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Biomedical subjects

M Rapp

Publications and source records attributed to M Rapp.

At least 37 records · Page 2Linked to original sources
PubMed1999

New immobilization method for SAW-biosensors: covalent attachment of antibodies via CNBr.

Surface acoustic wave devices (SAWs) based on horizontally polarized shear waves can be used as mass-sensitive immunosensors. If the standard material, aluminium, is used for the interdigital transducers (IDTs) of these devices a pretreatment with a shielding layer of polyimide is necessary to prevent corrosion processes. However, this layer also prevents an easy covalent attachment of the necessary receptive molecules. Therefore, we developed a new activation method of this inert polyimide surface by treating with cyanogenbromide using the established cyano-transfer-technique. With a subsequent covalent attachment of anti-glucose oxidase (anti-GOD) a very sensitive SAW-immunosensor for glucose oxidase (GOD) can be obtained.

Aluminum↗
PubMed1999

Study of implanted biomaterial functionality by diphosphonate molecules labeled with radioactive 99mTc.

An implanted biomaterial can be transformed into young bone after some months, but it has not necessary reached full biofunctionality. Mineral concentration kinetics and crystal-structure studies, still being carried out in our group, are completed here by biofunctionality determinations. A natural coral is implanted in vivo at the cortical level of the femoral diaphyoff++ in rabbits. Diphosphonates molecules labeled with radioactive 99mTc are then injected in rabbits and the fixation of the radioactivity is analyzed in several sites for 8 mo after the implantation. Nuclear instruments and methods are used for the measurements. Four successive cycles of osseous remodeling are determined before reaching a biofunctional phase.

Animals↗
PubMed1999

Focal transcriptional activity of murine cytomegalovirus during latency in the lungs.

Interstitial pneumonia is a frequent and critical manifestation of human cytomegalovirus (CMV) disease in immunocompromised patients, in particular in recipients of bone marrow transplantation. Previous work in the murine CMV infection model has identified the lungs as a major organ site of CMV latency and recurrence. It was open to question whether the viral genome is transcriptionally silent or active during latency. Transcription could be latency associated and thus be part of the latency phenotype. Alternatively, transcriptional activity could reflect episodes of reactivation. We demonstrate here that transcription of the immediate-early (IE) transcription unit ie1-ie3 selectively generates ie1-specific transcripts during latency. Notably, while the latent viral DNA was found to be evenly distributed in the lungs, transcription was focal and randomly distributed. This finding indicates that IE transcription is not a feature inherent to murine CMV latency but rather reflects foci of primordial reactivation. However, this reactivation did not initiate productive infection, since ie3 gene mRNA specifying the essential transactivator IE3 of murine CMV early gene expression was not detectable. Accordingly, transcripts encoding gB were absent during latency. By contrast, during induced virus recurrence, IE-phase transcription switched from focal to generalized and ie3-specific transcripts were generated. These data imply that latency and recurrence are regulated not only at the IE promoter-enhancer and that there exists an additional checkpoint at the level of precursor RNA splicing. We propose that focal transcription reflects random episodes of nonproductive reactivation that get terminated before IE3 is expressed and ignites the productive cycle.

Animals↗
PubMed1998

Covalent photolinker-mediated immobilization of an intermediate dextran layer to polymer-coated surfaces for biosensing applications.

A new method is presented for the covalent binding of dextran as an intermediate layer on surface acoustic wave (SAW) devices. For biosensing applications in aqueous media commercially available SAW devices require surface passivation to prevent corrosion of the aluminum device structures in electrolytes. Thin films of polyimide and parylene revealed exceptional passivation properties. They were used as a base for dextran immobilization. Covalent binding of dextran to polymer-coated surfaces was achieved by photoimmobilization. Aryldiazirine-functionalized bovine serum albumin served as the multifunctional light-activable linking agent (photolinker polymer). Dextran and photolinker polymer were mixed and photobonded to sensor surfaces. Essential photoimmobilization parameters were optimized. The binding of proteins to dextran applying carbodiimide chemistries was exemplified with antiurease antibodies and the feasibility of specific immunosensing was investigated on SAW sensors connected to a fluid handling system.

