Biomedical subjects

H Haas

Publications and source records attributed to H Haas.

At least 55 records · Page 3Linked to original sources

PubMed2000

xylP promoter-based expression system and its use for antisense downregulation of the Penicillium chrysogenum nitrogen regulator NRE.

A highly inducible fungal promoter derived from the Penicillium chrysogenum endoxylanase (xylP) gene is described. Northern analysis and the use of a beta-glucuronidase (uidA) reporter gene strategy showed that xylP expression is transcriptionally regulated. Xylan and xylose are efficient inducers, whereas glucose strongly represses the promoter activity. Comparison of the same expression construct as a single copy at the niaD locus in P. chrysogenum and at the argB locus in Aspergillus nidulans demonstrated that the xylP promoter is regulated similarly in these two species but that the level of expression is about 80 times higher in the Aspergillus species. The xylP promoter was found to be 65-fold more efficient than the isopenicillin-N-synthetase (pcbC) promoter in Penicillium and 23-fold more efficient than the nitrate reductase (niaD) promoter in Aspergillus under induced conditions. Furthermore, the xylP promoter was used for controllable antisense RNA synthesis of the nre-encoded putative major nitrogen regulator of P. chrysogenum. This approach led to inducible downregulation of the steady-state mRNA level of nre and consequently to transcriptional repression of the genes responsible for nitrate assimilation. In addition, transcription of nreB, which encodes a negative-acting nitrogen regulatory GATA factor of Penicillium, was found to be subject to regulation by NRE. Our data are the first direct evidence that nre indeed encodes an activator in the nitrogen regulatory circuit in Penicillium and indicate that cross regulation of the controlling factors occurs.

Aspergillus nidulans↗

PubMed2000

Synthetic hydrogel capacity to induce formation of foreign-body giant multinucleate cells differs in vivo and in vitro.

The granulomatous reaction accompanied with MGC formation represents the most striking feature of the non-favourable biological tolerance of implanted devices. We compared MGC formation in the course of the granulomatous reaction in vitro and in vivo employing three types of hydrogels whose biocompatibility had been well studied earlier. The efficiency of the in vitro assay for the granulomatous reaction, including MGC formation, was verified employing the nematode Nippostrongylus brasiliensis, a well-known inductor of MGC formation in vitro. The in vitro results demonstrated a very low level of MGC formation in reaction against all three types of hydrogels without polymer-specific differences in comparison with the nematode experiment characterized by a high extent of MGC formation. On the other hand, the extent of MGC formation was implant type-specific in vivo: pHEMA-co-DMAEMA > pHEMA > pHEMA-co-NaMA. These results indicate that in the in vitro assay it was not possible to discriminate among the types of polymers used in the experiment in comparison with the animal experiment. They also indicate potential differences between granuloma formation induced by parasites and by foreign bodies.

Animals↗

PubMed1999

Analysis of the histone acetyltransferase B complex of maize embryos.

Purified histone acetyltransferase B (HAT-B) from maize consists of two subunits, p50 and p45. Cloning of the cDNA and genomic DNA encoding the catalytic subunit p50 revealed a consensus motif reminiscent of other acetyltransferases. Internal peptide sequences and immunological studies identified p45 as a protein related to the Retinoblastoma associated protein Rbap. Antibodies against recombinant p50 were able to immunoprecipitate the enzymatic activity of p50 as well as p45. Consistent with the idea that HAT-B is involved in acetylation of newly synthesized histone H4 during DNA replication, mRNA and protein levels are correlated with S-phases during embryo germination. Inhibition of histone deacetylases by HC toxin or Trichostatin A caused a decrease of the in vivo expression of HAT-B mRNA. Regardless of its predominant cytoplasmic localization, a significant proportion of HAT-B-p50 is present in nuclei, irrespective of the cell cycle stage, suggesting an additional nuclear function.

Acetyltransferases↗

PubMed1999

Purification of morphologically and functionally intact human basophils to near homogeneity.

Certain studies on basophils require highly purified functionally intact cell preparations. Here, a three-step procedure is described that meets these requirements. The procedure consists of a Ficoll density gradient step, counterflow elutriation and negative selection by magnetic cell sorting (MACS). The mean purity of basophils obtained from 30 donors was 97.6+/-3.96% with a viability of 99.6+/-0.83%. The recovery rate was 49.7+/-15.6%. The cells had a normal morphological appearance as assessed by May-Grünwald-stained cytospins and were functionally intact as shown by their unaltered capacity to release histamine and interleukin 4 (IL-4) following immunological activation. This procedure is a clear improvement over currently available techniques and should facilitate future investigations on basophils.

