Biomedical subjects

B Dittrich

Publications and source records attributed to B Dittrich.

At least 19 recordsLinked to original sources

PubMed2006

Fed-batch mode in shake flasks by slow-release technique.

Most industrial production processes are performed in fed-batch operational mode. In contrast, the screenings for microbial production strains are run in batch mode which results in completely different physiological conditions than relevant for production conditions. This may lead to wrong selections of strains. Silicone elastomer discs containing glucose crystals were developed to realize fed-batch fermentation in shake flasks. No other device for feeding was required. Glucose was fed in this way to Hansenula polymorpha cultures controlled by diffusion. Two strains of H. polymorpha were investigated in shake flasks: the wild-type strain (DSM 70277) and a recombinant strain pC10-FMD (P(FMD)-GFP). The oxygen transfer rate (OTR) and respiratory quotient (RQ) of the cultures were monitored online in shake flasks with a Respiration Activity Monitoring System (RAMOS). Formation of biomass and green fluorescent protein (GFP), pH-drift and the metabolite dynamics of glucose, ethanol and acetic acid were measured offline. With the slow-release technique overflow metabolism could be reduced leading to an increase of 85% in biomass yield. To date, 23.4 g/L cell dry weight of H. polymorpha could be achieved in shake flask. Biomass yields of 0.38-0.47 were obtained which are in the same magnitude of laboratory scale fermentors equipped with a substrate feed pump. GFP yield could be increased by a factor of 35 in Syn6-MES mineral medium. In fed-batch mode 88 mg/L GFP was synthesized with 35.9 g/L fed glucose. In contrast, only 2.5 mg/L with 40 g/L metabolized glucose was revealed in batch mode. In YNB mineral medium over 420-fold improvement in fed-batch mode was achieved with 421 mg/L GFP at 41.3 g/L fed glucose in comparison to less than 1 mg/L in batch mode with 40 g/L glucose.

Biomass↗

PubMed2006

Introduction and validation of an invariom database for amino-acid, peptide and protein molecules.

A database of invarioms for structural refinement of amino-acid, oligopeptide and protein molecules is presented. The spherical scattering factors of the independent atom or promolecule model are replaced by ;individual' aspherical scattering factors that take into account the chemical environment of a bonded atom. All amino acids were analysed in terms of their invariom fragments. In order to generate 73 database entries that cover this class of compounds, 37 model compounds were geometry-optimized and theoretical structure factors were calculated. Multipole refinements were then performed on these theoretical structure factors to yield the invariom database. Validation of this database on an extensive number of experimental small-molecule crystal structures of varying quality and resolution shows that invariom modelling improves various figures of merit. Differences in figures of merit between invariom and promolecule models give insight into the importance of disorder for future protein-invariom refinements. The suitability of structural data for application of invarioms can be predicted by Cruickshank's diffraction-component precision index [Cruickshank (1999), Acta Cryst. D55, 583-601].

Amino Acids↗

PubMed2006

Invarioms for improved absolute structure determination of light-atom crystal structures.

The determination of molecular absolute configuration from an X-ray analysis for structures that contain only light elements is challenging owing to the weak anomalous dispersion signal. The achievable precision of the Flack x parameter for such structures is therefore limited, especially when the independent-atom model is employed. Invariom modelling can improve this situation. Invarioms are theoretically predicted pseudoatoms within the Hansen & Coppens multipole formalism. They are transferable from one molecule to another and provide generalized aspherical atomic form factors. It is shown that, by application of the invariom approach, the precision and standard uncertainty of the Flack x parameter and therefore the reliability of deducing molecular chirality in an absolute structure determination can be improved.

Crystallography, X-Ray↗

PubMed2006

Atomic and bond topological properties of the tripeptide L-alanyl-L-alanyl-L-alanine based on its experimental charge density obtained at 20 K.

A 20 K high resolution X-ray data set of L-Ala-L-Ala-L-Ala*1/2 H2O was measured using an ultra-low temperature laboratory setup, that combines area detection and a closed cycle helium cryostat. The charge density determination includes integration of atomic basins and topological analysis according to Bader's quantum theory of atoms in molecules. Two tripeptide units are found in the asymmetric unit, allowing the assessment of transferability of bond topological and atomic properties taking also into consideration previous data of oligopeptides. With respect to invariom modeling the limits of such transferability are investigated and the results of this study show the validity of the nearest/next-nearest neighbour approximation and support the use of database approaches for electron density modeling of macromolecules.

Crystallography, X-Ray↗

PubMed2005

The invariom model and its application: refinement of D,L-serine at different temperatures and resolution.

