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Biomedical subjects

H Haas

Publications and source records attributed to H Haas.

At least 91 records · Page 5Linked to original sources
PubMed1995

Sequence and structure of Penicillium chrysogenum phoG, homologous to an acid phosphatase-encoding gene of Aspergillus nidulans.

A Penicillium chrysogenum (Pc) gene (phoG), homologous to an Aspergillus nidulans (An) gene which confers phosphate-non-repressible acid phosphatase (APase) activity, has been cloned and sequenced. The 2.9-kb genomic sequence corresponds to two ORFs of 149 and 1630 bp encoding a protein of 593 amino acids (aa). As verified by cDNA sequencing, the coding region is interrupted by an 85-bp intron. The deduced aa sequence of phoG reveals 61% aa identity to the translated long ORF of the An APase-encoding gene. Northern blot analysis indicated a 2.3-kb transcript in approximately equivalent amount in mycelia grown under different phosphate concentrations.

Acid Phosphatase↗
PubMed1995

Effect of data compression on quantitative coronary measurements.

Digital coronary and left ventricular angiography demand high transfer rates and very large data storage if all the clinical data are to be achieved. If appropriate compression schemes were available without compromising the quality and resolution of the image data, such demands could be lessened. In this study we compared the influence of different compression factors of the Adaptive Real Time Image Compression (ARTIC) scheme used on the Philips DCI-SX systems on coronary measurements assessed with the Automated Coronary Analysis (ACA) package. Loss-free acquired images of size 512(2) x 8 bits, which had been stored digitally on tape, were reloaded into the DCI with compression factors of 2, 3, and 4; only the factor 2 is loss free. To evaluate the effect of the different data compressions on the accuracy of the measurements, the diameters of a vessel phantom (tube sizes ranging from 0.687 to 5.062 mm) were determined. To evaluate the reproducibility of the results, the intraobserver variability was determined for the different compression factors from 40 coronary obstructions. The differences in the reference diameter measurements of the vessel phantom were -0.03 +/- 0.06 mm, 0.01 +/- 0.07 mm, and 0.04 +/- 0.08 mm for the compression factors 2 (loss free), 3, and 4, respectively. The results were not statistically significantly different. The intraobserver variabilities in the obstruction diameter measurements of the coronary obstructions were -0.04 +/- 0.13 mm, 0.00 +/- 0.14 mm, and 0.02 +/- 0.13 mm for the compression factors 2, 3 and 4, respectively. The intraobserver variabilities in the reference diameter measurements were -0.02 +/- 0.12 mm, 0.01 +/- 0.09 mm, and 0.03 +/- 0.09 mm for the compression factors 2, 3, and 4, respectively. The intraobserver variabilities of the percent diameter stenosis were 0.96 +/- 4.19%, -0.01 +/- 4.88% and -0.04 +/- 4.68% for the compression factors 2, 3, and 4, respectively. None of these differences were statistically significant. Both from a qualitative and quantitative point of view, data compression factors 3 and 4 are acceptable in digital coronary arteriography.

Coronary Angiography↗
PubMed1995

Molecular cloning and analysis of nre, the major nitrogen regulatory gene of Penicillium chrysogenum.

We have isolated the Penicillium chrysogenum nre gene which is homologous to the major nitrogen regulatory genes areA from Aspergillus nidulans and nit-2 from Neurospora crassa. Overall, nre shows 60% identity to areA and 30% identity to nit-2 at the amino-acid level. The gene encodes a protein of 835 amino-acid residues and contains a single Cys2/Cys2-type zinc finger with an adjacent basic region and a putative acidic activation region. In the DNA-binding domain, 98% of the amino-acid residues are identical in nre, areA and nit-2. The nre gene has been shown to be functional in N. crassa by heterologous complementation of a nit-2 mutant. Growth tests indicated that transformants could utilize nitrate, amino-acids, purines and amides as sole nitrogen sources. Nitrate reductase activity assays performed with transformants demonstrated that nitrogen control was completely normal. Complementation of N. crassa nit-2 mutants with 5'-deletion clones of nre suggests the possible presence of an internal promoter within the coding region. Northern analysis and ribonuclease protection assays of total cellular RNA indicated that nre encodes a 3.2-kb transcript which is reduced in content under conditions of nitrogen repression.

