Animal testing in infectiology. Introduction.
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Animal Testing Alternatives↗
Biomedical subjects
A Schmidt
Publications and source records attributed to A Schmidt.
Structural and mechanistic investigation of 3-deoxy-D-manno-octulosonate-8-phosphate synthase by solid-state REDOR NMR.
15N¿(31)P¿ REDOR NMR experiments were applied to lyophilized binary complexes of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS), with each of its natural substrates, phosphoenolpyruvate (PEP) and arabinose-5-phsophate (A5P), and with a mechanism-based inhibitor (K(i) = 0.4 microM), directly characterizing the active site basic residues involved in the binding of their carboxylate and phosphate moieties. KDO8PS was labeled uniformly with (15)N or [eta-(15)N(2)]Arg, and the ligands were selectively labeled with (13)C and (15)N. The NMR data established that PEP is bound by KDO8PS via a preserved set of structurally rigid and chemically unique Arg and Lys residues, with 5 A (upper limit) between epsilon-(15)N of this Lys and (31)P of PEP. A5P is bound in its cyclic forms to KDO8PS via a different set of Lys and Arg residues. The two sets arise from adjacent subsites that are capable of independent and sufficiently strong binding. The inhibitor is best characterized as an A5P-based substrate analogue inhibitor of KDO8PS. Five mutants in which highly conserved arginines were replaced with alanines were prepared and kinetically characterized. Our solid-state NMR observations complement the crystallographic structure of KDO8PS, and in combination with the mutagenesis results enable tentative assignment of the NMR-identified active site residues. Lys-138 and Arg-168 located at the most recessed part of the active site cavity are the chemically distinct and structurally rigid residues that bind PEP phosphate; R168A resulted in 0.1% of wild-type activity. Arg-63, exposed at the opening of the active site barrel, is the flexible residue with a generic chemical shift that binds A5P; R63A resulted in complete deactivation. The mechanistic implications of our results are discussed.
Aldehyde-Lyases↗
Time-of-day variations of indicators of attention: performance, physiologic parameters, and self-assessment of sleepiness.
BACKGROUND: A study was performed to analyze time-of-day variations of different indicators of attention and their interrelations. METHODS: After a sufficiently long all-night sleep 12 healthy non-sleep-deprived subjects ran through a test battery (Stanford Sleepiness Scale, Visual Analogue Scale, Critical Flicker Fusion Test [CFF], Visualization Test, Number Facility Test, Reaction Time, Pupillometry, and modified Multiple Sleep Latency Test) every 2 hours from 7:00 AM until 11:00 PM. Time-of-day variations were tested nonparametrically with Friedman's test for repeated measurements. Principal component factor analysis (of individually standardized values) was used to identify variable complexes with the same pattern of time-of-day variation. RESULTS: Statistically significant time-of-day variations were found for all variables, except for Fusion Frequency in CFF and Reaction Time. In factor analysis the physiologic parameters (pupillometric variables and sleep latencies) load on one factor, whereas the self-assessment scales, the Visualization Test, Number Faculty Test, and CFF load on the second factor. The variables that load primarily on factor 1 show peak levels of alertness immediately after getting up (at 7:00 AM) and again at 9:00 PM. Those variables that load primarily on factor 2 indicate a peak level of alertness around noon (11:00 AM-3:00 PM). CONCLUSIONS: Different aspects of attention follow different time-of-day variations. It is discussed, that these findings can be attributed to underlying circadian and homeostatic factors.
Adult↗
HEAT repeats mediate plasma membrane localization of Tor2p in yeast.
The subcellular distribution of Tor1p and Tor2p, two phosphatidylinositol kinase homologs and targets of the immunosuppressive drug rapamycin in Saccharomyces cerevisiae, was analyzed. We found that Tor protein is peripherally associated with membranes. Subcellular fractionation and immunofluorescence studies showed that Tor1p and Tor2p associate with the plasma membrane and a second fraction that is distinct from Golgi, vacuoles, mitochondria, and nucleus and may represent vesicular structures. Pulse-chase experiments showed that association of Tor protein with plasma membrane and the second compartment is fast, does not appear to involve components of endocytic, secretory, or Golgi to vacuole transport pathways, and is not affected by the immunosuppressive drug rapamycin. Deletion analysis reveals that two domains within Tor2p independently mediate localization to both compartments. These domains are composed of HEAT repeats that are thought to act as protein-protein interaction surfaces. Our studies therefore place Tor proteins at the site of action of their known downstream effectors and suggest that they may be part of a multiprotein complex.
