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Biomedical subjects

A Schmidt

Publications and source records attributed to A Schmidt.

At least 73 records · Page 4Linked to original sources
PubMed2002

Biosensing of arteriosclerotic nanoplaque formation and interaction with an HMG-CoA reductase inhibitor.

Proteoheparan sulphate can be adsorbed to a methylated silica surface in a monomolecular layer via its transmembrane hydrophobic protein core domain. As a result of electrostatic repulsion, its anionic glycosaminoglycan side chains are stretched out into the blood substitute solution, thereby representing one receptor site for specific lipoprotein binding through basic amino acid-rich residues within their apolipoproteins. The binding process was studied by ellipsometric techniques suggesting that high-density lipoprotein (HDL) has a high binding affinity and a protective effect on interfacial heparan sulphate proteoglycan layers with respect to low-density lipoprotein (LDL) and Ca2+ complexation. Low-density lipoprotein was found to deposit strongly at the proteoheparan sulphate-coated surface, particularly in the presence of Ca2+, apparently through complex formation 'proteoglycan-LDL-calcium'. This ternary complex build-up may be interpreted as arteriosclerotic nanoplaque formation on the molecular level responsible for the arteriosclerotic primary lesion. On the other hand, HDL bound to heparan sulphate proteoglycan protected against LDL deposition and completely suppressed calcification of the proteoglycan-lipoprotein complex. In addition, HDL was able to decelerate the ternary complex deposition. Therefore, HDL attached to its proteoglycan receptor sites is thought to raise a multidomain barrier, selection and control motif for transmembrane and paracellular lipoprotein uptake into the arterial wall. Although much remains unclear regarding the mechanism of lipoprotein depositions at proteoglycan-coated surfaces, it seems clear that the use of such systems offers possibilities for investigating lipoprotein deposition at a 'nanoscopic' level under close to physiological conditions. In particular, Ca2+-promoted LDL deposition and the protective effect of HDL even at high Ca2+ and LDL concentrations agree well with previous clinical observations regarding risk and beneficial factors for early stages of atherosclerosis. Considering this, the system was tested on its reliability in a biosensor application in order to unveil possible acute pleiotropic effects of the lipid lowering drug fluvastatin. The very low-density lipoprotein (VLDL)/intermediate-density lipoprotein (IDL)/LDL plasma fraction from a high risk patient with dyslipoproteinaemia and type 2 diabetes mellitus showed beginning arteriosclerotic nanoplaque formation already at a normal blood Ca2+ concentration, with a strong increase at higher Ca2+ concentrations. Fluvastatin, whether applied to the patient (one single 80 mg slow release matrix tablet) or acutely in the experiment (2.2 micromol L-1), markedly slowed down this process of ternary aggregational nanoplaque complexation at all Ca2+ concentrations used. This action resulted without any significant change in lipid concentrations of the patient. Furthermore, after ternary complex build-up, fluvastatin, similar to HDL, was able to reduce nanoplaque adsorption and size. These immediate effects of fluvastatin have to be taken into consideration while interpreting the clinical outcome of long-term studies.

Adsorption↗
PubMed2002

Growth failure in a child showing characteristics of Seckel syndrome: possible effects of IGF-I and endogenous IGFBP-3.

Seckel syndrome is an autosomal-recessive disorder with a frequency of less than 1/10 000 births in which there are multiple malformations including severe short stature. We report on a patient with Seckel syndrome with a current body height of -7.5 SDS. Laboratory investigations at the age of 19 months revealed high levels of IGF-I, IGF-II and IGFBP-3. These data suggested the existence of IGF-I resistance possibly caused by impairment of the IGF-I receptor (IGF-IR) or altered IGFBPs. The purpose of this investigation was to examine whether the growth retardation in a Seckel syndrome patient is related to an alteration in the IGF system. Analysis of IGF-IR mRNA of patient's and control fibroblasts by solution hybridization/RNase protection assay did not show differences of IGF-IR transcript expression or size. Affinity crosslinking studies using [125I]-IGF-I showed normal-sized IGF-IR-ligand complexes. Mutation analysis of the complete coding regions of the IGF-I and IGF-IR genes showed no evidence of genetic alterations. Ligand blot analysis of IGFBPs secreted by the patient's fibroblasts showed stronger signals than control cells. Quantitative measurement of IGFBP-3 in cell-conditioned media was performed by radioimmunoassay (RIA) and revealed a sixfold increase when compared to control fibroblasts. We conclude that in this patient with Seckel syndrome and severe growth impairment IGF-I resistance is possibly related to altered production of IGFBP-3.

