Activity control of the ClpC adaptor McsB in Bacillus subtilis.

Controlled protein degradation is an important cellular reaction for the fast and efficient adaptation of bacteria to ever-changing environmental conditions. In the low-GC, Gram-positive model organism Bacillus subtilis, the AAA+ protein ClpC requires specific adaptor proteins not only for substrate recognition but also for chaperone activity. The McsB adaptor is activated particularly during heat stress, allowing the controlled degradation of the CtsR repressor by the ClpCP protease. Here we report how the McsB adaptor becomes activated by autophosphorylation on specific arginine residues during heat stress. In nonstressed cells McsB activity is inhibited by ClpC as well as YwlE.

Variability of non-clinical behavioral CNS safety assessment: An intercompany comparison.

INTRODUCTION: Irwin/FOB testing is routinely conducted to investigate the neurofunctional integrity of laboratory animals during preclinical development of new drugs, however, the study design frequently varies to meet specific needs. Representatives of several European-based pharmaceutical companies performed a "state-of-the-art" assessment of how they conduct their CNS safety evaluation using Irwin/FOB tests. METHODS: This assessment consisted of (1) a survey of current/historical practice, (2) an evaluation of historical studies with reference compounds (amphetamine, chlorpromazine) to determine intercompany reproducibility of results, and (3) an interlaboratory test using reference compounds (MK-801, chlorpromazine) to determine whether partially standardized conditions (animals, sex, doses, vehicles, administration route, observation time points, systemic exposure) might reduce variability of results. RESULTS: Our survey revealed several similarities, e.g., main endpoints of home cage and openfield observations, species, and positive control substances, but also a high level of heterogeneity between different companies with regard to behavioral endpoints during handling and reflex testing, scoring, group size, and timing of studies. Analysis of heterogeneously designed historical studies with amphetamine and chlorpromazine showed the anticipated behavioral changes, albeit with quantitative variability, and identified more robust (e.g., activity, posture, muscle tone, startle reflex, body temperature) and less robust (piloerection, stereotypical behavior, palpebral closure, respiration) Irwin/FOB parameters. A partially standardized interlaboratory test with MK-801 and chlorpromazine showed the expected behavioral changes and principally confirmed the historically-based more/less robust Irwin/FOB parameters, however, it also showed exposure variability and did not show a markedly reduced quantitative variability of behavioral results. DISCUSSION: Our survey and intercompany test results demonstrate certain heterogeneity in design and conduct of Irwin/FOB tests by pharmaceutical companies. Although the general behavioral profiles for the reference compounds were consistently found, quantitative variability of results remained even under partially standardized conditions. This suggests the importance of a high level of standardization with regard to the Irwin/FOB test modification used, scoring system, and observer training, in order to achieve an improved intercompany comparability of Irwin/FOB results.

Toxicological properties of metaflumizone.

Metaflumizone is a new insecticide developed for crop protection and urban pest control by BASF. Its mammalian toxicological profile was assessed by conducting multiple toxicity studies in the rat, mouse, and dog, covering all relevant endpoints. Metaflumizone is characterized by very low acute toxicity, is not irritating to the eye or the skin and does not possess a potential to induce skin sensitization. The substance also shows relatively low toxicity following subchronic oral or dermal exposure to mammals. In addition, metaflumizone demonstrates low toxicological potential following chronic oral exposure to rats, mice, and dogs. Overall, the lowest no observed adverse effect level (NOAEL) is 12mg/(kgday) from the 1-year chronic dog study. In a battery of in vitro and in vivo mutagenicity assays, the weight-of-the-evidence indicates a lack of potential genotoxicity for metaflumizone. Furthermore, the compound demonstrated a lack of potential oncogenicity in long-term toxicity studies in rats and mice. Results from the rat multi-generation reproductive toxicity study as well as the rat and rabbit developmental toxicity studies indicate that metaflumizone is not selectively toxic to the offspring or fetus, as compared to the parents. Also, metaflumizone is not teratogenic in the rat or rabbit. Lastly, no neurotoxicity could be detected in acute and subchronic neurotoxicity studies in rats.

Pilot study for comparison of reticulocyte-micronulei with lymphocyte-micronuclei in human biomonitoring.