Amino Acid Sequence↗
Oxylipin profiling reveals the preferential stimulation of the 9-lipoxygenase pathway in elicitor-treated potato cells.
Lipoxygenases are key enzymes in the synthesis of oxylipins and play an important role in the response of plants to wounding and pathogen attack. In cultured potato cells treated with elicitor from Phytophthora infestans, the causal agent of late blight disease, transcripts encoding a linoleate 9-lipoxygenase and a linoleate 13-lipoxygenase accumulate. However, lipoxygenase activity assays and oxylipin profiling revealed only increased 9-lipoxygenase activity and formation of products derived therefrom, such as 9-hydroxy octadecadienoic acid and colneleic acid. Furthermore, the 9-lipoxygenase products 9(S),10(S),11(R)-trihydroxy-12(Z)-octadecenoic and 9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoic acid were identified as novel, elicitor-inducible oxylipins in potato, suggesting a role of these compounds in the defense response against pathogen attack. Neither 13-lipoxygenase activity nor 13-lipoxygenase products were detected in higher amounts in potato cells after elicitation. Thus, formation of products by the 9-lipoxygenase pathway, including the enzymes hydroperoxide reductase, divinyl ether synthase, and epoxy alcohol synthase, is preferentially stimulated in cultured potato cells in response to treatment with P. infestans elicitor. Moreover, elicitor-induced accumulation of desaturase transcripts and increased phospholipase A(2) activity after elicitor treatment suggest that substrates for the lipoxygenase pathway might be provided by de novo synthesis and subsequent release from lipids of the endomembrane system.
Cells, Cultured↗
Cytotoxic activity of recombinant bFGF-rViscumin fusion proteins.
A fusion protein (bFGF-rMLA), containing the mitogen basic fibroblast growth factor (bFGF) and the cytotoxic component of rViscumin (recombinant mistletoe lectin), the enzymatic A-chain (rMLA), was expressed in Escherichia coli, purified, and functionally characterized. bFGF-rMLA is cytotoxic for mouse B16 melanoma cells expressing the FGF receptor with an IC(50) value of approximately 1 nM. rMLA shows no significant effect on the viability of the B16 cells up to a concentration of 141 nM. Additionally, bFGF-rMLA was associated with the rViscumin B-chain (rMLB) in an in vitro folding procedure. The IC(50) value of bFGF-rMLA/rMLB to B16 cells in the presence of lactose-to block rMLB lectin activity-was 134 pM. Thus, it was possible to enhance the efficacy of a rViscumin A-chain mitotoxin through addition of rMLB. We conclude that rViscumin fusion proteins may be generally applicable for the receptor-specific inactivation of target cells and point out their potential in drug development.
Animals↗
Suppression of maternal virus load with zidovudine, didanosine, and indinavir combination therapy prevents mother-to-fetus HIV transmission in macaques.
Recently, we developed a maternal-fetal macaque model using a highly pathogenic HIV-2 strain, HIV-2287, to study the time course of HIV transmission in utero. Most pregnant macaques (Macaca nemestrina) infected with HIV-2287 (10-103 infective doses) transmitted HIV to their fetuses, as verified by positive identification of virus-infected mononuclear cells and free viral RNA in fetal blood. To determine whether an antiretroviral drug combination therapy composed of two dideoxynucleosides, azidothymidine (15 mg/kg) and dideoxyinosine (15 mg/kg), and a protease inhibitor, indinavir (25 mg/kg), could completely inhibit mother-to-fetus HIV transmission, we administered these drugs orally through gastric catheters to five pregnant macaques infected with 10 infective doses of HIV-2287. Beginning 30 minutes after HIV inoculation, the dams were given the combination antiviral therapy three times daily until delivery by cesarean section. Drug treatment reduced the maternal virus load to a minimally detectable level but did not prevent primary HIV-2287 infection. All fetal and infant blood samples were virus negative by internally controlled RNA polymerase chain reaction (QC-RNA-PCR) and virus coculture assays. Fetal and infant CD4+ T-cell levels remained normal throughout the experiment. These findings strongly suggest that combination chemotherapy with azidothymidine, dideoxyinosine, and indinavir can suppress maternal viral load enough to prevent mother-to-fetus transmission of HIV.