Amino Acid Sequence↗
PubMed1995

NRE, the major nitrogen regulatory protein of Penicillium chrysogenum, binds specifically to elements in the intergenic promoter regions of nitrate assimilation and penicillin biosynthetic gene clusters.

NRE, the nitrogen regulatory protein of Penicillium chrysogenum, contains a single Cys2/Cys2-type zinc-finger motif followed immediately by a highly basic region. The zinc-finger domain was expressed to Escherichia coli as a fusion protein with beta-galactosidase. In order to test the putative DNA-binding ability of NRE, the intergenic promoter region of the nitrate reductase/nitrite reductase gene cluster (niiA-niaD) of Penicillium was sequenced. Our results show that NRE is a DNA-binding protein and binds to the intergenic promoter regions of the P. chrysogenum niiA-niaD and acvA-pcbC gene cluster, encoding the first two enzymes in penicillin biosynthesis. Three of the four high-affinity NRE-binding sites contained two GATA core elements. In one of the recognition sites for NRE, one GATA motif was replaced by GATT. The two GATA elements showed all possible orientations, head-to-head, head-to-tail and tail-to-tail, and were separated by between 4 and 27 bp. Missing-contact analysis showed that all three purines in both of the GATA core sequences and the single adenine residue in each of the complementary TATC sequences were involved in the binding of NRE. Moreover, loss of purines in the flanking regions of the GATA elements also affect binding of NRE, as their loss causes reduced affinity.

Amino Acid Sequence↗
PubMed1995

GEDNAP IV and V. The 4th and 5th Stain Blind Trials using DNA technology.

In the collaborative exercise GEDNAP IV one EDTA blood sample (2 ml) and 5 bloodstains (0.5 ml on cotton) were investigated and in GEDNAP V, a total of 8 bloodstains (0.5 ml on cotton), including 2 mixed bloodstains. DNA typing was carried out using the RFLP systems YNH24/Hinf I and MS43a/Hinf I and the PCR systems HLA DQ alpha, D1S80, ApoB and YNZ22. In both exercises approximately 20 laboratories obtained results using the RFLP systems. Of the PCR systems, D1S80 was the most commonly used (14 labs in GEDNAP IV; 18 labs in GEDNAP V). The interlaboratory standard deviation for YNH24 in both exercises was approx. 0.6%, for MS43a 0.7-2.2% (GEDNAP IV) and 0.4-1.4% (GEDNAP V), depending on the fragment size. The fragment size calculation performed in each laboratory yielded a standard deviation twice that obtained when the fragment size calculation was performed centrally (IfR, Münster). In GEDNAP III, a system-specific corridor was developed to define the limits of deviation; this was modified for the present study by combining the fragment size ranges of YNH24 and MS43a. In both studies a subgroup of laboratories was involved in preliminary exercises using three PCR VNTRs and the system HLA DQ alpha. Owing to the substantial variation in experience of the participating laboratories with PCR typing the results obtained in these two studies do not fulfil the basic quality criteria of the GEDNAP studies.

Apolipoproteins B↗
PubMed1995

In vitro synthesis of human IgE is suppressed by human IgG.

Recently it has been shown that intravenous immunoglobulin (IVIG) preparations suppress the in vitro synthesis of IgG, IgA, and IgM. In this paper we demonstrate that IVIG and IgG purified from a single donor's serum also suppress the in vitro synthesis of IgE. We had noticed this effect when we added human serum (HS) to in vitro cultures for IgE synthesis. The interleukin-4 (IL-4)-induced IgE synthesis from human peripheral blood mononuclear cells (PBMC) in the presence of fetal calf serum (FCS) was suppressed by HS in a dose-dependent fashion. The following results indicate that this suppression is mediated by IgG: (1) IVIG preparations, which consist mainly of IgG, suppressed the IgE synthesis from IL-4-stimulated PBMC in a dose-dependent way; (2) when HS was fractionated by protein G sepharose or anti-IgG sepharose, the eluate fractions (containing IgG), but not the effluent fractions (void of IgG) suppressed IgE synthesis, whereas the opposite was found when HS was fractionated by FCS-coupled sepharose. We conclude from these data that human IgG preparations suppress the in vitro synthesis not only of the IgG, IgA, and IgM isotypes, but also of the IgE isotype.