Abnormalities, Multiple↗
PubMed2002

Animal models of aspergillosis - also useful for vaccination strategies?

The attempt to establish vaccination strategies against infections caused by Aspergillus fumigatus seems to be questionable. Invasive aspergilloses are opportunistic diseases of the immunocompromised host and only a passive immunization with immunoglobulins could be taken into consideration. Until now there have been no preclinical and/or clinical data available concerning the efficacy of specific immunoglobulins; animal experiments could offer an approach for the preclinical assessment of this topic. Generally, A. fumigatus is an opportunistic pathogen. Birds show a relatively high susceptibility to infections caused by A. fumigatus. In laboratory animal species, rabbits seem to have the highest susceptibility followed by mice, rats and guinea-pigs. Mice are easy to handle in all-day laboratory use, and infections are mostly established by the intravenous, intranasal or intraperitoneal route. The main target organs of infection are the kidneys by all three infection routes. Forty clinical isolates of A. fumigatus tested showed a comparable virulence in systemic infections in the intravenously infected mouse model. By using histopathological techniques, we also observed infectious lesions within the central nervous system in all cases. Only A. fumigatus strains lacking green pigmentation showed a significantly lower virulence. Histopathological examinations are of great benefit in the study of these animal models as they give detailed information about the infectious process. Measuring colony-forming units in tissues is only of minor use in prediction as it cannot discriminate between infective tissue lesions and cavity-infections/persistence, e.g. in the kidney pelvis. Quantitative methods for measuring fungal organ burdens, e.g. by chitin-enzyme-linked immunosorbent assays, have also been described and offer an alternative towards solely measuring colony-forming units in tissues.

Animals↗
PubMed2002

Lumbar ependymoma presenting with paraplegia following attempted spinal anaesthesia.

Neurological deterioration from intraspinal haematoma following insertion of a spinal needle is extremely rare. We present the case of a 28-yr-old female, who presented with complete paraplegia following attempted spinal anaesthesia for delivery of her third child. Space-occupying iatrogenic spinal haemorrhage from a previously undiagnosed lumbar ependymoma was found to be the precipitating cause. Following laminotomy with blood clot and tumour removal her neurological function improved.

Adult↗
PubMed2002

A modified hepatitis B virus core particle containing multiple epitopes of the Plasmodium falciparum circumsporozoite protein provides a highly immunogenic malaria vaccine in preclinical analyses in rodent and primate hosts.

Despite extensive public health efforts, there are presently 200 to 400 million malaria infections and 1 to 2 million deaths each year due to the Plasmodium parasite. A prime target for malaria vaccine development is the circumsporozoite (CS) protein, which is expressed on the extracellular sporozoite and the intracellular hepatic stages of the parasite. Previous studies in rodent malaria models have shown that CS repeat B-cell epitopes expressed in a recombinant hepatitis B virus core (HBc) protein can elicit protective immunity. To design a vaccine for human use, a series of recombinant HBc proteins containing epitopes of Plasmodium falciparum CS protein were assayed for immunogenicity in mice [A. Birkett, B. Thornton, D. Milich, G. A. Oliveira, A. Siddique, R. Nussenzweig, J. M. Calvo-Calle, and E. H. Nardin, abstract from the 50th Annual Meeting of the American Society of Tropical Medicine and Hygiene 2001, Am. J. Trop. Med. Hyg. 65(Suppl. 3):258, 2001; D. R. Milich, J. Hughes, J. Jones, M. Sallberg, and T. R. Phillips, Vaccine 20:771-788, 2001]. The present paper summarizes preclinical analyses of the optimal P. falciparum HBc vaccine candidate, termed ICC-1132, which contains T- and B-cell epitopes from the repeat region and a universal T-cell epitope from the C terminus of the CS protein. The vaccine was highly immunogenic in mice and in Macaca fascicularis (cynomolgus) monkeys. When formulated in adjuvants suitable for human use, the vaccine elicited antisporozoite antibody titers that were logs higher than those obtained in previous studies. Human malaria-specific CD4(+)-T-cell clones and T cells of ICC-1132-immunized mice specifically recognized malaria T-cell epitopes contained in the vaccine. In addition to inducing strong malaria-specific immune responses in naïve hosts, ICC-1132 elicited potent anamnestic antibody responses in mice primed with P. falciparum sporozoites, suggesting potential efficacy in enhancing the sporozoite-primed immune responses of individuals living in areas where malaria is endemic.

