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A Witte

Publications and source records attributed to A Witte.

At least 19 recordsLinked to original sources
PubMed2006

The lysogenic region of virus phiCh1: identification of a repressor-operator system and determination of its activity in halophilic Archaea.

phiCh1 is a temperate virus infecting the haloalkaliphilic archaeon Natrialba magadii. As for all temperate viruses, a control of the lysogenic state versus the lytic life cycle is essential. Two open reading frames (ORFs) have been identified as putative repressor encoding genes: ORF48 and ORF49. The protein of ORF48 showed sequence similarities to putative repressor molecules. ORF49 was identified by the analysis of a mutant of phiCh1: the lysogenic strain carrying mutant phiCh1-1 showed a different lysis behavior than wild type virus phiCh1, indicating a dysfunction in the regulation of gene expression. Here, we show that the intergenic region between ORF48 and ORF49 comprises a promoter/operator sequence that is a transcriptionally active region in the model system Haloferax volcanii. Transcription from this region can be repressed by the activity of the ORF48 gene product. Gp43/gp44 has an enhancing effect on this regulatory sequence. Evidence is given for a possible binding site of Rep and gp43/gp44 within the coding region of the rep gene.

Archaeal Viruses↗
PubMed2005

Type I interferon correlates with serological and clinical manifestations of SLE.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disease affecting multiple organ systems triggered by the production of autoantibodies. Previous clinical studies in humans and murine models suggest that type I interferons (IFNs) are important for the initiation and potentiation of SLE activity. METHODS: 65 consecutive patients with SLE were identified from the University of California, San Francisco Lupus Clinic with moderate-severe disease activity. 94 serological samples were collected. Type I IFN levels and the ability of plasma to induce expression of several surface markers of dendritic cell maturation were measured. RESULTS: Type I IFN levels correlated with the presence of cutaneous manifestations, and there was a trend towards correlation with renal disease. No correlation was found between type I IFN levels and neurological disease. Type I IFN levels correlated positively with the SLEDAI score and anti-dsDNA levels and inversely with C3 levels. Interestingly, type I IFN levels were highest in African American patients. SLE plasma also induced the expression of MHC class I, CD38, and CD123 on monocytes, and was blocked by the addition of a monoclonal antibody to IFNAR1. CONCLUSIONS: The pathogenic role of type I IFN is suggested by the induction of cell surface markers for dendritic cell maturation. The potential therapeutic utility of antibodies directed to either type I IFN or IFNAR1/IFNAR2 may be of interest in further studies.

Adult↗
PubMed2004

Inversion within the haloalkaliphilic virus phi Ch1 DNA results in differential expression of structural proteins.

The sequence of phi Ch1 contains an open reading frame (int1) in the central part of its genome that belongs to the lambda integrase family of site-specific recombinases. Sequence similarities to known integrases include the highly conserved tetrad R-H-R-Y. The flanking sequences of int1 contain several direct repeats of 30 bp in length (IR-L and IR-R), which are orientated in an inverted direction. Here, we show that a recombination active region exists in the genome of phi Ch1: the number of those repeats, non-homologous regions within the repeat clusters IR-L and IR-R and the orientation of the int1 gene vary in a given virus population. Within this study, we identified circular intermediates, composed of the int1 gene and the inwards orientated repeat regions IR-L and IR-R, which could be involved in the recombination process itself. IR-L and IR-R are embedded within ORF34 and ORF36 respectively. As a consequence of the inversion within this region of phi Ch1, the C-terminal parts of the proteins encoded by ORF34 and 36 are exchanged. Both proteins, expressed in Escherichia coli, interact with specific antisera against whole virus particles, indicating that they could be parts of phi Ch1 virions. Expression of the protein(s) in Natrialba magadii could be detected 98 h after inoculation, which is similar to other structural proteins of phi Ch1. Taken together, the data show that the genome of phi Ch1 contains an invertible region that codes for a recombinase and structural proteins. Inversion of this segment results in a variation of these structural proteins.