Biomonitoring tries to determine the consequences for humans of exposures to environmental or pharmaceutical agents. Different end points have been employed to assess the burden of genomic damage. This is the first report comparing a recently introduced new end point, the reticulocyte-micronuclei analyzed by flow cytometry with the widely used lymphocyte-micronucleus assay, applied to two exposure scenarios leading to enhanced genomic damage. Radioiodine therapy was chosen to represent a short time exposure and hemodialysis treatment in end-stage renal failure was chosen to represent a chronic exposure. The results show that iodine radiation induced measurable genomic damage in the lymphocyte-micronucleus assay as well as in the reticulocyte-micronucleus test. Of two groups of patients under hemodialysis treatment, a reduced genomic damage was found with the lymphocyte-micronucleus test, but not with the reticulocyte-micronucleus test in the group undergoing daily hemodialysis, which removes uremic toxins more efficiently as compared to conventional hemodialysis, the treatment applied in the other group. The limited life-span of reticulocytes may make them less suitable for accumulation of chronic low level damage than lymphocytes. In conclusion, the lymphocyte-micronucleus test may be applicable to more exposure situations (including low chronic exposure), but the reticulocyte-micronucleus assay may be easier to perform in a clinical setting. The latter reflects a more rapid reduction of genomic damage after an acute exposure.

Quantitative investigation on the urinary excretion and metabolism of 3,4-dimethoxyphenylethylamine in schizophrenics and normal individuals.

A quantitative method for the detection of DMPEA in urine was developed. It is based on the fluorometric determination of DMPEA in the form of its phosphopyridoxyl derivate. The limit of detection is 2 microgram DMPEA per 1 g creatinine. The DMPEA content was measured in urine from healthy persons, from schizophrenics, and from psychiatric patients without schizophrenia hospitalized with the schizophrenics. From each person five to ten 24-hr urine samples were investigated. DMPEA could be found neither in schizophrenics nor in controls or healthy persons. Finally, the urinary excretion of parenterally applied 14C-DMPEA was determined in three healthy volunteers and in three rats. In man about 25% of the label was excreted as DMPEA. The main metabolite in urine was homoveratric acid. Both compounds were excreted as conjugates.

Increased histidine and histamine content in the brain of chronic uremic rats. Cause of enhanced cerebral cyclic adenosine monophosphate in uremia?

In rats with experimental chronic renal insufficiency (90% nephrectomy) the histidine content in brain was increased (+ 35%) in spite of normal plasma values and decreased concentrations in the striated muscle (-23%). The finding of a raised histidine level in the brain seems to be a uremia specific disorder, probably cuased by a local disturbance in histidine metabolism. In addition an increase of the histidine decarboxylation product histamine could be observed in the brain of rats with chronic renal insufficiency, as compared to pair-fed controls. This increase was directly related to the severity of azotemia. In the pathogenesis of the histamine alteration the increased histidine content in the brain of uremic rats must be considered, since the specific histidine decarboxylase is not saturated by the normal endogenous level of the amino acid precursor. Probably the increased histamine contributes to the raised cerebral cyclic AMP in the brain of uremic rats.

Isolation and quantification of class 1 MHC gene mutants in mouse T cells by immunoselection with a magnetic cell sorter (MACS).

A method is described for determining the frequency of cells with a mutation in an autosomal gene coding for a membrane protein. Using a monoclonal antibody to H-2Kk surface antigen and magnetic cell separation (MACS) more than 10(4)-fold enrichment of the H-2Kk negative population was achieved, as tested with artificial mixtures containing a known number of antigen-negative cells. After a second magnetic sorting mutant frequencies as low as 10(-6) could be measured. The number of clonogenic mutants was evaluated by limiting dilution cloning and verification of the mutant phenotype by FACScan (flow cytometry) analysis in a representative number of clones. The spontaneous frequency of H-2Kk deficient mutants was 0.80 x 10(-6), and this increased after irradiation with 6 Gy X rays to 3.38 x 10(-6) within the next 8 weeks. About 50 mutant clones were screened for the presence of other class 1 antigens on the cell surface by FACScan analysis. All mutants continued to express other class 1 antigens.

Quantification of thioguanine-resistant lymphocytes from mice irradiated in vivo.