Acoustics↗
PubMed1998

Recombinant mouse-human chimeric antibodies as calibrators in immunoassays that measure antibodies to Toxoplasma gondii.

In the present study, we examined the feasibility of using recombinant antibodies containing murine variable regions and human constant regions as calibrators or controls in immunoassays. As a model system, we chose the Abbott IMx Toxo immunoglobulin M (IgM) and Toxo IgG assays designed to detect antibodies to Toxoplasma gondii. Two mouse monoclonal antibodies were selected based on their reactivity to the T. gondii antigens P30 and P66. Heavy- and light-chain variable-region genes were cloned from both hybridomas and transferred into immunoglobulin expression vectors containing human kappa and IgG1 or IgM constant regions. The constructs were stably transfected into Sp2/0-Ag14 cells. In the IMx Toxo IgG assay, immunoreactivity of the anti-P30 chimeric IgG1 antibody paralleled that of the positive human plasma-derived assay calibrators. Signal generated with the anti-P66 chimeric IgG1 antibody was observed to plateau below the maximal reactivity observed for the assay calibrator. Examination of the IgM chimeric antibodies in the IMx Toxo IgM assay revealed that both the anti-P30 and anti-P66 antibodies matched the assay index calibrator manufactured with human Toxo IgM-positive plasma. When evaluated with patient samples, the correlation between results obtained with the chimeric antibody calibrators and the positive human plasma calibrators was > or =0.985. These data demonstrate that chimeric mouse-human antibodies are a viable alternative to high-titer positive human plasma for the manufacture of calibrators and controls for diagnostic assays.

Amino Acid Sequence↗
PubMed1998

Disposition and metabolism of a novel antineoplastic agent, 4-tert-butyl-[3-(2-chloroethyl)ureido]benzene, in mice.

1-Aryl-3-(2-chloroethyl)ureas are new agents that have shown promising cytotoxic and antineoplastic activities. In this work, we studied the disposition and metabolism of one of these molecules, 4-tert-butyl-[3-(2-chloroethyl)ureido]benzene (tBCEU). tBCEU was labeled with 14C and 13C in the urea function and in the chloroethyl moiety. After ip administration of the molecule labeled in the urea function, radioactivity was widely distributed in the whole organism, including the brain. HPLC analysis of plasma showed that tBCEU was extensively metabolized, with <20% being found in the plasma as unchanged tBCEU 1 hr after administration. One main metabolite was identified by NMR and MS analysis as N-[4-(2-hydroxy-1, 1-dimethylethyl)phenyl]urea, widely conjugated to glucuronic acid. The same metabolite was found in the urine. After administration of tBCEU labeled in the chloroethyl moiety, the same tissue affinities were observed, but the decrease of total radioactivity in blood and tissues was slower than that observed for the molecule labeled in the urea function. HPLC analysis of urine showed the presence of two main metabolites, identified by MS as thiodiacetic acid and its sulfoxide. From these results, we can deduce that the metabolic pathway of tBCEU involves N-dealkylation of the urea portion of the molecule and hydroxylation of the tert-butyl group. The strong cytochrome P450 reactivity of the carbon adjacent to the urea portion of tBCEU is probably related to particular sensitivity to oxidation at this position, based on the chemical structure of tBCEU. These results can explain the fact that the cytotoxic effect of tBCEU is not due to DNA alkylation, in contrast to that of its parent molecule, chloroethylnitrosourea.

Animals↗
PubMed1997

Enhancement by O6-benzyl-N2-acetylguanosine of N'-[2-chloroethyl]-N-[2-(methylsulphonyl)ethyl]-N'-nitrosourea therapeutic index on nude mice bearing resistant human melanoma.