Amino Acid Sequence↗
PubMed2003

[Radio-frequency-ablation (RFA) with wet electrodes in the treatment of primary and secondary liver tumours].

INTRODUCTION: The majority of patients with primary and secondary tumours of the liver cannot be treated curatively by surgery. The treatment of these patients with radio-frequency thermoablation using perfused needle applicators (wet electrodes) was evaluated in a feasibility study. METHOD: Patients with primary and secondary tumours of the liver and contraindications against surgery or LTX were included into the feasibility study. RFA was performed percutaneously under ultrasound guidance. The patients were followed up sonographically and by computed tomography. RESULTS: 20 patients (9 male, 11 female) with 35 lesions were treated with RFA. 12 patients (22 tumour locations) suffered from HCC and 8 patients (13 tumour masses) had liver metastases (colorectal, breast, pancreas, carcinoid). The median age was 65.6 years (36 to 83 years). The median tumour size was 33.5 mm. 59 RFTA applications (1.7 applications per tumour mass) were performed. The mean duration of RFTA per patient was 16.2 minutes. During the procedure isotonic saline was injected at a mean flow rate of 6.63 ml/min. All patients received local anaesthesia. In 33 sessions an additional analgosedation was necessary (average dose 63.8 mg Pethidine and 1.4 mg Midazolam). In 2 cases a reduction of the haemoglobin level, occurred, necessitating a blood transfusion. 3/4 of the treated metastases could be eradicated completely. Within a median follow-up of 145 days no intrahepatic local recurrence but 4 distant metastases occurred. 2/3 of the treated HCC could initially be brought into complete remission (CR). After a median follow-up of 329 days 5 of the 8 initially successfully treated patients with HCC were still in complete remission. In 3 cases an intrahepatic local recurrence developed. CONCLUSION: RFA with wet electrodes is a safe, effective and inexpensive treatment for primary and secondary tumours of the liver, measuring less than 4 cm in diameter.

Adult↗
PubMed2002

[Suprarenal leiomyoma of the vena cava. A rare differential adrenal gland tumor diagnosis].

Leiomyomas are benign tumors that can arise in the smooth muscle and can appear practically everywhere; hence, they must be taken into consideration as a rare possibility in the differential diagnosis of numerous tumors. While leiomyomas of the peripheral vessels are a relatively common finding, they are rarely found in the central vessels. Only a few cases of leiomyomas in the vena cava are known. In contrast to malignant leiomyosarcomas, leiomyomas usually grow towards the lumen. We report on the rare case of a leiomyoma in the inferior vena cava that appeared in the image to be located in the adrenal gland. Therefore, transperitoneal extirpation of the site was undertaken. It was only during surgery that a tumor emanating from the vessel wall became apparent. Thus, after an initial laparoscopic approach it became necessary to change to open resection of the tumor with cavotomy and resection of the vessel wall. The histopathological work-up revealed a benign leiomyoma and further imaging diagnostics gave no indication for the presence of metastases.

Adrenal Gland Neoplasms↗
PubMed2002

Natrialba magadii virus phiCh1: first complete nucleotide sequence and functional organization of a virus infecting a haloalkaliphilic archaeon.

The double-stranded (ds)DNA virus phiCh1 infects the haloalkaliphilic archaeon Natrialba magadii. The complete DNA sequence of 58 498 bp of the temperate virus was established, and the probable functions of 21 of 98 phiCh1-encoded open reading frames (ORFs) have been assigned. This knowledge has been used to propose functional modules each required for specific functions during virus development. The phiCh1 DNA is terminally redundant and circularly permuted and therefore appears to be packaged by the so-called headful mechanism. The presence of ORFs encoding homologues of proteins involved in plasmid replication as well as experimental evidence indicate a plasmid-mediated replication strategy of the virus. Results from nanosequencing of virion components suggest covalent cross-linking of monomers of at least one of the structural proteins during virus maturation. A comparison of the phiCh1 genome with the partly sequenced genome of Halobacterium salinarum virus phiH revealed a close relationship between the two viruses, although their host organisms live in distinct environments with respect to the different pH values required for growth.