Amino Acid Sequence↗
PubMed2002

Abnormal bone architecture and biomechanical properties with near-lifetime treatment of rats with PTH.

Skeletal effects are described for near-lifetime treatment of young, female rats with recombinant human PTH (1-34) (PTH). Rats (5-8 wk of age) were administered 0, 5, 30, or 75 microg/kg x d sc PTH for up to 2 yr, as part of an oncogenicity evaluation, which is required by regulatory agencies for potential chronic therapies. Proliferative lesions were observed in the skeleton as described in Vahle et al. (1 ); in this paper, we describe the quantitative bone data for this study. In the appendicular skeleton, PTH stimulated trabecular and endocortical mineral apposition to the near exclusion of marrow spaces at 5 microg/kg, with some periosteal apposition at 30 microg/kg, followed by considerable periosteal apposition and altered geometry at 75 microg/kg. Increased bone mass was observed for all treatment groups that substantially exceeded normal levels attained by vehicle controls and exceeded skeletal efficacy reported previously for similar doses in shorter-term studies. Dose-dependent increases in osteocalcin levels and a linear increase in wet weight of femora were observed for the entire treatment duration, suggesting nearly continuous PTH stimulation of osteoblasts and skeletal growth throughout life. Histology showed many osteocytes and prominent osteoblasts, but a conspicuous absence of osteoclasts. Morphometry showed a lack of distinction between trabecular and cortical bone. Biomechanics of vehicle controls showed that optimal mechanical integrity for the normal skeleton is observed at about 11 months of age. PTH greatly strengthened and stiffened vertebra and femora; however, the midshaft showed reduced toughness and increased brittleness with treatment, which was not the case for vertebra. Related studies of 6 and 9 months duration showed that the optimal duration for PTH skeletal efficacy was about 6 months in rats, based on toughness, strength, ultimate displacement, and architecture, especially for cortical bone. Therefore, treatment duration is an under appreciated aspect of PTH pharmacology; and PTH skeletal effects are a complex function of dose and duration. Comparative analyses showed that short-term treatment (6 months or less) is more advantageous than near-lifetime treatment, because PTH stimulates skeletal growth throughout life, resulting in abnormal architecture and untoward biomechanical properties in rats.

Absorptiometry, Photon↗
PubMed2002

A new experimental murine aspergillosis model to identify strains of Aspergillus fumigatus with reduced virulence.

Experimental animals are an obligate screen to investigate microorganism pathogenicity. Numerous animal models have been used to analyse the virulence of the opportunistic human pathogen Aspergillus fumigatus but none of the experimental models used previously have been satisfactory. This report discuss these models and presents a murine model of pulmonary aspergillosis that is very easy and the most adapted to compare the pathogenicity of A. fumigatus strains. Strains to be tested are inoculated intranasally and synchronously to mice and strains isolated from the lung of mice killed by the infection are typed. The number of colonies recovered is directly correlated to the virulence of the strain.

Animals↗
PubMed2001

Integration of information from both eyes by single neurons of nucleus rotundus, ectostriatum and lateral neostriatum in the zebra finch (Taeniopygia guttata castanotis Gould).