Three X-ray data sets of the same D,L-serine crystal were measured at temperatures of 298, 100 and 20 K. These data were then evaluated using invarioms and the Hansen & Coppens aspherical-atom model. Multipole populations for invarioms, which are pseudoatoms that remain approximately invariant in an intermolecular transfer, were theoretically predicted using different density functional theorem (DFT) basis sets. The invariom parameters were kept fixed and positional and thermal parameters were refined to compare the fitting against the multi-temperature data at different resolutions. The deconvolution of thermal motion and electron density with respect to data resolution was studied by application of the Hirshfeld test. Above a resolution of sin theta/lambda approximately 0.55 A-1, or d approximately 0.9 A, this test was fulfilled. When the Hirshfeld test is fulfilled, a successful modeling of the aspherical electron density with invarioms is achieved, which was proven by Fourier methods. Molecular geometry improves, especially for H atoms, when using the invariom method compared to the independent-atom model, as a comparison with neutron data shows. Based on this example, the general applicability of the invariom concept to organic molecules is proven and the aspherical density modeling of a larger biomacromolecule is within reach.

Crystallography, X-Ray↗

PubMed2005

Oxidative stress related markers in the "VITA" and the centenarian projects.

Oxidative stress seems to play an important role in the pathophysiology of Alzheimer's disease (AD). At present there are no easily accessible biochemical markers for AD. We performed activity assays for platelet MAO-B and erythrocyte Cu/Zn-SOD as well as Western blotting for these two proteins. Moreover, we assessed plasma lactoferrin and performed RFLP-analysis for the MAO-B-intron-13-polymorphism in patients from the Vienna-Transdanube Aging (VITA) and from the so called centenarian project. The first one, VITA, is a community-based cohort study of all 75 years old inhabitants of a geographical region of Vienna. The centenarian project investigates chronic care in-old patients suffering from AD. In both sexes platelet MAO-B activity increased significantly in the AD group, and Cu/Zn-SOD activity decreased, but the latter effect was significant only in females. No significant difference was found regarding plasma lactoferrin. No correlation was found between MAO-Bi13 and MAO-B platelet activity or allele MAO-Bi13 and disease frequency. These results point to the possibility that a combination of MAO-B and SOD activity levels might be useful tools for an early diagnosis of AD.

Aged↗

PubMed2004

Atomic volumes and charges in a system with a strong hydrogen bond: L-tryptophan formic acid.

A high-resolution X-ray diffraction data set was collected within 48 h at 100 K with synchrotron radiation and area detection. A full topological analysis was applied to the resulting electron-density model. This analysis was followed by a Bader partitioning making use of the zero-flux surfaces of the electron-density gradient vector field. The atomic and bonding properties obtained were compared with the results of previous experimental studies, and with theoretical calculations for the title complex and free tryptophan as reported in the literature. The agreement between experiment and theory is similar to the agreement between different theoretical calculations. There is no charge transfer via the strong hydrogen bond; however, its strength is indicated by the very small atomic volume of the H atom involved.

Crystallography, X-Ray↗

PubMed2002

Reproducability and transferability of topological properties; experimental charge density of the hexapeptide cyclo-(D,L-Pro)2-(L-Ala)4 monohydrate.

The charge density of a hexapeptide was determined from high-resolution CCD area-detector experiments at 100 K. Two datasets, one from a rotating anode and a second one from synchrotron radiation, were measured and the results are compared. The data are interpreted in terms of the 'rigid pseudoatom' model. The topology of the experimental density is analyzed and compared with the topology of the constituting amino acids, and shows good agreement. All critical points of the electron density at the covalent and hydrogen bonds, as well as those of the Laplacian, were located. With respect to the transferability of electronic and bond topological properties the six peptide bonds were compared with values given in the literature.

Models, Molecular↗

PubMed2002

Intra- and intermolecular topological properties of amino acids: a comparative study of experimental and theoretical results.

The charge densities rho(r) of the six amino acids L-Asn.H(2)O, DL-Glu.H(2)O, DL-Lys.HCl, DL-Pro.H(2)O, DL-Ser, and DL-Val were determined from high-resolution X-ray diffraction experiments at 100 K using synchrotron radiation and area detection (CCD) techniques. Bond topological parameters derived from these densities and from those of six additional amino acids published earlier are compared to each other and to the results of ab initio calculations. Experimental and theoretical properties for each chemically equivalent bond are in a fair agreement, and their variances are of similar magnitude. A noticeable outlier is the positive curvature of the density at the bond critical point, for which no correlation between the experimental and theoretical values can be established. The location of nonbonded valence shell charge concentrations derived from the crystalline densities scatter in a wider range than those obtained for the isolated molecules.