Adult mice were Co-60 gamma irradiated, and 7 months later splenocytes were isolated, cultured in microwells, and the frequency of hprt-deficient mutants was determined by measuring the cloning efficiency in media with 6-thioguanine. The mutant frequency at 2, 4, and 6 Gy was 1.6 x 10(-5), 4.4 x 10(-5), and 12.7 x 10(-5), respectively. The frequency of spontaneous mutants was 2.5 x 10(-6). The effect of metabolic cooperation on the cloning efficiency of thioguanine-resistant T-cells in selective medium was evaluated in co-cultures with wild-type T-cells. We found that the growth of hprt-deficient T-cells is supported in the presence of thioguanine-inactivated wild-type splenocytes up to a cell density of 5 x 10(5) cells per well. When cell density was higher, cell growth was inhibited. Possibilities and limitations of cloned lymphocytes for the analysis of somatic mutations that occur in vivo are discussed.

An experiment in surgical education--the first international exchange of residents. The letters of Halsted, Küttner, Heuer, and Landois.

Dr William Halsted firmly believed that the young physician achieved greater surgical maturity by observing the practice of surgery in countries in addition to his own. To promote this belief, Halsted initiated the concept of exchanging residents between training programs in different lands. This article presents a review of that historic first international exchange of residents. This glimpse into the past is accomplished by presenting previously unpublished letters of Halsted; Hermann Küttner, director of a surgical clinic in Germany; George Heuer, a resident from The Johns Hopkins Hospital, Baltimore; and Felix Landois, a resident from Küttner's clinic.

Rapid alterations in cAMP accumulation in brain nuclei of rats following microinjections of vasoactive intestinal polypeptide (VIP) into the lateral ventricle.

The in vivo effect of exogenous vasoactive intestinal polypeptide (VIP) on the accumulation of cAMP in 21 microdissected brain nuclei was investigated 3 and 7 min after intraventricular injections in rats. VIP elicited significant (up to 20-fold) increases in cAMP levels. This effect is region specific varying considerably among the brain regions investigated. VIP dramatically increased the cAMP content of the lateral septal nucleus, several hypothalamic nuclei, the habenula, the midbrain central gray and the locus coeruleus. Smaller increases were observed elsewhere including some VIP-rich brain areas such as the cerebral cortex and the hippocampus.

Myelin basic protein in cerebrospinal fluid of patients with multiple sclerosis and other neurological diseases.

Cerebrospinal fluid (CSF) from 105 patients was analyzed by radioimmunoassay for the presence of material cross-reactive with peptide 89-169 of bovine myelin basic protein (BP). In a group of 72 multiple sclerosis patients, 52 showed higher BP content than the control group, i.e. more than 2 ng/ml CSF. Increased BP or BP fragments could be detected in CSF from almost all patients who recently (within 2 weeks) had had an acute episode, or after deterioration in the progressive form of the disease. Fifteen to 30 days after the onset of exacerbation or in a stable period, BP content decreases and in the slowly progressive form was in the range of the control group with one exception. BP content was also elevated in the CSF of patients with other neurological diseases. The presence of BP in the CSF from patients with isolated retrobulbar neuritis is of particular interest. Thus the presence of material cross-reactive with BP fragment 89-169 is not specific for multiple sclerosis, but is a useful parameter in diagnosis and evaluation of MS.

Inactivation and excretion of dopamine by the cat kidney in vivo.

14C-Dopamine at a dose between 0.16 and 400 nmol per kg body weight was injected locally into the renal artery and urinary excretion of the label was followed for a period of up to 75 min. During the first renal passage the injected kidney excreted 28.2+/-8.3% (n = 8) of the activity applied. As shown by column chromatography the 14C-activity in urine was mainly present as 3,4-dihydroxyphenyl acetic acid (40%), homovanillic acid (15%) and dopamine (app. 20%). Excretion rate and the pattern of dopamine metabolites in urine was independent of the administered dose. Thus, the excretion of dopamine by the cat kidney is linked to an inactivation by the kidney enzymes MAO and COMT. From the literature it is known that in dog and chicken kidney catecholamines are not metabolized to such a large extent during renal excretion.

Decreased cAMP content of the hypothalamus of genetically hypertensive rats.

In genetically hypertensive rats an altered catecholamine content of hypothalamic structures has been reported. In the present study cAMP was estimated in the hypothalamus and cortex of genetically hypertensive rats and compared with normotensive controls of the same strain. It is shown, that the cAMP content of the hypothalamus of the hypertensive animals was decreased to about 60% of control values, whereas there was no difference of the cAMP content in the cortical regions of the same animals. These results indicate an alteration of the adenyl cyclase-cAMP-phosphodiesterase system in hypothalamic structures of genetically hypertensive rats.

