary between centers and are influenced by sex and age. Eliminating the latter 2 factors by standardization of data by age and sex, the present analysis addresses possible differences between centers remaining after elimination of these confounders, and analyzes other factors which might influence rates, e.g., the MOAHL index. Overall standardized rates were well within the range reported in previous studies and may be regarded as representing the rates of the "patch test population" in Central Europe (e.g., nickel sulfate 12.9%, fragrance mix 10.5%, balsam of Peru 7.3%, thimerosal 5.6%). For this analysis, data of those departments which contributed more than 2000 patients, or of those with extreme proportions concerning sex, age and occupational cases were selected. Patients from these 10 departments differed considerably with regard to the items of the MOAHL index and with regard to standardized rates. The items of the MOAHL index proved to be suitable for describing different patch test populations and for explaining some differences between centers. Only 'atopic dermatitis' seems to have little influence on (standardized) rates. Face dermatitis is not yet represented in the MOAHL index, but should be included, together with age > 40 years, in an extended index (acronym: MOAHLFA). Regional allergen exposure (with striking differences between East Germany, West Germany and, to a lesser extent, Austria) seems to have a great influence on the sensitization pattern observed in a department. In addition, sociological factors may influence sensitization rates, which is exemplified by high rates of nickel allergy in a socially defined subgroup. Future studies should focus on these factors, as well as on factors concerning patch test practices and quality control.

Adult↗
PubMed1984

Behavioral sensitivity to purinergic drugs parallels ethanol sensitivity in selectively bred mice.

Behavioral responses to an adenosine receptor agonist and antagonist were examined in mice genetically selected for differential sensitivity to the soporific effects of ethanol. Both ethanol and the adenosine receptor agonist L-phenylisopropyladenosine had greater sedative and hypothermic effects in ethanol-sensitive "long-sleep" mice than in ethanol-insensitive "short-sleep" mice. Long-sleep mice were also more sensitive to the excitatory behavioral effects of theophylline, an adenosine receptor antagonist. These data suggest that adenosine may be an endogenous mediator of responses to ethanol.

Adenosine↗
PubMed1984

Effect of cell-cell interactions on drug sensitivity and growth of drug-sensitive and -resistant tumor cells in spheroids.

Multicellular spheroids were grown from mixtures of rat brain tumor cells sensitive (9L) and resistant (R3) to 1,3-bis(2-chloroethyl)-1-nitrosourea. Percentages of each cell subpopulation in these spheroids were estimated with the sister chromatid exchange assay and were found to be approximately the same as those used to initiate spheroids. Spheroids grown from 9L cells alone had a higher growth rate than spheroids grown from R3 cells alone. However, the growth rate of mixed-cell spheroids was essentially the same as that of pure 9L spheroids and was independent of the percentages of R3 cells in mixed-cell spheroids. The sensitivity of 9L cells in mixed-cell spheroids treated with 1,3-bis(2-chloroethyl)-1-nitrosourea, estimated by changes in the number of sister chromatid exchanges per metaphase induced by treatment, decreased as the percentage of R3 cells increased. These effects are probably the result of an interaction between the two cell subpopulations held in three-dimensional contact, a situation similar to that in tumors in situ. The results suggest why one cell subpopulation of tumors does not become dominant during growth and indicate that interactions between cell subpopulations can influence the sensitivity of one subpopulation to 1,3-bis(2-chloroethyl)-1-nitrosourea.

Animals↗
PubMed1977

Sensitivity of Various Bacteria, Including Actinomycetes, and Fungi to Cadmium and the Influence of pH on Sensitivity.

A variety of microorganisms, including gram-negative and gram-positive eubacteria, actinomycetes, yeasts, and filamentous fungi, were tested for their sensitivity to cadmium (Cd). In general, the actinomycetes were more tolerant to Cd than were the eubacteria; gram-negative eubacteria were more tolerant to Cd than were gram-positive eubacteria. The period of exponential growth of the eubacteria and actinomycetes was extended in the presence of Cd. Wide extremes in sensitivity to Cd were noted among the fungi; there was no correlation between the class of fungus and tolerance to Cd. Fungal sporulation was more sensitive to Cd than was mycelial growth, as spore formation was inhibited at Cd concentrations that were noninhibitory to mycelial proliferation. The toxicity of Cd to the eubacteria, actinomycetes, and fungi appeared to be pH dependent, as toxicity was generally potentiated at pH 8 or 9.