Adult↗
PubMed1995

X-ray diffraction of a protein crystal anchored at the air/water interface.

We report the first successful in situ x-ray diffraction experiment with a 2D protein array at the lipid/water interface and demonstrate that the order can be controlled via lateral pressure or density. A protein (streptavidin) was bound to a monolayer of biotinylated lipid at the air/water interface, and diffraction of the protein layer could be measured to many orders. Compression of the monolayer changed the diffraction pattern drastically, indicating that the protein structure can be strongly influenced by external parameters like lateral pressure or density. From the width of the peaks, we find that aggregates consisting of as few as 100 monomers contribute to the diffraction. This indicates that the structure of even low order aggregates can be studied in situ. Grazing incidence diffraction can become a strong new method to study the crystallization and the interactions between proteins free from artifacts by staining or sample preparation.

Air↗
PubMed1995

Possible active site of the sweet-tasting protein thaumatin.

Epitopes on thaumatin and monellin were studied using the PEPSCAN-technology. The antibodies used were raised against thaumatin. Only antibodies that, in an ELISA, both recognized thaumatin and monellin were used in the PEPSCAN-analyses. On thaumatin two major overlapping epitopes were identified. On monellin no epitopes could be identified. The identified epitope region on thaumatin shares structural features with various peptide and protein sweeteners. It contains an aspartame-like site which is formed by Asp21 and Phe80, tips of the two extruding loops KGDAALDAGGR19-29 and CKRFGRPP77-84, which are spatially positioned next to each other. Furthermore, sub-sequences of the KGDAALDAGGR19-29 loop are similar to peptide-sweeteners such as L-Asp-D-Ala-L-Ala-methyl ester and L-Asp-D-Ala-Gly-methyl ester. Since the aspartame-like Asp21-Phe80 site and the peptide-sweetener-like sequences are also not present in non-sweet thaumatin-like proteins it is postulated that the KGDAALDAGGR19-29- and CKRFGRPP77-84 loop contain important sweet-taste determinants. This region has previously not been implicated as a sweet-taste determinant of thaumatin.

Amino Acid Sequence↗
PubMed1995

In vitro cultivation of third-stage larvae of Brugia malayi to the young adult stage.

The in vitro cultivation of the filarial nematode Brugia malayi from the infective stage to the fourth and the young adult stage is described. Different culture conditions including cell-free systems and co-culture with different human lymphatic cell lines were compared. Cell-free systems reported by others to promote the in vitro development of the parasites to the adult stage failed to work, i.e. the parasite development stopped at the L4 stage and the larvae died after approximately 3 weeks. Cocultivation with each of the cell lines used enhanced the survival of the parasites. The best results were obtained employing the human T cell leukemia line Jurkat and human dermal fibroblasts as feeder cells in RPMI 1640 supplemented with 10% heat-inactivated human serum. This culture system allowed the cultivation of B. malayi for more than 7 weeks with an average growth of the larvae by factor 6.4 (0.77 +/- 0.035 cm) and a maximum growth by factor 10 (1.2 cm). 69% of the initially cultivated larvae (which corresponded to 100% larvae alive at that time) reached the fourth larval stage after 14 days, and 2.6% of the initially cultivated larvae (which corresponded to 17% of the parasites alive at that day) had reached the young adult stage by day 37 of culture. Parasites remained alive up to 52 days. During the first four weeks of culture, both the length and the periods of moulting of the in vitro cultivated filariae closely resembled those observed with B. malayi in vivo in rodent hosts.

Aedes↗
PubMed1994

Aluminium reduces glutamate-activated currents of rat hippocampal neurones.