The exposure of cells to O6-benzyl-N2-acetylguanosine (BNAG) and several guanine derivatives is known to reduce the activity of O6-alkylguanine-DNA alkyltransferase (MGMT) and to enhance the sensitivity of Mer+ (methyl enzyme repair positive) tumour cells to chloroethylnitrosoureas (CENUs) in vitro and in vivo. High water solubility and the pharmacokinetic properties of BNAG make it a candidate for simultaneous administration with CENUs by the i.v. route in human clinical use. In vivo we have shown previously that BNAG significantly increases the efficiency of N'-[2-chloroethyl]-N-[2-(methylsulphonyl)ethyl]-N'-nitrosourea (cystemustine) against M4Beu melanoma cells (Mer+) through its cytostatic activity by the i.p. route, but also increases its toxicity. To investigate the toxicity of BNAG and cystemustine when administered simultaneously in mice, we compared the maximum tolerated dose and LD50 doses of cystemustine alone or in combination with 40 mg kg(-1) BNAG by the i.p. route. The toxicity of cystemustine was enhanced by a factor of almost 1.44 when combined with BNAG. To compare the therapeutic index of cystemustine alone and the cystemustine/BNAG combination, pharmacological tests were carried out in nude mice bearing Mer+ M4Beu human melanoma cells. Isotoxic doses were calculated using the 1.44 ratio. The treatments were administered three times by the i.v. route on days 1, 5 and 9 after s.c. inoculation of tumour cells. Although the toxicities of the treatments were equal, BNAG strongly enhanced tumour growth inhibition. These results demonstrate the increase of the therapeutic index of cystemustine by BNAG and justify the use of BNAG to enhance nitrosourea efficiency in vivo by i.v. co-injection.

Animals↗
PubMed1997

Tissue distribution and metabolism in rat of an ethynesulphonamide with filaricidal activity.

1. The tissue distribution and metabolism of a new filaricidal agent P903 (N-[(2-phenylethynyl)sulfonyl]morpholine) were studied in rat. 2. After s.c. administration of 14C and 13C P903, the Tmax in the blood was observed on day 2. Elimination was slow and > 95% was bound to protein. Radioactivity was distributed in the whole organism but particularly in erythrocytes and the lymphatic channel. Four days later, > 60% of the radioactivity was excreted in urine and faeces at equal amounts and 15% remained at the injection point. 3. In all biological fluids tested no P903 was found but only its metabolites. 4. One principal metabolite, the N-[(2-phenyloxo-2-ethane) sulphonyl] morpholine or oxosulphonamide was identified in blood, urine and faeces as compared with the reference compound by GC/MS and NMR. This latter molecule was detected following hydrolysis by hydrochloric acid but not with beta glucuronidase/sulphatase. 5. Unconjugated and conjugated oxosulphonamide represented > 85% of the radioactivity at all times tested in blood but only 38 and 35% respectively of urinary and faecal radioactivity on day 1 after the administration of the labelled drug. 6. Thus, P903 is rapidly converted to a reactive metabolite, probably an oxirene, which is then conjugated with endogenous components to form conjugated oxosulphonamide and an unknown metabolite. The role of this reactive metabolite in antifilarial activity seems to be very important in understanding the mechanism of action of P903.

Animals↗
PubMed1997

Disposition and metabolism of 2,6-dimethylbenzamide N-(5-methyl-3-isoxazolyl) (D2916) in male and female rats.

Disposition and metabolism of the new anticonvulsant 2,6-dimethylbenzamide N-(5-methyl-3-isoxazolyl) (D2916) was studied in male and female rats after oral administration of 14C-labeled material. D2916 was well absorbed in both sexes and distributed to all tissues, with maximal drug concentrations found in elimination and metabolization organs, as well as in fatty tissues. Striking differences in pharmacokinetic parameters of total radioactivity were observed between males and females; females had higher brain concentrations and longer blood and tissue half-lives. The study of blood, bile, urine, and brain metabolites showed that D2916 follows two degradation pathways related to hydroxylation of methyl groups. Males prefer to hydroxylate one of the methyl groups of the phenyl ring, and females prefer to hydroxylate the methyl of the isoxazolyl ring forming the active metabolite D3187. These findings suggest a sex difference in the location of the hydroxylation of the D2916 molecule and can explain the longer anticonvulsant effect observed in the female rat that is related both to an orientation of the metabolism toward the formation of the active metabolite and to a better ability to this metabolite to cross the blood-brain barrier, compared with the unchanged drug.