Journal Article↗
PubMed1976

Selective activation of classical and alternative pathways of human complement by "promptly serum-sensitive" and "delayed serum-sensitive" strains of Serratia marcescens.

Chelation of fresh human serum with 0.01 M MgCl2 (Mg) plus 0.01 M ethylene glycol tetraacetic acid failed to abrogate the bactericidal activity against "delayed serum-sensitive" strains of Serratia marcescens, whereas previously "promptly serum-sensitive" strains of S. marcescens and control strain Escherichia coli C were killed after an extended period of incubation. The addition of 0.01 M ethylenediametetracetate to fresh human serum neutralized bactericidal activity against S. marcescens of either serum sensitivity category.

Blood Bactericidal Activity↗
PubMed1976

Temperature-sensitive mutant of Schizosaccharomyces pombe exhibiting enhanced radiation sensitivity.

A conditional lethal and radiation-sensitive mutant of Schizosaccharomyces pombe is described in which both characteristics result from a single gene mutation. Confirmation of the pleiotropic nature of this mutant was obtained by tetrad analysis and by testing the radiation sensitivity of a large number of revertants that grew normally at the restrictive temperature. The colony-forming ability of the mutant after ultraviolet radiation, gamma radiation, and ethyl methane sulfonate treatment is considerably altered by the post-treatment incubation temperature, showing higher survival at 25 than at 30degreesC. The radiosensitivity of the mutant is also influenced by the stage of growth. The difference in radiation sensitivity between the wild type and mutant is greater when log-phase cultures are compared. The characteristics of this mutant suggest that it is defective in a step common to both deoxyribonucleic acid replication and repair.

Ascomycota↗
PubMed1983

Physiological properties of cold-sensitive suppressor mutations of a temperature-sensitive dnaZ mutant of Escherichia coli.

Suppressors of a temperature-sensitive dnaZ polymerization mutant of Escherichia coli have been identified by selecting temperature-insensitive revertants. Those suppressed strains which concomitantly became cold sensitive were chosen for further study. Intragenic suppressor mutations, which caused cold-sensitive defects in DNA polymerization, were located in dnaZ by transduction with lambda dnaZ+ phages. Extragenic suppressor mutations were mapped within the initiation gene dnaA. These suppressor-containing strains were defective in initiation at low temperature as determined by measurements of DNA synthesis in vivo and in toluene-treated cells. The occurrence of suppressor mutations of dnaZ(Ts) within the dnaA gene is considered evidence that the dnaA and dnaZ products interact in vivo. A second indication of a dnaA-dnaZ protein-protein interaction was provided by the observation that the introduction of additional copies of the dnaZ+ gene into a strain carrying the dnaA suppressor mutation was lethal [whether the strain was dnaZ+ or dnaZ(Ts)].

Bacterial Proteins↗
PubMed2005

Mutants with temperature-sensitive defects in the Escherichia coli mismatch repair system: sensitivity to mispairs generated in vivo.

We have used direct selections to generate large numbers of mutants of Escherichia coli defective in the mismatch repair system and have screened these to identify mutants with temperature-sensitive defects. We detected and sequenced mutations that give rise to temperature-sensitive MutS, MutL, and MutH proteins. One mutation, mutS60, results in almost normal levels of spontaneous mutations at 37 degrees C but above this temperature gives rise to higher and higher levels of mutations, reaching the level of null mutations in mutS at 43 degrees C. However, at 37 degrees C the MutS60 protein can be much more easily titrated by mispairs than the wild-type MutS, as evidenced by the impaired ability to block homologous recombination in interspecies crosses and the increased levels of mutations from weak mutator alleles of mutD (dnaQ), mutC, and ndk. Strains with mutS60 can detect mispairs generated during replication that lead to mutation with much greater sensitivity than wild-type strains. The findings with ndk, lacking nucleotide diphosphate kinase, are striking. An ndk mutS60 strain yields four to five times the level of mutations seen in a full knockout of mutS. These results pose the question of whether similar altered Msh2 proteins result from presumed polymorphisms detected in tumor lines. The role of allele interactions in human disease susceptibility is discussed.

2-Aminopurine↗
PubMed1995

Sequential addition of temperature-sensitive missense mutations into the PB2 gene of influenza A transfectant viruses can effect an increase in temperature sensitivity and attenuation and permits the rational design of a genetically engineered live influenza A virus vaccine.