Animals↗
Structures of three inhibitor complexes provide insight into the reaction mechanism of the human methylenetetrahydrofolate dehydrogenase/cyclohydrolase.
Enzymes involved in tetrahydrofolate metabolism are of particular pharmaceutical interest, as their function is crucial for amino acid and DNA biosynthesis. The crystal structure of the human cytosolic methylenetetrahydrofolate dehydrogenase/cyclohydrolase (DC301) domain of a trifunctional enzyme has been determined previously with a bound NADP cofactor. While the substrate binding site was identified to be localized in a deep and rather hydrophobic cleft at the interface between two protein domains, the unambiguous assignment of catalytic residues was not possible. We succeeded in determining the crystal structures of three ternary DC301/NADP/inhibitor complexes. Investigation of these structures followed by site-directed mutagenesis studies allowed identification of the amino acids involved in catalysis by both enzyme activities. The inhibitors bind close to Lys56 and Tyr52, residues of a strictly conserved motif for active sites in dehydrogenases. While Lys56 is in a good position for chemical interaction with the substrate analogue, Tyr52 was found stacking against the inhibitors' aromatic rings and hence seems to be more important for proper positioning of the ligand than for catalysis. Also, Ser49 and/or Cys147 were found to possibly act as an activator for water in the cyclohydrolase step. These and the other residues (Gln100 and Asp125), with which contacts are made, are strictly conserved in THF dehydrogenases. On the basis of structural and mutagenesis data, we propose a reaction mechanism for both activities, the dehydrogenase and the cyclohydrolase.
Amino Acid Sequence↗
[Calcaneus pseudarthrosis: a clinical rarity].
The calcaneus pseudoathrosis is rarely seen in clinical workday and we couldn't find any case be reported. In two cases the authors present the diagnosis and therapy of a non-union after a calcaneus fracture. The authors point especially at anatomical characteristics and biomechanical reasons and their meaning for aetiology of pseudoathrosis after a calcaneus fracture.
Adolescent↗
Rapid shift from virally infected cells to germinal center-retained virus after HIV-2 infection of macaques.
Lymphoid tissues are the primary target during the initial virus dissemination that occurs in HIV-1-infected individuals. Recent advances in antiretroviral therapy and techniques to monitor virus load in humans have demonstrated that the early stages of viral infection and host response are major determinants of the outcome of individual infections. Relatively little is known about immunopathogenic events occurring during the acute phase of HIV infection. We analyzed viral dissemination within lymphoid tissues by in situ hybridization and by combined immunohistochemistry/in situ hybridization during the acute infection phase (12 hours to 28 days) in pig-tailed macaques (Macaca nemestrina), challenged intravenously with a virulent strain of HIV-2, HIV-2(287). Two stages in viral dissemination were clearly evident within the first 28 days after HIV-2(287) infection. First, a massive increase in individual HIV-2-infected cells, mostly CD3+ T lymphocytes and a smaller percentage of macrophages and interdigitating dendritic cells, was identified within lymph nodes which peaked on the 10th day after HIV-2 infection. A shift of HIV-2 distribution was demonstrable between day 10 and day 14 after HIV-2 infection. Coincident with a marked reduction in individual HIV-2 RNA+ cells by day 14 postinfection, there was a dramatic increase in germinal center-associated HIV-2 RNA. High concentrations of HIV-2 RNA persisted in germinal centers in all animals by days 21 and 28 postinfection. Thus, HIV-2 appears to go through an initial, highly disseminated cellular phase followed by localization in the follicular dendritic cell network with relatively few infected cells. In this nonhuman primate model of HIV-associated immunopathogenesis, using a virus derived from a human pathogen, we identified a significant shift in the pattern of HIV-2 localization within a narrow time frame (day 10 to day 14). This shift in virus localization and behavior indicates that there may be a discrete but remarkably narrow window for therapeutic interventions that interrupt this stage in the natural course of HIV infection. Reproducibility and the accelerated time course of disease development make this model an excellent candidate for such intervention studies.