Base Sequence↗
PubMed2002

Electrophysiologic investigation in Brugada syndrome; yield of programmed ventricular stimulation at two ventricular sites with up to three premature beats.

INTRODUCTION: Numerous reports on the inducibility of ventricular tachyarrhythmias (VT) in patients with atypical right bundle branch block and right precordial ST-elevation (Brugada syndrome) are based on multicentre studies that have used different stimulation protocols. Therefore, we prospectively investigated the inducibility of VT in these patients using a uniform protocol. METHODS: In 41 consecutive patients (29 males) showing a pattern of right bundle branch block and ST-elevation, programmed ventricular stimulation was performed in the right ventricular apex with up to three premature stimuli at sinus rhythm and at four different paced cycle lengths (500, 430, 370, and 330 ms) until refractoriness was reached or reproducible induction of a sustained (>30s) VT occurred. If a VT was not reproducibly inducible, the same protocol was repeated in the right ventricular outflow tract. RESULTS: A history of life-threatening events defined as syncope (n=17) or aborted sudden cardiac death (n=13) was present in 30 patients (73%); 11 individuals were asymptomatic. Inducibility (68%) was similar between symptomatic (n=21, 70%) and asymptomatic patients (n=7, 64%). In 16 (39%) patients, VT were reproducibly inducible. If patients were only stimulated in the right ventricular apex, inducibility rate decreased to 39%. If only two premature beats at two sites were used it was as low as 32%. The mean coupling intervals of the second and third premature stimuli inducing sustained VT were short: 189+/-21 ms vs 186+/-22 ms, respectively. Forty-four percent of all patients (i.e. 64% of the inducible patients) had inducible VT only with coupling intervals shorter than 200 ms. CONCLUSIONS: The stimulation protocol markedly influences the extent of inducibility of VT in patients with right bundle branch block and ST-segment elevation. These findings question the significance of previous multicentre studies using different stimulation protocols and should have implications for further studies.

Adult↗
PubMed2001

S-layer variation in Bacillus stearothermophilus PV72 is based on DNA rearrangements between the chromosome and the naturally occurring megaplasmids.

Bacillus stearothermophilus PV72 expresses different S-layer genes (sbsA and sbsB) under different growth conditions. No stretches of significant sequence identity between sbsA and sbsB were detected. In order to investigate S-layer gene regulation in B. stearothermophilus PV72, we characterized the upstream regulatory region of sbsA and sbsB by sequencing and primer extension analysis. Both genes are transcribed from unique but different promoters, independently of the growth phase. Localization of sbsB in the sbsA-expressing strain PV72/p6 revealed that the coding region of the second S-layer gene sbsB is located not on the chromosome but on a natural megaplasmid of the strain, whereas the upstream regulatory region of sbsB was exclusively detected on the chromosome of PV72/p6. For sbsB expression, the coding region has to be integrated into the chromosomally located expression site. After the switch to sbsB expression, the sbsA coding region was removed from the chromosome but could still be detected on the plasmid of the sbsB-expressing strain PV72/p2. The sbsA upstream regulatory region, however, remained on the chromosome. This is the first report of S-layer variation not caused by intrachromosomal DNA rearrangements, but where variant formation depends on recombinational events between the plasmid and the chromosome.

Bacterial Proteins↗
PubMed2000

The structural protein E of the archaeal virus phiCh1: evidence for processing in Natrialba magadii during virus maturation.