Basophils↗

PubMed1999

Expression of atrC - encoding a novel member of the ATP binding cassette transporter family in Aspergillus nidulans - is sensitive to cycloheximide.

A new member of the ABC superfamily of transmembrane proteins in Aspergillus nidulans has been cloned and characterized. The topology of conserved motifs subgroups AtrC in the P-glycoprotein cluster of ABC permeases, the members of this subfamily, are known to participate in multidrug resistance (MDR) in diverse organisms. Alignment results display significant amino acid similarity to AfuMDR1 and AflMDR1 from Aspergillus fumigatus and flavus, respectively. Northern analysis reveals that atrC mRNA levels are 10-fold increased in response to cycloheximide. Evidence for the existence of eight additional hitherto unpublished ABC transporter proteins in A. nidulans is provided.

ATP-Binding Cassette Transporters↗

PubMed1999

The Aspergillus nidulans GATA factor SREA is involved in regulation of siderophore biosynthesis and control of iron uptake.

A gene encoding a new GATA factor from Aspergillus nidulans, sreA, was isolated and characterized. SREA displays homology to two fungal regulators of siderophore biosynthesis: about 30% overall identity to SRE from Neurospora crassa and about 50% identity to URBS1 from Ustilago maydis over a stretch of 200 amino acid residues containing two GATA-type zinc finger motifs and a cysteine-rich region. This putative DNA binding domain, expressed as a fusion protein in Escherichia coli, specifically binds to GATA sequence motifs. Deletion of sreA results in derepression of L-ornithine-N5-oxygenase activity and consequently in derepression of the biosynthesis of the hydroxamate siderophore N,N',N"-triacetyl fusarinine under sufficient iron supply in A. nidulans. Transcription of sreA is confined to high iron conditions, underscoring the function of SREA as a repressor of siderophore biosynthesis under sufficient iron supply. Nevertheless, overexpression of sreA does not result in repression of siderophore synthesis under low iron conditions, suggesting additional mechanisms involved in this regulatory circuit. Consistent with increased sensitivity to the iron-activated antibiotics phleomycin and streptonigrin, the sreA deletion mutant displays increased accumulation of 59Fe. These results demonstrate that SREA plays a central role in iron uptake in addition to siderophore biosynthesis.

Amino Acid Sequence↗

PubMed1999

Dietary lectins can induce in vitro release of IL-4 and IL-13 from human basophils.

Dietary lectins, present in beans and other edible plant products, pose a potential threat to consumers due to their capacity to induce histamine release from basophils. In this study, we analyzed the capacity of 16 common, in particular dietary, lectins to induce human basophils to secrete IL-4 and IL-13, the key promoters of Th2 responses and IgE synthesis. Several of the lectins, especially concanavalin A, lentil lectin, phytohemagglutinin, Pisum sativum agglutinin and Sambucus nigra agglutinin, triggered basophils to release IL-4 at concentrations of up to 1 ng/10(6) basophils. Lectins with high IL-4-inducing capacity also stimulated the release of IL-13 and histamine. Lectin-induced IL-4 and IL-13 release reached a maximum after 4-6 h and more than 18 h, respectively. Affinoblotting revealed that lectins with the capacity to induce mediator release bind to IgE, suggesting IgE binding as initial step of signal generation. In conclusion, several dietary lectins can trigger human basophils to release IL-4 and IL-13. Since lectins can enter the circulation after oral uptake, they might play a role in inducing the so-called early IL-4 required to switch the immune response towards a Th2 response and type I allergy.

Basophils↗

PubMed1999

Factors related to the prescription of antibiotics for young children with viral pharyngitis by general practitioners and paediatricians in southeastern France.