Animals↗
Upregulation of the interleukin-8 system in hypercholesterolemic patients. Does inhibition of the mevalonate pathway lower interleukin-8 levels in the vessel wall?
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Antibodies, Monoclonal↗
Differences in the effects of HMG-CoA reductase inhibitors on proliferation and viability of smooth muscle cells in culture.
We investigated the influence of lovastatin, simvastatin and pravastatin on proliferation and viability of vascular smooth muscle cells (SMC) in vitro and studied the effects of lovastatin on a mouse SMC line transgenic for a temperature-sensitive mutant of SV40 large T antigen (TAg), known to inhibit the function of p53 and pRb family members. We found that lovastatin and simvastatin inhibited cell proliferation by provoking G0/G1 phase arrest with concomitant depression of the proliferation antigen Ki-67/MIB-1. Lovastatin at high concentrations of 20 micromol/l caused cell death in the presence of serum but not under serum starved conditions, which was verified on the basis of increased DNA strand breaks, decreased DNA content and morphological alterations seen by electron microscopy. Cell death was also found for simvastatin, whereas pravastatin did not exhibit antiproliferative or cytotoxic effects. Mouse SMC transgenic for TAg did not show any impaired sensitivity to the antiproliferative and cell death inducing effect of lovastatin, but both effects could be antagonized by the supplementation of mevalonate. The data indicate that antiproliferative and cytotoxic effects of lovastatin are caused by the using up of products of mevalonate metabolism and do not require the presence of p53 or pRb.
Animals↗
Use of minimally modified antisense oligonucleotides for specific inhibition of gene expression.
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Animals↗
Lipids, lipid modification and lipid-protein interaction in membrane budding and fission--insights from the roles of endophilin A1 and synaptophysin in synaptic vesicle endocytosis.
Studies on the endocytosis of synaptic vesicles have provided two novel insights into the mechanism of vesicle formation from donor membranes, both of which concern lipids. One is the essential role of endophilin, a cytosolic protein converting lysophosphatidic acid by addition of the fatty acid arachidonate into phosphatidic acid. The other is the essential role of membrane cholesterol, which specifically interacts with synaptophysin, the major transmembrane protein of synaptic vesicles. These findings reveal novel modes of membrane lipid modification and lipid-protein interaction in vesicle biogenesis.
Adaptor Proteins, Signal Transducing↗
Parathyroid hormone and prostaglandin E2 preferentially increase luciferase levels in bone of mice harboring a luciferase transgene controlled by elements of the pro-alpha1(I) collagen promoter.
Type I collagen is the major extracellular protein in bone, tendons, ligaments, and skin. DNA elements of the mouse pro-alpha1 (I) collagen promoter were shown to drive the bone-selective expression of a luciferase transgene. We examined whether this expression can be used to evaluate the effect of anabolic agents on bone formation in vivo. Treatment of either intact males, intact females, or ovariectomized (ovx) mice with 80 microg/kg/day of human parathyroid hormone (hPTH), for 5 to 11 days increased luciferase levels in tibiae by two- to threefold compared with vehicle-treated mice. The increases were tissue specific, as no changes in skin luciferase expression were observed. Treatment with prostaglandin E2, a potent bone anabolic agent, for 11 days also increased expression of the transgene in bone, but not in skin. Treatment with dihydrotestosterone (DHT) for 11 days increased luciferase activity in skin, but not in bone. Histomorphometric analysis revealed that 28-day treatment with PTH increased bone formation; 60-day treatment of OVX mice with DHT did not. These findings show a correlation between bone formation and the expression of a transgene driven by DNA elements of the mouse pro-alpha1 (I) collagen promoter, suggesting that this expression can be used as an indicator and provide a faster readout for the ability of agents to stimulate bone formation in this mouse strain.
Animals↗
Enhanced replication of HIV-1 in vivo in pigtailed macaques (Macaca nemestrina).