Animals↗
PubMed1996

Structure-activity relationships in a series of substituted indolocarbazoles: topoisomerase I and protein kinase C inhibition and antitumoral and antimicrobial properties.

A series of compounds structurally related to staurosporine, rebeccamycin, and corresponding aglycones was synthesized, and their activities toward protein kinase C and topoisomerases I and II were tested together with their in vitro antitumor efficiency against murine B16 melanoma and P388 leukemia cells. Their antimicrobial activities were also examined against a Gram-negative bacterium (Escherichia coli), a yeast (Candida albicans), and three Gram-positive bacteria (Bacillus cereus, Streptomyces chartreusis, and Streptomyces griseus). To avoid side effects expected with protein kinase C inhibitors, we introduced substitution on the maleimide nitrogen and/or a sugar moiety linked to one of the indole nitrogens to obtain specific inhibitors of topoisomerase I with minimal activities on protein kinase C. As expected, these structures were inefficient on topoisomerase II, and some of them exhibited a strong activity against topoisomerase I. Generally, dechlorinated compounds were found to be more active than chlorinated analogues against both purified topoisomerase I and protein kinase C. On the other hand, opposite results were obtained in the cell antiproliferative assays. These results suggest lack of cell membrane permeability in the absence of the chlorine residue or cleavage of carbon-chlorine bonds inside the cell.

Animals↗
PubMed1996

Modeling back propagating action potential in weakly excitable dendrites of neocortical pyramidal cells.

Simultaneous recordings from the soma and apical dendrite of layer V neocortical pyramidal cells of young rats show that, for any location of current input, an evoked action potential (AP) always starts at the axon and then propagates actively, but decrementally, backward into the dendrites. This back-propagating AP is supported by a low density (-gNa = approximately 4 mS/cm2) of rapidly inactivating voltage-dependent Na+ channels in the soma and the apical dendrite. Investigation of detailed, biophysically constrained, models of reconstructed pyramidal cells shows the following. (i) The initiation of the AP first in the axon cannot be explained solely by morphological considerations; the axon must be more excitable than the soma and dendrites. (ii) The minimal Na+ channel density in the axon that fully accounts for the experimental results is about 20-times that of the soma. If -gNa in the axon hillock and initial segment is the same as in the soma [as recently suggested by Colbert and Johnston [Colbert, C. M. & Johnston, D. (1995) Soc. Neurosci. Abstr. 21, 684.2]], then -gNa in the more distal axonal regions is required to be about 40-times that of the soma. (iii) A backward propagating AP in weakly excitable dendrites can be modulated in a graded manner by background synaptic activity. The functional role of weakly excitable dendrites and a more excitable axon for forward synaptic integration and for backward, global, communication between the axon and the dendrites is discussed.

Animals↗
PubMed1996

Effects of fluoxetine and olfactory bulbectomy on mouse circadian activity rhythms.

Olfactory bulbectomy (OBX) in SWR outbred male mice lengthened the free-running period and delayed the phase of a circadian rhythm for wheel-running activity. OBX also increased mean levels of activity. Two weeks of daily intraperitoneal injections of Fluoxetine (8 mg/kg), a serotonin re-uptake inhibitor, reversed the effects of bulbectomy on the mean level of activity and significantly shortened the free-running period of the activity rhythm. The phase of the activity rhythm was not significantly affected by the Fluoxetine treatment. These results are consistent with a hyposerotonergic mediation of the effects of OBX on circadian period and activity level.