We have previously described a strategy for the recovery of a synthetic influenza A virus wild-type (wt) PB2 gene (derived from influenza A/Ann Arbor/6/60 [AA] virus) into an infectious virus. It was possible to introduce an attenuating temperature-sensitive (ts) mutation at amino acid residue 265 of the AA wt PB2 gene and to rescue this mutant gene into infectious virus. Application of this new technology to influenza A virus vaccine development requires that multiple attenuating mutations be introduced to achieve a satisfactorily attenuated virus that retains the attenuation (att) phenotype following replication in vivo. In this report, we demonstrate that putative ts mutations at amino acids 112, 556, and 658 each indeed specify the ts and att phenotypes. Each of these mutations was introduced into a cDNA copy of the AA mutant mt265 PB2 gene to produce three double-mutant PB2 genes, each of which was rescued into an infectious virus. In general, the double-mutant PB2 transfectant viruses were more ts and attenuated in the lower respiratory tracts of hamsters than the single-mutant transfectant viruses, and the ts phenotype of two of three double-mutant PB2 transfectant viruses was stable even after prolonged replication in the upper respiratory tracts of immunocompromised mice. Two triple-mutant PB2 transfectant viruses with three predicted amino acid substitutions resulting from five nucleotide substitutions in the cDNA were then generated. The triple-mutant PB2 transfectant viruses were more ts and more attenuated than the double-mutant PB2 transfectant viruses. These results indicate that sequential introduction of additional ts mutations into the PB2 gene can yield mutants that exhibit a stepwise increase in temperature sensitivity and attenuation compared with the preceding mutant(s) in the series. Furthermore, the level of temperature sensitivity of the transfectant viruses correlated significantly with the level of attenuation of these viruses in hamsters. Although the triple-mutant PB2 transfectant viruses were attenuated in hamsters, intranasal administration of these viruses elicited a vigorous serum hemagglutination-inhibiting antibody response, and this was associated with resistance of the lower respiratory tract to subsequent wt virus challenge. These observations suggest the feasibility of using PB2 reverse genetics to generate a live influenza A virus vaccine donor strain that contains three attenuating mutations in one gene. It is predicted that reassortant viruses derived from such a donor virus would have the properties of attenuation, genetic stability, immunogenicity, and protective efficacy against challenge with wt virus.

Animals↗
PubMed1983

Transfer of sensitivity to tumor promoters by transfection of DNA from sensitive into insensitive mouse JB6 epidermal cells.

Sensitivity to promotion of transformation by tumor promoters in mouse epidermal JB6 cells appears to have a genetic basis since the phenotypes of both promotable and nonpromotable JB6 cells derived from a common parent line are stable. Hybridization of promotable (P(+)) and nonpromotable (P(-)) cells previously indicated that promotability appears to behave as a dominant trait. These results suggest that it should be possible to find DNA sequences which specify sensitivity to promotion of anchorage independence by 12-o-tetradecanoyl-phorbol-13-acetate (TPA). Cellular DNA isolated from one of two P(+) lines, JB6 Cl 41 or JB6 Cl 22, was CaPO(4) precipitated and used to transfect the P(-) cell line JB6 Cl 30. At 7 days posttransfection, the cells were suspended in agar with or without TPA at 1.6 x 10(-8) M and assayed 10 days later for TPA-dependent colony formation. Untreated or Cl 30 DNA-treated P(-) JB6 Cl 30 cells yielded 40 to 50 colonies per 10(5) cells. In contrast, transfection of Cl 30 cells with "P(+) DNA" derived from either Cl 41 or Cl 22 yielded 200 to 500 TPA-induced colonies per 10(5) cells, or a five- to eightfold enhancement of promotability. The enhanced promotability obtained after transfection with P(+) DNA was stable, as judged by the retention of promotability for at least eight passages in cell lines derived from TPA-induced agar colonies. Other transfectants showed irreversible transformation by TPA, as observed in the parental P(+) lines. When NIH 3T3 cells instead of the putative preneoplastic JB6 Cl 30 cells were used as recipients for transfection of P(+) DNA, no evidence for acquisition of promotability was obtained. P(-) JB6 Cl 25, like Cl 30, also permitted expression of transfected P(+) DNA. These results suggest that sensitivity to phorbol ester promotion of transformation in JB6 cells is determined by DNA sequence(s) present in the P(+) DNA and requires recipient cells of the appropriate phenotype for expression.

Animals↗