Influence of endogenous S-adenosylmethionine on the determination of catechol O-methyltransferase activity in red blood cells.

A double isotope method for the simultaneous determination of S-adenosylmethionine (SAM) and catechol O-methyltransferase (COMT) activity in red blood cells was developed. Healthy persons contained 7.9 +/- 1.9 nmol SAM per ml red blood cells and 1.9 to 3.8 nmol per ml plasma. Epinephrine increases the SAM biosynthesis rate in red blood cells. The possible influence of endogenous SAM on previous methods to determine COMT activity in red blood cells is discussed. The statement of Briggs and Briggs (Briggs, M.H. and Briggs, M. (1973) Experientia 29, 278--281) that COMT activity in red blood cells is lower during the last 3 months of pregnancy could not be verified by us.

Diagnostic meaning of the urinary output of Nepsilon-methylated lysines. Investigation of healthy individuals and patients with malignant diseases, myopathies or renal failure.

In the urine of 36 healthy persons the excretion of the three Nepsilon-methylated lysines and some other basic amino acids was determined. The following average values, related to 1 g creatinine, were found: Lys(Me) 16.2 mumol, Lys(Me2) 31.2 mumol, Lys(Me3) 40.5 mumol. The 24-hour excretion in 6 adults related to 1 kg body weight, had the following average values: Lys(Me) 0.37 mumol, Lys(Me2) 0.88 mumol, Lys(Me3) 0.92 mumol. In patients with degenerative or inflammatory myopathies (6 cases) as well as with generalized tumors (7 cases) urinary output of methyllysines was not significantly altered. In a patient with extremely impaired renal function, it was found that the plasma level and the excretion pattern of the methylated lysines were unequivocally altered. Metabolic stability and renal excretion of 3H-labelled l-Lys(Me3) were investigated in man. During a 24 hour period 65 per cent of Lys(Me3) was excreted into the urine unmetabolized after intravenous injection but not more than 20 per cent after oral administration.

Use of the recursion formula of the Gompertz function for the quantitation of PCR-amplified templates.

One common drawback of the currently used procedures to quantitate the polymerase chain reaction (PCR) is that the statistical evaluation of the experimental data depends on many, not just trivial, model assumptions. In the present study we report on an improvement in this crucial step of the quantitative PCR. The experimental design underlying the introduced method is exactly the same as in the case of the so-called PCR. However, by applying growth curve analysis based on the recursion formula of the Gompertz function the kinetics of the accumulation of the amplicon are estimated conjointly from data spanning both the and phases of the reaction. We demonstrate the method by determining the relative number of templates (a 206 bp segment spanning the exon 3 of the X-chromosomal murine Hprt-gene) contained in known orders of dilutions of DNA isolated from the spleen of the C57BL/6J-mouse. [32P]-dATP incorporation was used in duplicate experiments to quantify the amplicons as a function of amplification cycles. Our results: i) indicate that the accumulation of the PCR product as a function of PCR cycles follows a sigmoidal pattern compatible with the Gompertz growth model (P<0.0000001); ii) directly support the thesis that the kinetical pattern of accumulation of amplicons of a given DNA fragment does not depend on the number of corresponding DNA templates provided to the reaction; iii) permit a simple direct evaluation of the parallelity in the course of the accumulation of amplicons from different template numbers as a function of amplification cycles, which is a silent preposition in the evaluation of the so-called PCR; iv) allow an easy quantitation of the relative number of provided templates.

Mutant frequency at the H-2K class 1 and HPRT genes in T lymphocytes from the X-ray-exposed mouse.