phiCh1 is a lysogenic virus for the haloalkalophilic archaeon Natrialba magadii. The virus morphology resembles other members of Myoviridae infecting Halobacterium species. The gene of the major capsid protein E of virus phiCh1 was cloned and the DNA sequence was determined. Gene E was mapped to a 3.2-kbp ClaI fragment, localized to the 5'-end of the phiCh1 genome. The complete nucleotide sequence of this region was determined and the identity of gene E was confirmed by comparing the experimentally determined N-terminal amino acid sequence of the purified protein to the translated DNA sequence of its open reading frame. We present evidence that the gene E product is proteolytically cleaved between Lys(16) and Asn(17) to yield the 305 residue polypeptides found in the mature viral capsid. Processing of the protein itself during virus development was determined by 2D gel electrophoresis using protein E-specific antibodies. Sequence similarity studies revealed an 80% identity to capsid protein Hp32 of phiH, infecting Halobacterium salinarum. RT-PCR analysis as well as Western blot studies revealed gene E as a late gene. Transcripts and proteins could be detected shortly before onset of lysis of the lysogenic strain N. magadii L11.

Amino Acid Sequence↗
PubMed2000

Investigation of 0.2 µm filterable bacteria from the Western Mediterranean Sea using a molecular approach: dominance of potential starvation forms.

Although the existence of 0.2 µm filterable bacteria has been known since the early 80's, they are not taken into consideration when modeling microbial food webs, due to an overall lack of information concerning this specific size class. According to physiological studies on starvation forms and investigations on small bacterial cells in marine ecosystems, a 0.2 µm filtrate may consist of different phenotypes: starvation forms of typical marine bacteria, ultramicrobacteria or bacterial cells, even larger than 0.2 µm, but flexible enough to pass the nominal filter pore-size. In this pilot study we examined three filtered seawater fractions from the Western Mediterranean Sea (Bay of Calvi, Corsica/France) - the total bacterial population, the bacterial fraction above 0.2 µm and the 0.2 µm filtrate - to investigate the bacterial community structure of each of those fractions by the molecular approach of denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments. The analysis of the resulting DGGE profiles revealed different patterns of dominant bands for the 0.2 µm filterable and the total bacterial populations within the samples. Additionally the 0.2 µm filterable bacterial compartment exhibited obvious differences in band patterns for winter and summer samples, which were not observed for the total bacterial fraction. According to the current knowledge concerning the status of 0.2 µm filterable bacteria, DGGE patterns indicate that most of the fragments representing 0.2 µm filterable bacteria were rather starvation forms of marine bacteria than ultramicrobacteria. The sequencing of excised and cloned DNA bands of the DGGE profiles characterized the phylogenetic affiliation of the corresponding 0.2 µm filterable bacteria, clustering mainly with known, typical marine isolates of both alpha-subclass and gamma-subclass of the Proteobacteria and the Cytophaga-Flavobacterium-Bacteroides branch.

Journal Article↗
PubMed2000

The archaeal halophilic virus-encoded Dam-like methyltransferase M. phiCh1-I methylates adenine residues and complements dam mutants in the low salt environment of Escherichia coli.

The genome of the archaeal virus phiCh1, infecting Natrialba magadii (formerly Natronobacterium magadii), is composed of 58.5 kbp linear ds DNA. Virus particles contain several RNA species in sizes of 100-800 nucleotides. A fraction of phiCh1 genomes is modified within 5'-GATC-3' and related sequences, as determined by various restriction enzyme digestion analyses. High performance liquid chromatography revealed a fifth base, in addition to the four nucleosides, which was identified as N6-methyladenosine. Genetic analyses and subsequent sequencing led to the identification of a DNA (N6-adenine) methyltransferase (mtase) gene. The protein product was designated M.phiCh1-I. By the localization of the most conserved motifs (a DPPY motif occurring before FxGxG), the enzyme was placed within the beta-subgroup of the (N6-adenine) methyltransferase class. The mtase gene of phiCh1 was classified as a 'late' gene, as determined by measuring the kinetics of mRNA and protein expression in N. magadii during the lytic cycle of phiCh1. After infection of cells, M.phiCh1-I mRNA and protein could be detected in lower amounts than in the situation of virus induction from lysogenic cells. Consequently, only about 5% of the phiCh1 progeny genomes after infection of N. magadii carry the M.phiCh1-I methylation in contrast to 50% of virus genomes generated by induction of phiCh1-lysogenic N. magadii cells. Heterologous expression of the mtase from a halophile with 3 M cytoplasmic salt concentration showed an unexpected feature: the protein was active in the low environment of Escherichia coli and was able to methylate DNA in vivo. Interestingly, it seemed to exhibit a higher sequence specificity in E. coli that resulted in adenine methylation exclusively in the sequence 5'-GATC-3'. Additionally, expression of M.phiCh1-I in dam- E. coli cells led to a complete substitution of the function of M. Dam in DNA mismatch repair.