Although the optic nerve in birds crosses completely, visual information from the ipsilateral eye also reaches the ectostriatum, the telencephalic statibon of the tectofugal pathway, by recrossing fibers. These recrossing projections connect the contralateral tectum opticum with the ipsilateral nucleus rotundus, which in turn projects to the ectostriatum. The ectostriatum itself projects to the overlying lateral neostriatum, an area which serves an important role in sexual imprinting. This study shows that contralateral and ipsilateral information converges on single neurons within the nucleus rotundus, the ectostriatal region and the lateral neostriatum. In the three brain areas almost all isolated neurons exhibited responses to contralateral as well as bilateral visual stimuli. The number of neurons responding to ipsilateral stimuli increases from nucleus rotundus to the lateral neostriatum. We did not find any neurons driven exclusively by ipsilateral stimuli. The strength of ipsilateral responses is rather weak within the nucleus rotundus and ectostriatum, but shows a sharp increase in the lateral neostriatum. For most neurons of nucleus rotundus and ectostriatum, an additional ipsilateral stimulus did not significantly affect the response to a contralateral one. In contrast, a strong excitatory effect can be found in the neostriatum. The results are in agreement with previous evoked-potential studies and give new insights on the integration of ipsilateral and contralateral stimuli in zebra finch tectofugal visual pathway.

Action Potentials↗
PubMed2001

Influence of pressure in the first pumping stage on analyte desolvation and fragmentation in nano-ESI MS.

In ESI MS, some classes of biomolecules are detected only with low signal intensities due to difficulties in achieving efficient analyte desolvation, either because an analyte tends to fragment already at gentle desolvation conditions (i.e., noncovalent protein complexes or nucleotides) or because an analyte requires very strong activation in order to remove solvent molecules (i.e., carbohydrates). Even though the pressure in the first pumping stage of the ESI instrument is known to have an influence on the desolvation conditions, it has never been the focus of a detailed investigation. The role of the pressure in the first pumping stage is systematically interrogated in this study for several model substances. Ion signal intensities and signal-to-noise ratios are significiantly enhanced if the pressure in the first pumping stage is increased and adjusted, and analyte fragmentation can be substantially reduced. Thus, besides thermal heating and the acceleration in the nozzle-skimmer region, which are usually optimized, the pressure in the first pumping stage is an additional important desolvation parameter.

Concanavalin A↗
PubMed2001

Use of surface plasmon resonance for real-time analysis of the interaction of ZO-1 and occludin.

Surface plasmon resonance (SPR) spectroscopy was applied to study in real time, the interaction between the tight junction proteins ZO-1 and occludin. To imitate the morphology of tight junctions, a cytosolic tail of mouse occludin was immobilised at the sensor and guanylate kinase-like domain (Guk) was allowed to pass over the modified chip surface. The Guk domain of ZO-1 (residues 644-812) was found to bind to the cytoplasmic, carboxy-terminal region of occludin (residues 378-521). This interaction was systematically characterised with respect to the concentrations of both proteins and the binding conditions. Under the given experimental conditions, association and dissociation showed saturation kinetics, with affinity in micromolar range: k(a) = 4.14 +/- 0.52 x 10(3) M(-1) s(-1), k(d) = 3.04 +/- 0.38 x 10(-3) s(-1), K(D) = 639 +/- 51 nM. The results support the hypothesis that the Guk domain of ZO-1 is involved in the recruitment of the transmembrane protein occludin at tight junctions by interacting with the cytosolic carboxy-terminal sequence of occludin, located far from the cell membrane. We demonstrate the use of SPR spectroscopy as an effective approach for characterisation of the interactions of junction proteins.

Amino Acid Sequence↗
PubMed2001

Test of low-energy theorems for 1H(--> gamma, pi(0))(H)in the threshold hegion.

The photon asymmetry in the reaction 1H(--> gamma,pi(0))(1)H close to threshold has been measured for the first time with the photon spectrometer TAPS using linearly polarized photons from the tagged-photon facility at the Mainz Microtron MAMI. The total and differential cross sections were also measured simultaneously with the photon asymmetry. This allowed determination of the S-wave and all three P-wave amplitudes. The values obtained at threshold are E(0+) = [-1.33+/-0.08(stat)+/-0.03(syst)] x 10(-3)/m(pi(+)), P(1) = [9.47 +/- 0.08(stat) +/- 0.29(syst)] x 10(-3)q/m(2)(pi(+)), P(2) = [-9.46 +/- 0.1(stat) +/- 0.29(syst)] x 10(-3)q/m(2)(pi(+)), and P(3) = [11.48 +/- 0.06(stat) +/- 0.35(syst)] x 10(-3)q/m(2)(pi(+)).