This study evaluates the antibiotic-prescribing practices of physicians as well as other related issues in the context of viral pharyngitis. In a telephone interview, 535 physicians practising in southeastern France were submitted a clinical case description of an episode of acute pharyngitis in a 2-year-old child. Questions concerned antibiotic treatment and physicians' reasons for their treatment decision. The viral origin of the pharyngitis was more likely to be suspected by paediatricians than by general practitioners (92% vs. 78%, P<0.01); 57% of allopaths (compared with 26% of homeopaths/acupuncturists and 14% of paediatricians, P<0.001) declared they would prescribe an antibiotic in this situation (amoxicillin only in 42% of cases). This difference between allopaths and other physicians was still significant after controlling for knowledge regarding antibiotic therapy. In order to limit the risk of emerging resistant bacteria, it is urgent that training be upgraded for physicians, especially for allopaths.

Anti-Bacterial Agents↗

PubMed1999

Seasonal dynamics of bacterioplankton community structure in a eutrophic lake as determined by 5S rRNA analysis.

Community structure of bacterioplankton was studied during the major growth season for phytoplankton (April to October) in the epilimnion of a temperate eutrophic lake (Lake Plusssee, northern Germany) by using comparative 5S rRNA analysis. Estimates of the relative abundances of single taxonomic groups were made on the basis of the amounts of single 5S rRNA bands obtained after high-resolution electrophoresis of RNA directly from the bacterioplankton. Full-sequence analysis of single environmental 5S rRNAs enabled the identification of single taxonomic groups of bacteria. Comparison of partial 5S rRNA sequences allowed the detection of changes of single taxa over time. Overall, the whole bacterioplankton community showed two to eight abundant (>4% of the total 5S rRNA) taxa. A distinctive seasonal succession was observed in the taxonomic structure of this pelagic community. A rather-stable community structure, with seven to eight different taxonomic units, was observed beginning in April during the spring phytoplankton bloom. A strong reduction in this diversity occurred at the beginning of the clear-water phase (early May), when only two to four abundant taxa were observed, with one taxon dominating (up to 72% of the total 5S rRNA). The community structure during summer stagnation (June and July) was characterized by frequent changes of different dominating taxa. During late summer, a dinoflagellate bloom (Ceratium hirudinella) occurred, with Comamonas acidovorans (beta-subclass of the class Proteobacteria) becoming the dominant bacterial species (average abundance of 43% of the total 5S rRNA). Finally, the seasonal dynamics of the community structure of bacterioplankton were compared with the abundances of other major groups of the aquatic food web, such as phyto- and zooplankton, revealing that strong grazing pressure by zooplankton can reduce microbial diversity substantially in pelagic environments.

Animals↗

PubMed1999

Early interleukin-4: its role in the switch towards a Th2 response and IgE-mediated allergy.

The etiology of IgE-mediated allergies is complex and, thus far, not completely understood. A common feature, however, is the overproduction of IgE-inducing cytokines, e.g. interleukin-4(IL-4), compared to IgE-antagonistic cytokines, such as interferon-gamma or IL-12. IgE-inducing cytokines are produced by T helper type 2 (Th2) cells. The differentiation of naive T cells towards the Th2 phenotype seems to be crucially dependent upon the particular cytokines present in the early stages of an immune response. Concerning the factors driving Th2 differentiation, the so-called 'early IL-4' seems to play an important role, although there is some controversy over the degree of its requirement and its cellular source. We have recently demonstrated that basophils might be such a source, since they rapidly release IL-4 upon antigen-specific or nonantigen-specific stimuli, such as certain lectins. This makes lectins interesting candidates for inducing a Th2 response and IgE-mediated allergy in unsensitized individuals.

Humans↗

PubMed1999

Different cytokine patterns correlate with the extension of disease in pulmonary tuberculosis.

The relative amounts of different pro- and anti-inflammatory cytokines released at the site of infection by bronchoalveolar lavage (BAL) cells may influence the presentation of tuberculosis. To investigate this hypothesis the in situ release by BAL cells of the following cytokines was measured and correlated with the chest X-ray findings of 43 patients with pulmonary tuberculosis: interleukin (IL)-8, macrophage inflammatory protein-1alpha (MIP-1alpha), IL-6, tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), interferon-gamma (IFN-gamma), IL-2, IL-4 and IL-5. The release of IL-8 and IL-6 decreased with the progression of the disease, while the release of MIP-1alpha was increased in patients with advanced tuberculosis. The release of TNF-alpha and TGF-beta did not differ between patients with or without cavitary lesions. The Th1 (IFN-gamma and IL-2) and Th2 (IL-4 and IL-5) cytokine release exhibited a gradual increment with the advance of tuberculosis. Thus, our data provide evidence that a Th0 cytokine pattern is predominant at the site of pulmonary tuberculosis. In conclusion, immunoparalysis status could not be observed in our patients with severe tuberculosis.