Animals↗
PubMed1996

Circadian variation in O6-methylguanine-DNA methyltransferase activity in mouse liver.

Bifunctional chloroethylating cytostatic agents produce lethal DNA lesions, as a result of the formation of O6-alkylguanines. These lesions can be repaired by O6-methylguanine-DNA methyltransferase (MGMT). This ubiquitous nuclear and cytosolic enzyme removes the alkyl group by accepting it to the cysteine residue of its active site, thus preventing the formation of DNA interstrand cross-links. The role of the circadian organization in cellular protection against such DNA insults was examined in male B6D2F1 mice, synchronized with an alternation of 12 h of light and 12 h of darkness (LD12:12). MGMT activity was determined in liver of mice obtained at eight different circadian times, located 3 h apart. MGMT activity varied 5-fold along the 24 h time-scale, from 7 +/- 1 pmol/g of tissue at 7 h after light onset (HALO), during the rest span, up to 32 +/- 9 pmol/g at 19 HALO (second mid to late activity span). This large amplitude circadian rhythm in MGMT activity may be an important determinant of the susceptibility rhythms to alkylating agents. The greatest DNA repair activity occurred at night when mice were active, eat and drink, and thus are at a higher risk of being exposed to chemical insults.

Animals↗
PubMed1996

Syntheses and antimicrobial activities of five-membered ring heterocycles coupled to indole moieties.

Indole-substituted oxazolidinones, oxazolones, pyrrolidinone, imidazolidinone and imidazolones were synthesized. Their inhibitory potencies towards protein kinase C and protein kinase A were determined and their in vitro activities against Streptomyces chartreusis, Streptomyces griseus, Bacillus cereus, Candida albicans and Escherichia coli were examined. The inhibition of Streptomyces sporulation observed for some of them could not be linked to in vitro protein kinase C inhibition. All proved inactive against C. albicans but three of them exhibited a marked activity towards E. coli. This effect extends to other Gram-negative bacteria.

Alkaloids↗
PubMed1995

Characterization of a conserved gene block in the murine cytomegalovirus genome.

The nucleotide sequence between the lytic origin and the gH gene of the murine cytomegalovirus genome (map units 0.416-0.455) was determined. Five of the 15 open reading frames identified encode polypeptides, which exhibit significant homology to polypeptides of human cytomegalovirus (HCMV) (UL69, UL71, and UL73) and to polypeptides of several other herpesviruses (dUTPase and helicase/primase). The location in the genome of the five open reading frames and their direction of transcription is perfectly conserved between murine cytomegalovirus (MCMV) and HCMV. These data suggest that MCMV and HCMV have a highly related genome organization. Thus, MCMV offers a good model for molecular and functional analysis of cytomegalovirus genes in vitro and in vivo.

Amino Acid Sequence↗
PubMed1995

Antimicrobial activities of indolocarbazole and bis-indole protein kinase C inhibitors. II. Substitution on maleimide nitrogen with functional groups bearing a labile hydrogen.

New compounds, structurally related to the potent protein kinase C inhibitor staurosporine, and substituted on the imide nitrogen with a functional group bearing a labile hydrogen (hydroxymethyl, amino, hydroxy), were synthesized. Their in vitro inhibitory potencies towards protein kinase C and protein kinase A showed that N-hydroxymethyl and N-hydroxy substitution, unlike alkyl substitution, can provide efficient protein kinase C inhibitors. The antimicrobial activities of these new compounds against Streptomyces chartreusis and Streptomyces griseus, Bacillus cereus, Escherichia coli, Candida albicans and Botrytis cinerea were examined. They proved to be inactive against E. coli and two fungi. The results suggest that there is no link between in vitro inhibition of protein kinase C and inhibition of growth and sporulation of the two Streptomyces tested. Unlike indolocarbazole maleimides, bis-indole maleimides are active against the two Streptomyces species.

Carbazoles↗