Journal Article↗
PubMed2001

Identification of the cardiac ryanodine receptor channel in membrane blebs of sarcoplasmic reticulum.

Blebs of the sarcoplasmic reticulum (SR) membrane of heart muscle cells were generated after saponin perforation of the plasma membrane followed by complete hypercontraction of the cell. Although characteristic proteins of the plasma membrane, namely the beta1-adrenoreceptor and Galphai, were stained by monoclonal antibodies in the hypercontracted cells, these proteins could not be detected in the adjacent blebs. Monoclonal antibodies to the cardiac ryanodine receptor (RyR2), calsequestrin and SERCA2 bound at different amounts to surface components of the blebs and to components of the hypercontracted cells. From the immunofluorescence signals we conclude that the blebs are mainly constituted of corbular and junctional SR membrane, and only to a lesser extent of network SR membrane. Deconvolution microscopy revealed that the membrane location of RyR2, calsequestrin and SERCA2 in the bleb is comparable to native SR membrane. At the bleb membrane giga-ohm seals could be obtained and patches could be excised in a way that single-channel currents could be measured, although these are not completely identified.

Animals↗
PubMed2001

Systemic and intestinal immune responses to HIV-2287 infection in Macaca nemestrina.

Nonhuman primate models of human AIDS have been used successfully to evaluate candidate vaccines and infection intervention therapies. Successes of pathogenicity studies in primate models have been limited because of the varied infection outcomes and characteristic low number of study animals. The acutely pathogenic HIV-2(287)--Macaca nemestrina model has shown promise both in antiviral drug evaluation and in pathogenicity studies. Here we describe virus replication, spread, and host responses during the first 28 days of HIV-2(287) infection. Focusing on 18 macaques from a larger 27-macaque study, we report changing virus loads, CD4(+) cell depletions, and antibody responses both systemically and in the mucosa of the small intestine. After intravenous inoculation, blood and intestinal tissue were collected from pairs of macaques at 12 hr and 1, 2, 4, 6, 10, 14, 21, and 28 days postinfection. Specimens were examined for evidence of infection by quantitative cultures, in situ hybridization, lymphocyte subset monitoring, and antibody production. The data were presented serially as though all samples were collected from a single macaque. The highest blood virus loads were detected between days 10 and 14 and subsequently decreased through day 28. This coincided with a significant increase in ileum mucosa virus loads on day 10, which became undetectable by day 28. The lowest levels of CD4(+) cells were observed on days 21 and 28 in blood and ileum mucosa. CD4(+):CD8(+) cell ratios in blood and ileum dropped dramatically after day 10 to lowest levels by day 28. Intestinal virus loads were inversely correlated with CD4(+) cell and virus-specific antibody levels in the ileum after day 6. These results underscore the suitability of this model for pathogenicity studies as well as the importance of the intestinal lymphoid tissues as an initial site of virus replication and cell destruction during the acute, asymptomatic stage of AIDS development.

Animals↗
PubMed2001

Identification and characterization of a novel extracellular matrix protein nephronectin that is associated with integrin alpha8beta1 in the embryonic kidney.

The epithelial-mesenchymal interactions required for kidney organogenesis are disrupted in mice lacking the integrin alpha8beta1. None of this integrin's known ligands, however, appears to account for this phenotype. To identify a more relevant ligand, a soluble integrin alpha8beta1 heterodimer fused to alkaline phosphatase (AP) has been used to probe blots and cDNA libraries. In newborn mouse kidney extracts, alpha8beta1-AP detects a novel ligand of 70-90 kD. This protein, named nephronectin, is an extracellular matrix protein with five EGF-like repeats, a mucin region containing a RGD sequence, and a COOH-terminal MAM domain. Integrin alpha8beta1 and several additional RGD-binding integrins bind nephronectin. Nephronectin mRNA is expressed in the ureteric bud epithelium, whereas alpha8beta1 is expressed in the metanephric mesenchyme. Nephronectin is localized in the extracellular matrix in the same distribution as the ligand detected by alpha8beta1-AP and forms a complex with alpha8beta1 in vivo. Thus, these results strongly suggest that nephronectin is a relevant ligand mediating alpha8beta1 function in the kidney. Nephronectin is expressed at numerous sites outside the kidney, so it may also have wider roles in development. The approaches used here should be generally useful for characterizing the interactions of novel extracellular matrix proteins identified through genomic sequencing projects.