Bronchoalveolar Lavage Fluid↗

PubMed1999

[Nasopharyngeal carriage of resistance pneumococci in day-care centers in the Alps-Maritimes region].

To provide ongoing information on regional trends of antibiotic resistance prevalence to pneumococci, a cross selectional survey was conducted on a large representative sample of children attending day-care centers. Children were analyzed in spring (n = 378) and autumn (n = 379) for nasopharyngeal carriage. Streptococcus pneumoniae was detected in 149 children (39.4%) in Spring and 204 (59.8%) in Autumn. Half of these isolated strains showed penicillin insensitivity or resistance. A high proportion of children (43.6% in spring and 47.5% in autumn) had been treated with antibiotics during the 3 months prior to sample collection; 21.6% of isolated strains were serotype 6B, 20.1% type 23F, 18.9% type 19A and 19F, 11.5% type 14. Reduced susceptibility was frequently noted in serotype 23F, 14 and 19F, representing 93%, 94% and 46% of identified serotypes, respectively. Acquisition of a strain of PRP was correlated with prescription of antibiotics during the previous three months (p < 0.05). This type of survey on children in day-care centers can contribute to the understanding of regional variations in antibiotic resistance and provide information for epidemiological surveillance.

Animals↗

PubMed1998

Cutaneous leishmaniasis due to Leishmania infantum. Case reports and literature review.

BACKGROUND: Leishmania infantum recently has been identified as a possible agent of cutaneous leishmaniasis (CL). This species has been isolated from cutaneous lesions of patients from the Mediterranean Basin. However, little is known about the clinical, biological, or therapeutic features of this newly recognized CL. OBSERVATIONS: Six patients aged 9 months to 85 years in southeastern France were found to have autochthonous leishmaniasis. Parasitological identification showed that the agent was L infantum, zymodemes Montpellier-1 for 2 patients and Montpellier-24 for 1 patient. Five patients who underwent testing with a Western blot assay were found to have antibodies against 4 antigens with molecular masses of 18, 21, 23, and 31 kd. Five patients were successfully treated with local injections of N-methylglucamine, and 1 patient was successfully treated with topical paromomycin sulfate. No patient had visceral disease at diagnosis or after follow-up. CONCLUSIONS: Recent data provide increasing evidence that L infantum is an important agent of CL. In southwestern Europe, this species is the only agent that has long been identified from autochthonous CL. Leishmania infantum should be considered an agent of CL in areas in which visceral leishmaniasis is endemic. Western blot assay could be a useful test for the diagnosis, but precise parasitological identification is important to having a better knowledge of the disease. The relationships between CL and the visceral disease have to be explored.

Administration, Cutaneous↗

PubMed1998

Staining pattern of seven monoclonal anti-CD26 antibodies in leprosy: implications for the use of CD26 as a surrogate marker of a human Th1-like reaction.

In a previous study using the monoclonal anti-CD26 antibody MIB-DS2/7 in leprosy and other granulomatous diseases, it was shown that CD26 may be a candidate for use as an operational marker of a human Th1-like reaction. In this follow-up study, we compared seven different monoclonal anti-CD26 antibodies with respect to their staining pattern in lepromatous and tuberculoid leprosy tissues. Three distinct staining patterns became apparent in this anti-CD26 antibody panel: staining of T-lymphocytes and of connective tissue; staining of T-lymphocytes, connective tissue and macrophages; and almost no staining of T-lymphocytes but staining of connective tissue and macrophages. The two antibodies assigned to the first staining pattern, including MIB-DS2/7, were found to be most suitable for the operational discrimination between Th1-like and Th2-like reactions in leprosy. The antibodies assigned to staining patterns 2 and 3 did not allow this discrimination. Although all seven monoclonal antibodies investigated were specific for CD26, only two were found to be useful in identifying a Th1-like immune reaction in human tissue.

Antibodies, Monoclonal↗

PubMed1998

Isolation and characterization of a new gene, sre, which encodes a GATA-type regulatory protein that controls iron transport in Neurospora crassa.