Non-human primate models for acquired immunodeficiency syndrome (AIDS) are important for studies of prevention and intervention strategies. Ideally, such models would make use of human immunodeficiency virus type 1 (HIV-1) and animals that are readily available for research. HIV-1 was obtained from an infected macaque, and passaged sequentially in three groups of two Macaca nemestrina neonates each. Evidence for enhanced viral replication was first found in one of the group 2 animals, and in both group 3 animals. Observations that underlie this conclusion are sustained viral recovery from peripheral blood mononuclear cells (PBMCs), increased and accelerated production of antiviral antibodies, and the ability to detect plasma viral ribonucleic acid (RNA) months after infection. There was no evidence of CD4 depletion in any of the animals during the follow-up period. These data suggest that a useful non-human primate model for AIDS can be attained in pigtailed macaques (M. nemestrina).
Acquired Immunodeficiency Syndrome↗
Characterization of a sulfurtransferase from Arabidopsis thaliana.
A database search for similarities between sequenced parts of the Arabidopsis thaliana genome with known sulfurtransferase sequences from Escherichia coli and mammals was undertaken to obtain information about plant sulfurtransferase-like proteins. One gene and several homologous EST clones were identified. One of the EST clones was used for screening an Arabidopsis cDNA library. The isolated full-length clone consists of 1134 bp and encodes a 42.6 kDa protein that includes a putative transit peptide sequence of about 7.1 kDa. Sequence comparisons with known sulfurtransferases from different organisms confirmed high homology between them and the existence of several highly conserved regions. Results of a Southern blot performed with genomic Arabidopsis DNA showed the occurrence of at least two sulfurtransferase-like isozymes in Arabidopsis. Recombinant proteins with and without the putative transit peptide were expressed in E. coli with an N-terminal His6-tag, purified by affinity chromatography and tested for enzyme activity using different sulfur donors and acceptors. Both recombinant proteins catalyzed the formation of SCN- from thiosulfate and cyanide as a rhodanese per definition; however, both recombinant proteins preferred 3-mercaptopyruvate to thiosulfate. A monospecific antibody produced by using the mature recombinant protein as an antigen recognized a single protein band in total extracts of Arabidopsis plants equating to the full-length protein size. A single band equating to the size of the mature protein was detected from purified Arabidopsis mitochondria, but there was no antigenic reaction with any protein from chloroplasts. The function of the protein is still speculative. Now tools are available to elucidate the roles and substrates of this sulfurtransferase in higher plants.
Amino Acid Sequence↗
Characterization of two sulfurtransferase isozymes from Arabidopsis thaliana.
Sulfurtransferases transfer a sulfane atom from a donor substrate to a thiophilic acceptor molecule. Recently a sulfurtransferase specific for the substrate 3-mercaptopyruvate was isolated from Arabidopsis thaliana [Papenbrock, J. & Schmidt, A. (2000) Eur. J. Biochem. 267, 145-154]. In this study a second sulfurtransferase from Arabidopsis was characterized and compared to the enzyme described previously. Sequences of the mature proteins had an identity of 77.7%. The plant sulfurtransferases formed a distinct group within the known eukaryotic sulfurtransferases. When Southern blots were hybridized with labelled cDNA fragments from each of the plant sulfurtransferases the same pattern of bands was obtained indicating the existence of only these two closely related sulfurtransferases. The new sulfurtransferase was expressed in Escherichia coli fused with an N-terminal His6-tag, purified and tested for enzyme activity. Like the first enzyme, the newly isolated protein preferred 3-mercaptopyruvate to thiosulfate as substrate. The Km of both enzymes determined for 3-mercaptopyruvate and cyanide were almost identical. As a result of database searches it became obvious that sulfurtransferase proteins from higher plants showed high similarities to small senescence- and stress-induced proteins. To prove the involvement of sulfurtransferases in senescence-associated processes 3-mercaptopyruvate sulfurtransferase activity was determined in crude protein extracts from Arabidopsis plants of different ages. 3-mercaptopyruvate sulfurtransferase activity and steady-state RNA levels of sulfurtransferases increased with increasing age. However, steady-state protein levels as measured by using an antibody against the sulfurtransferase protein expressed previously decreased. Putative roles of sulfurtransferases in senescence-associated processes are discussed.
Amino Acid Sequence↗