The actions of aluminum on glutamate-activated currents of acutely isolated hippocampal neurones were investigated. N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) and glutamate mediated currents were reduced by 50% in the presence of 1.4 micrograms ml-1 aluminium. Higher concentrations (> or = 2.7 micrograms ml-1) inhibited all currents completely and irreversibly. Additionally, successive application of agonists in the presence of 2.7 micrograms ml-1 aluminium resulted in non-specific membrane currents followed by the loss of the seal resistance. Application of aluminium per se had no influence on resting membrane current or voltage-activated sodium currents. The estimation of the concentration-response relationship of the action of aluminium on NMDA-activated currents revealed a threshold concentration < 0.27 micrograms ml-1. Our data indicate that glutamate receptors are putative sites of action in aluminium neurotoxicity.

Aluminum↗
PubMed1994

Horizontal polyacrylamide gel electrophoresis for the separation of DNA fragments.

A discontinuous borate/formate buffer system is presented for horizontal polyacrylamide gel electrophoresis of DNA fragments. The resolution potential of the system could be altered by changing the total monomer concentration (5-9%T), the concentration of the crosslinker piperazine diacrylamide (2-5%CPDA), as well as the concentration of formate in the gel (40-120 mM), the leading ion of the buffer system. The separation of DNA fragments would be improved by increasing the migration distance from 22 to 28 cm. This discontinuous polyacrylamide gel electrophoresis system proved highly reproducible.

Buffers↗
PubMed1994

Miscibility of lipoteichoic acid in dipalmitoylphosphatidylcholine studied by monofilm investigations and fluorescence microscopy.

The miscibility of the bacterial amphiphile lipoteichoic acid, a constituent of the cytoplasmic membrane of Gram-positive bacteria, in dipalmitoylphosphatidylcholine has been investigated by classic monofilm measurements and fluorescence microscopy at the air-water interface of monofilms obtained by spreading mixtures of both amphiphiles on a water subphase. The isotherms indicated miscibility of both lipids at concentrations up to 30 mol% lipoteichoic acid, whereas at higher concentrations immiscibility was detected. Increasing the lateral pressure over a certain value, lipoteichoic acid is squeezed out of the monofilm. By fluorescence microscopy the influence of lipoteichoic acid on the domain shape of condensed dipalmitoylphosphatidylcholine phases has been studied. The balance between hydrophobic and hydrophilic forces in the mixtures of both amphiphiles is discussed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

[The Th1/Th2 concept--its importance for regulation of IgE].

T helper type 2 (TH2) cells are a major source of interleukin- (IL-)4 which plays a critical role in the induction of the IgE synthesis. Their counterplayers, the T helper type 1 (TH1) cells produce interferon- (IFN-)gamma which inhibits the IL-4-induced IgE synthesis. Thus, the TH2/TH1 ratio plays an important role in the regulation of the IgE synthesis. Moreover, IL-4 seems to be the crucial factor controlling the switch of T helper precursor (THp) cells to the TH2 phenotype. However, the primary cellular source of IL-4 that skews an immune response towards the TH2 phenotype is not yet known. For future prophylactic and therapeutic interventions, e.g. in allergy, it will be crucial to know the type of cell involved and the factors that are activating this cell, i.e. the exact mechanism that is controlling the switch to the TH2 phenotype.

Animals↗
PubMed1993

Extracellular pH modulates aluminium-blockade of mammalian voltage-activated calcium channel currents.

The pH-dependence of aluminium (Al) blockade of voltage activated calcium channels (VACCs) was investigated. Using cultures of rat dorsal root ganglion (DRG) neurones, whole-cell patch-clamp experiments were performed. Various concentrations of Al were extracellularly applied within solutions of different pH-values. The block of VACC currents was highly pH-dependent. At pH 7.3-7.8, the concentration-response curve shifted slightly to higher concentrations, whereas at pH 6.4-6.9 a pronounced shift to lower concentrations was observed. This effect could be due to changes of the chemical equilibria of the different Al species or to altered properties of the VACCs. Thus, pH-shifts may influence the interactions of Al with VACCs making them more susceptible to the effects of Al and therefore contribute to its toxicity.

Aluminum↗
PubMed1993

Cloning and structural organization of a xylanase-encoding gene from penicillium chrysogenum.