The frequency of H-2Kk and HPRT-deficient T cells was measured in the H-2Kb, kDd,k genotype mouse 8-10 weeks after X-ray exposure at doses up to 6 Gy to compare the mutant frequency (MF) of an autosomal gene with that of an X-chromosomal gene. H-2K mutants were enriched by magnetic cell separation (MACS) using the H-2Kk-specific monoclonal antibody H100.5/28 and were isolated by limiting dilution cloning. Finally, the mutant phenotype was verified by flow cytometric analysis in a representative number of clones. The frequency of HPRT-deficient T cells rises from 2.5 x 10(-6) at 0 Gy to a maximum of 1.13 x 10(-4) at 4 Gy, and decreases to 2.9 x 10(-5) at 6 Gy. The H-2K- MF in the non-irradiated mouse was 8.4 x 10(-7). It increases with dose to a maximum of 8.1 x 10(-6) at 4 Gy and declines to 3.3 x 10(-6) at 6 Gy. The H-2K- MF measured depends on the monoclonal antibody used for the isolation of mutants. In a pilot study with another H-2Kk-specific monoclonal antibody (11.4.1), the spontaneous MF was four times higher than in experiments with the H100.5/28 monoclonal antibody. The expression of other class 1 antigens was investigated in H-2K- clones. The H-2Dd antigen had also disappeared in six of 41 clones from irradiated animals. This gene is situated at a distance of 1500 kb from the K-locus. The H-2Kb antigen was present in every investigated clone. In the discussion a model is presented that explains the shape of the dose-response curve of MF by selection against mutants in vivo systems under homeostasis. The results of the present investigation indicate that observed X-ray mutagenicity depends on many factors and that several genes have to be explored before reliable risk estimates are possible.

Improved determination of variant erythrocytes at the glycophorin A (GPA) locus and variant frequency in patients treated with radioiodine for thyroid cancer.

Red blood cells from individuals with the blood group MN express each form of the allelic GPA protein (GPAM and GPAN) on their cell surface. Variant cells have lost one form of the protein. Their frequency is about 10(-5) in blood from unexposed persons. The BR6 assay is currently the most widely used assay to determine variant frequency (VF) by immunolabelling and flow cytometry. The precision of the BR6 assay is mainly limited by the Poisson error because only small numbers of variant cells are detected in each assay. The BR6 assay has been improved by magnetic cell separation (MACS) of variant erythrocytes prior to their determination by this assay. This new version of the assay is named 'MACS-BR6'. It allows enumeration of variant cells from 2 x 10(8) or more blood cells instead of 5 x 10(6) in the BR6 assay with a precision which is about 5 times higher than that of the BR6 assay. The MACS-BR6 assay was used to determine the VF of GPAN/0 and GPAN/N variant cells in 12 healthy adults and 11 patients treated with radioiodine for thyroid cancer 2 to 16 years before. The average red bone marrow dose was 347 mGy. In healthy adults the mean VF of GPAN/0 and GPAN/N variant cells was 16.1 x 10(6) and 5.3 x 10(-6) respectively. In patients the corresponding mean VF was 25.4 x 10(6) and 11.9 x 10(-6), respectively. The patients GPAN/0 VF was significantly higher than that of controls. In patients VF increases linearly with the dose. The linear regression parameters of VF were 16.6 x 10(-6) (intercept), 23.7 x 10(-6) GY-1 (slope) and 6.3 x 10(-6) (intercept), 12.9 x 10(-6) Gy-1 (slope) for GPAN/0 and GPAN/N variant cells, respectively.

Dose and dose-rate dependence of the frequency of HPRT deficient T lymphocytes in the spleen of the 137Cs gamma-irradiated mouse.

The frequency of hypoxanthine phosphoribosyl transferase (HPRT) deficient splenic T lymphocytes was measured in the 137Cs gamma-irradiated mouse by the T cell cloning method. Doses from 0.3 to 6 Gy were applied at the dose-rates 0.5 Gy/min, 1 Gy/day and 1 Gy/week. Mutants were determined 8-10 and 30-40 weeks after the end of exposure. Radiation-induced mutant frequency (MFi) was calculated by subtracting the age corrected spontaneous mutant frequency (MFsp) from total mutant frequency (MF) found in irradiated animals. Data were fitted to linear and linear-quadratic dose-response models. MFi depended markedly on dose, dose-rate and time after exposure. When mutants were determined 8-10 weeks after acute irradiation (0.5 Gy/min) the dose-effect curve fitted the linear-quadratic equation MFi = 6.9 x 10(-6) Gy + 1.2 x 10(-6) Gy2, whereas in low dose-rate experiments (1 Gy/day, 1 Gy/week) the dose-effect curves were linear. The slope of the linear regression was about 3 x 10(-6). When low dose-rate-irradiated animals were killed 30-40 weeks after irradiation, MFi was about one-third of that observed after 8 weeks. The dose dose-rate effectiveness factor (DDREF) for radiation mutagenicity was calculated in animals that had been exposed 8-10 weeks previously. For doses < 2 Gy the reduction in effectiveness was about 1.5 when the irradiation dose-rate was < or = 1 Gy/day. For higher doses DDREF was 3-5.