Alkaline Phosphatase↗
PubMed2001

Direct observation of a rho decay of the D13(1520) baryon resonance.

The reaction gammap-->pi(+)pi(0)n has been measured at MAMI for photon energies up to 820 MeV. Invariant mass spectra of the particles in the final state (pi(+)n), (pi(0)n), (pi(+)pi(0)) have been determined for several bins of incident photon energy. Differences in pi(+)pi(0) and simultaneously measured pi(0)pi(0) invariant mass distributions are assigned to a rho branch of the D13(1520) nucleon resonance.

Journal Article↗
PubMed2001

Three-dimensional structure of 2-amino-3-ketobutyrate CoA ligase from Escherichia coli complexed with a PLP-substrate intermediate: inferred reaction mechanism.

2-Amino-3-ketobutyrate CoA ligase (KBL, EC 2.3.1.29) is a pyridoxal phosphate (PLP) dependent enzyme, which catalyzes the second reaction step on the main metabolic degradation pathway for threonine. It acts in concert with threonine dehydrogenase and converts 2-amino-3-ketobutyrate, the product of threonine dehydrogenation by the latter enzyme, with the participation of cofactor CoA, to glycine and acetyl-CoA. The enzyme has been well conserved during evolution, with 54% amino acid sequence identity between the Escherichia coli and human enzymes. We present the three-dimensional structure of E. coli KBL determined at 2.0 A resolution. KBL belongs to the alpha family of PLP-dependent enzymes, for which the prototypic member is aspartate aminotransferase. Its closest structural homologue is E. coli 8-amino-7-oxononanoate synthase. Like many other members of the alpha family, the functional form of KBL is a dimer, and one such dimer is found in the asymmetric unit in the crystal. There are two active sites per dimer, located at the dimer interface. Both monomers contribute side chains to each active/substrate binding site. Electron density maps indicated the presence in the crystal of the Schiff base intermediate of 2-amino-3-ketobutyrate and PLP, an external aldimine, which remained bound to KBL throughout the protein purification procedure. The observed interactions between the aldimine and the side chains in the substrate binding site explain the specificity for the substrate and provide the basis for a detailed proposal of the reaction mechanism of KBL. A putative binding site of the CoA cofactor was assigned, and implications for the cooperation with threonine dehydrogenase were considered.

Acetyltransferases↗
PubMed2001

Hormone-induced conformational change of the purified soluble hormone binding domain of follitropin receptor complexed with single chain follitropin.

Human follicle-stimulating hormone receptor (hFSHR) belongs to family I of G protein-coupled receptors. FSHR extracellular domain (ECD) is predicted to have 8-9 alphabeta or leucine-rich repeat motif elements. The objective of this study was to identify elements of the FSHR ECD involved in ligand binding. Preincubation of recombinant hFSHR ECD with rabbit antisera raised against synthetic peptides of hFSHR ECD primary sequence abolished follitropin binding primarily in the region of amino acids 150-254. Accessibility of hFSHR ECD after hormone binding, captured by monoclonal antibodies against either ECD or FSH, was decreased for the region of amino acids 150-220 but additionally for amino acids 15-100. Thus, when hFSH bound first, accessibility of antibody binding was decreased to a much larger extent than if antibody was bound first. This suggestion of a conformational change upon binding was examined further. Circular dichroism spectra were recorded for purified single chain hFSH, hFSHR ECD, and hFSHR ECD-single chain hFSH complex. A spectral change indicated a small but consistent conformational change in the ECD.FSH complex after hormone binding. Taken together, these data demonstrate that FSH binding requires elements within the leucine-rich repeat motifs that form a central region of hFSHR ECD, and a conformational change occurs upon hormone binding.

Animals↗