Amino Acids↗

PubMed2000

Topological properties of the peptide bond in glycyl-L-threonine dihydrate based on a fast synchrotron/CCD-diffraction experiment at 100 K.

The charge density of glycyl-L-threonine dihydrate is extracted from a synchrotron data set of 98405 reflections collected at 100 K with a Bruker CCD area detector up to a resolution of d=0.38 A (sintheta/lambda = 1.32 A 1). The data are interpreted in terms of the "rigid pseudoatom" model. The topology of the experimental density is analyzed and compared with the topology obtained experimentally for the constituting amino acids and to that derived from Hartree-Fock calculations for the isolated molecule. All critical points of the electron density at the covalent and hydrogen bonds, as well as those of the Laplacian, were located, thereby deriving quantitative topological data for the peptide and side chain bonds. Bond topological indices in the dipeptide compare well with those of the corresponding bonds in the building amino acids, thus suggesting transferability of electronic properties of atoms and functional groups when these are derived by Bader's partitioning. Discrepancies between theoretical and experimental results could be attributed to crystal field effects.

Dipeptides↗

PubMed2000

DL-arginine monohydrate at 100 K.

In the title compound, C(6)H(14)N(4)O(2).H(2)O, the alpha-amino group is neutral. The molecular side chain including the guanidinium group is not fully extended, having a near gauche-gauche conformation [chi(3) = 59.0(1)degrees; chi(4) = 72.8(1)degrees]. The network of hydrogen bonds stabilizing the crystal lattice includes those formed between the deprotonated and negatively charged alpha-carboxylate groups and the positively charged amino groups of the guanidinium group of neighbouring molecules. N-H...O=C and water-mediated N-H...O hydrogen bonds link individual molecules to produce pairs of spiral motifs laterally connected by N-H...O and C-H...O hydrogen bonds.

Arginine↗

PubMed2000

Racemic dipeptide glycyl-DL-leucine at 120 K.

The structure of glycyl-DL-leucine, C(8)H(16)N(2)O(3), has been determined at 120 K by single-crystal X-ray diffraction. In addition to three N-H.O-type hydrogen bonds of the positively charged RNH(3)(+) group of the zwitterionic molecule, an intermolecular N-H. O contact exists between the peptide bond and the carboxylate group. Four hydrogen-bond cycles were identified, giving a complex pattern.

Crystallography, X-Ray↗

PubMed1999

A 28-kb deletion spanning D15S63 (PW71) in five families: a rare neutral variant?

Methylation analysis with probe PW71 (D15S63) is an established procedure to test patients suspected of having Prader-Willi syndrome or Angelman syndrome. Using this test, we have identified a 28-kb deletion spanning D15S63 in five independent families. Sequence analysis revealed identical breakpoints in all the families. The haplotype data are compatible with a common ancestral origin of the deletion in at least two families. The deletion was not found in 1, 000 unrelated controls. Although the deletion maps within the imprinting-center region, neither maternal nor paternal inheritance of the deletion appears to affect imprinting in proximal 15q. We conclude that the deletion is a rare neutral variant that can lead to false-positive results in the PW71-methylation test.

Adolescent↗

PubMed1999

The chromosome 15 imprinting centre (IC) region has undergone multiple duplication events and contains an upstream exon of SNRPN that is deleted in all Angelman syndrome patients with an IC microdeletion.

Imprinting of the Prader-Willi/Angelman syndrome region on human chromosome 15 is regulated by an imprinting centre (IC), which spans 5' exons of the gene encoding the small nuclear ribonucleoprotein N ( SNRPN ). The IC/ SNRPN transcripts are initiated at two alternative start sites, which share a high degree of sequence similarity with each other and with two newly identified sites 63 and >700 kb further upstream. Three of these sites are hypermethylated on the maternal chromosome, whereas one displays an oppositemethylation pattern. We have also identified novel splice variants of the IC/ SNRPN transcripts and hitherto undetected exons. One of these exons, which we designate u5, is deleted in all Angelman syndromepatients with a microdeletion of the IC. We conclude that elements of the IC region have undergone multiple duplication events and that u5 or a sequence close by may play a role in maternal imprinting.

Angelman Syndrome↗

PubMed1998

Sporadic imprinting defects in Prader-Willi syndrome and Angelman syndrome: implications for imprint-switch models, genetic counseling, and prenatal diagnosis.

The Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) are caused by the loss of function of imprinted genes in proximal 15q. In approximately 2%-4% of patients, this loss of function is due to an imprinting defect. In some cases, the imprinting defect is the result of a parental imprint-switch failure caused by a microdeletion of the imprinting center (IC). Here we describe the molecular analysis of 13 PWS patients and 17 AS patients who have an imprinting defect but no IC deletion. Heteroduplex and partial sequence analysis did not reveal any point mutations of the known IC elements, either. Interestingly, all of these patients represent sporadic cases, and some share the paternal (PWS) or the maternal (AS) 15q11-q13 haplotype with an unaffected sib. In each of five PWS patients informative for the grandparental origin of the incorrectly imprinted chromosome region and four cases described elsewhere, the maternally imprinted paternal chromosome region was inherited from the paternal grandmother. This suggests that the grandmaternal imprint was not erased in the father's germ line. In seven informative AS patients reported here and in three previously reported patients, the paternally imprinted maternal chromosome region was inherited from either the maternal grandfather or the maternal grandmother. The latter finding is not compatible with an imprint-switch failure, but it suggests that a paternal imprint developed either in the maternal germ line or postzygotically. We conclude (1) that the incorrect imprint in non-IC-deletion cases is the result of a spontaneous prezygotic or postzygotic error, (2) that these cases have a low recurrence risk, and (3) that the paternal imprint may be the default imprint.

Angelman Syndrome↗

PubMed1997

Identification of novel exons 3' to the human SNRPN gene.

The gene for the small nuclear ribonucleoprotein N (SNRPN) maps to human chromosome 15 and has 10 exons. Using cDNA cloning, direct cDNA selection, and exon-connection reverse transcriptase (RT)-PCR, we have identified three novel 3' exons of SNRPN, which have no protein coding potential. Like the other SNRPN exons, the novel exons are expressed from the paternal allele only. In contrast to several cDNA clones and RT-PCR products, however, the 3.4-kb transcript detected by Northern blot hybridization with a probe for the novel exons does not contain SNRPN. It is possible that the steady-state RNA observed in fetal tissues and in adult testis, ovary, brain, and muscle is initiated at an as yet unidentified transcription start site downstream of SNRPN or is generated by endonucleolytic cleavage of a precursor transcript, as has been shown for another imprinted gene, the insulin-like growth factor II gene.

Adult↗

PubMed1997

Imprinting mutations on human chromosome 15.

Genomic imprinting is an epigenetic process by which the male and the female germline of viviparous taxa confer a sex-specific mark (imprint) on certain chromosomal regions. The imprint is reset in the germline of each generation, inherited through somatic cell divisions during postzygotic development and used to regulate parent-of-origin specific expression of susceptible genes. Aberrant imprinting leading to aberrant gene expression patterns represents a novel class of mutations and was first identified in patients with Angelman syndrome and Prader-Willi syndrome. The finding of inherited cis-acting mutations in some of these cases has led to the identification of an imprinting center, which is involved in resetting of the imprint during gametogenesis. Other mutations may interfere with the somatic inheritance of the imprint during postzygotic development.

Chromosomes, Human, Pair 15↗

PubMed1997

Different mechanisms and recurrence risks of imprinting defects in Angelman syndrome.

Angelman syndrome (AS) is a neurogenetic disorder that appears to be caused by the loss of function of an imprinted gene expressed from maternal chromosome 15 only. Approximately 6% of patients have a paternal imprint on the maternal chromosome. In the few cases, this is due to an inherited microdeletion, in the 15q11-q13 imprinting center (IC), that blocks the paternal-->maternal imprint switch in the maternal germ line. We have determined the segregation of 15q11-q13 haplotypes in nine families with AS and with an imprinting defect. One family, with two affected siblings, has a microdeletion affecting the IC transcript. In the other eight patients, no mutation was found at this locus. In two families, the patient and a healthy sibling share the same maternal alleles. In one of these families and in two others, grandparental DNA samples were available, and the chromosomes with the imprinting defect were found to be of grandmaternal origin. These findings suggest that germ-line mosaicism or de novo mutations account for a significant fraction of imprinting defects, among patients who have an as-yet-undetected mutation in a cis-acting element. Alternatively, these data may indicate that some imprinting defects are caused by a failure to maintain or to reestablish the maternal imprint in the maternal germ line or by a failure to replicate the imprint postzygotically. Depending on the underlying cause of the imprinting defect, different recurrence risks need to be considered.

Angelman Syndrome↗