Multiple GATA factors - regulatory proteins with consensus zinc finger motifs that bind to DNA elements containing a GATA core sequence - exist in the filamentous fungus Neurospora crassa. One GATA factor, NIT2. controls nitrogen metabolism, whereas two others, WC-1 and WC-2, regulate genes responsive to blue light induction. A gene encoding a new GATA factor, named SRE, was isolated from Neurospora using a PCR-mediated method. Sequence analysis of the new GATA factor gene revealed an ORF specifying 587 amino acids, which is interrupted by two small introns. Unlike all previously known Neurospora GATA factors, which possess a single zinc-finger DNA-binding motif, SRE contains two GATA-type zinc fingers. The deduced amino acid sequence of SRE shows significant similarity to URBSI of Ustilago and SREP of Penicillium. A loss-of-function mutation was created by the RIP procedure. Analysis of sre+ and sre- strains revealed that SRE acts as a negative regulator of iron uptake in Neurospora by controlling the synthesis of siderophores. Siderophore biosynthesis is repressed by high iron concentrations in the wild-type strain but not in sre- mutant cells. The sre promoter contains a number of GATA sequences; however, expression of sre mRNA occurs in a constitutive fashion and is not regulated by the concentration of iron available to the cells.

Amino Acid Sequence↗

PubMed1997

Overexpression of nreB, a new GATA factor-encoding gene of Penicillium chrysogenum, leads to repression of the nitrate assimilatory gene cluster.

To investigate the mechanism of nitrogen metabolite repression in the biotechnologically important fungus Penicillium chrysogenum a polymerase chain reaction approach was employed to identify transcription factors involved in this regulatory circuit, leading to the isolation of a new gene (nreB) encoding a 298 amino acid protein. Despite a low overall amino acid sequence identity of approximately 30%, it shares several features with Dal80p/Uga43p and Gzf3p/Nil2p, both repressors in nitrogen metabolism in Saccharomyces cerevisiae. All three proteins contain an N-terminal GATA-type zinc finger motif, displaying 86% amino acid sequence identity, and a putative leucine zipper motif in the C terminus. Northern blot analysis revealed the presence of two nreB transcripts, 1.8 and 1.5 kilobases in length, that differ in polyadenylation sites. The steady state level of both transcripts is subject to nitrogen metabolite repression. The putative DNA binding domain of NREB, expressed as a fusion protein in Escherichia coli, binds in vitro to GATA sites of its own 5'-upstream region as well as in the promoter of the nitrate assimilation gene cluster. Consistent with a role in the regulation of nitrogen metabolism, overexpression of nreB leads to repression of nitrate assimilatory genes. Hence, the simple view of nitrogen regulation by four GATA factors in yeast, but only one key regulator in filamentous ascomycetes seems no longer valid.

Amino Acid Sequence↗

PubMed1997

Identification of maize histone deacetylase HD2 as an acidic nucleolar phosphoprotein.

The steady state of histone acetylation is established and maintained by multiple histone acetyltransferases and deacetylases, and this steady state affects chromatin structure and function. The identification of a maize complementary DNA encoding the chromatin-bound deacetylase HD2 is reported. This protein was not homologous to the yeast RPD3 transcriptional regulator. It was expressed throughout embryo germination in correlation with the proliferative activity of cells. Antibodies against recombinant HD2-p39 immunoprecipitated the native enzyme complex, which was composed of phosphorylated p39 subunits. Immunofluorescence microscopy and sequence homologies suggested nucleolar localization. HD2 is an acidic nucleolar phosphoprotein that might regulate ribosomal chromatin structure and function.

Acetylation↗

PubMed1997

Molecular analysis of a Penicillium chrysogenum GATA factor encoding gene (sreP) exhibiting significant homology to the Ustilago maydis urbs1 gene.

Employing a PCR-aided strategy, a Penicillium chrysogenum gene (sreP) encoding a putative GATA-transcription factor has been cloned and characterized. Comparison of the genomic and cDNA sequences revealed the presence of an open reading frame (ORF) encoding a protein of 532 amino acids (aa) which is interrupted by two introns. The deduced aa sequence of sreP reveals 50% identity to a regulator of siderophore biosynthesis (URBS1) from Ustilago maydis over a stretch of 200 aa containing two GATA-type zinc finger motifs and a Cys-rich intervening sequence. Northern blot analysis indicated two transcripts of 2.2 and 2.7 kb in approximately equivalent amount. due to two major transcription start sites.

Amino Acid Sequence↗