The filamentous fungus, Penicillium chrysogenum, is able to grow on xylan as a sole carbon source. Under these conditions, high levels of a xylanase (XYLP) are secreted into the medium. After purification and characterization of this enzyme, we have isolated both the encoding cDNA and the genomic sequence by using oligodeoxyribonucleotides derived from partial amino acid (aa) sequences of the purified enzyme. The gene is approximately 1.6 kb in length, and comparison of the nucleotide (nt) sequence of the genomic and the cDNA clone revealed the presence of ten exons and nine introns. All intron/exon splice junctions exactly follow the GT/AG rule, except for the seventh intron which shows atypical AT/AC splice sites. The immediate 5'-flanking region of the first exon contains one putative CCAAT consensus sequence and a perfect TATA box. Primer extension analysis revealed two transcription start points located 38 and 34 nt upstream from the ATG start codon. A sequence of 23 aa representing a typical signal peptide is present at the N terminus of the deduced aa sequence. Northern blot analysis of total cellular RNA indicated that xylP encodes a 1.3-kb transcript which is induced by xylan. The aa sequence of XYLP shows considerable homology to high-M(r) acidic xylanases (Xln) and cellulases from different bacteria, yeasts and fungi.

Acid Phosphatase↗
PubMed1992

Purification, characterization and partial amino acid sequences of a xylanase produced by Penicillium chrysogenum.

An extracellular xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8, endo 1,4-beta-xylanase) was found to be the major protein in the culture filtrate of Penicillium chrysogenum when grown on 1% xylan. In contrast to other microorganism no xylanase multiplicity was found in P. chrysogenum under the conditions used. This enzyme was purified to homogeneity by high performance anion-exchange and size-exclusion chromatography. It had an M(r) of 35,000 as estimated by SDS-PAGE and was shown to be active as a monomer. No glycosylation of the protein could be detected neither by a sensitive glycostain nor by enzymatic deglycosylation studies. The enzyme hydrolyzed oat spelt and birchwood xylan randomly, yielding xylose and xylobiose as major end products. It had no cellulase, CMCase, beta-xylosidase or arabinogalactanase activity but acted on p-nitrophenylcellobioside. The pH and temperature optima for its activity were pH 6.0 and 40 degrees C, respectively. Eight peptides obtained after endoproteinase LysC digestion of xylanase have been sequenced, six of them showed considerable amino acid similarity to glucanases and high M(r)/acidic xylanases from different bacteria, yeasts and fungi.

Amino Acid Sequence↗
PubMed1992

Calcium-dependent and calcium-independent signals in the conglutinin-binding assay (KgBa) for immune complexes. Influence of anti-collagen-antibodies.

A solid phase ELISA conglutinin-binding assay (KgBa) was evaluated for the detection of circulating immune complexes. ELISA wells were coated with purified bovine conglutinin and incubated with test sera. Bound IgG was detected with enzyme labelled anti-immunoglobulin. Heat aggregated IgG which had been "solubilized" (i.e., complement treated by incubation with serum) was employed as a reference. The binding of the complement-reacted IgG to solid phase conglutinin was found to be calcium-dependent and inhibitable with N-acetyl-D-glucosamine (GlcNAc). Prolonged incubation (4 days) of aggregated IgG with serum at 37 degrees C abolished the binding to conglutinin, a finding consistent with the complete degradation of deposited C3b to C3c and C3d. The solubilized IgG that bound to solid phase conglutinin was found by gel chromatography to be of high molecular weight (greater than 600 kDa). Binding of IgG to solid phase bovine conglutinin was also observed to a variable degree in normal and pathological sera. However, in this situation the IgG binding was largely calcium-independent, was not inhibited by GlcNAc and did not decrease after prolonged incubation of the serum at 37 degrees C. The reactive IgG eluted on gel chromatography at the position of monomeric IgG suggesting binding via the antigen binding sites. Binding of this IgG was inhibited by both collagen type II and purified conglutinin. These observations suggest that the assay detects cross-reacting autoantibodies against collagen epitopes, or, alternatively, antibodies against the dietary antigen, bovine conglutinin.

Acetylgalactosamine↗