Amino Acid Sequence↗
PubMed1999

Extended recombinant bacterial ghost system.

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, peri-plasma and internal lumen of the ghosts can be fully utilized. This extended recombinant ghost system represents a new strategy for adjuvant free combination vaccines.

Adjuvants, Immunologic↗
PubMed1999

Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene E expression in Escherichia coli.

Cell lysis of Gram-negative bacteria can be efficiently achieved by expression of the cloned lysis gene E of bacteriophage PhiX174. Gene E expression is tightly controlled by the rightward lambda pR promoter and the temperature-sensitive repressor cI857 on lysis plasmid pAW12. The resulting empty bacterial cell envelopes, called bacterial ghosts, are currently under investigation as candidate vaccines. Expression of gene E is stringently repressed at temperatures up to 30 degrees C, whereas gene E expression, and thus cell lysis, is induced at temperatures higher than 30 degrees C due to thermal inactivation of the cI857 repressor. As a consequence, the production of ghosts requires that bacteria have to be grown at 28 degrees C before the lysis process is induced. In order to reflect the growth temperature of pathogenic bacteria in vivo, it seemed favorable to extend the heat stability of the lambda pR promoter/cI857 repressor system, allowing pathogens to grow at 37 degrees C before induction of lysis. In this study we describe a mutation in the lambda pR promoter, which allows stringent repression of gene E expression at temperatures up to 36 degrees C, but still permits induction of cell lysis at 42 degrees C.

Bacteriolysis↗
PubMed1999

New strategies for combination vaccines based on the extended recombinant bacterial ghost system.

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts have been produced from a great variety of bacteria and are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extents the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens, immunomodulators or other substances. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in bacterial candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying inserts of foreign epitopes of up to 600 amino acids within the flexible surface loop areas of the S-layer further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts do not need the addition of adjuvants to induce immunity in experimental animals they can also be used as carriers or targeting vehicles or as adjuvants in combination with subunit vaccines. Matrixes like dextran which can be used to fill the internal lumen of ghosts can be substituted with various ligands to bind the subunit or other materials of interest. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of ghosts and recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in the production of ghosts. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. As carriers of foreign antigens there is no limitation in the size of foreign antigens to be inserted and the capacity of all spaces including the membranes, periplasma and internal lumen of the ghosts can be fully utilized. Using the different building blocks and combining them into the recombinant ghost system represents a new strategy for adjuvant free combination vaccines.

Adjuvants, Immunologic↗
PubMed1999

Polyphasic classification of 0.2 microm filterable bacteria from the western Mediterranean Sea

The 0.2 microm filtration of sea water samples from the Mediterranean Sea (Bay of Calvi, Corsica), collected from 10 m and 35 m depth led to the isolation of several gram-negative bacterial strains able to grow on full-strength media as well as on diluted media. The analysis of the 16S rRNA gene sequences and estimation of the phylogenetic relationships of these facultative oligotrophic bacteria indicated that they grouped into two phylogenetic branches. The strains RE10F/2, RE10F/5 (10 m depth samples) and RE35/F12 (35 m depth samples) were assigned to the gamma-subclass, while RE35F/1 (35m depth sample) was assigned to the alpha-4-subclass of the Proteobacteria. The strains RE10/F2 and RE10/F5 were most closely related to species and strains of the Pseudoalteromonas group, whereas the strain RE35F/12 placed adjacent to the family Vibrionaceae. The phylogenetic analysis of strain RE35F/1 revealed that this bacterium clusters with marine strains and species of the aerobic anoxygenic phototrophic bacteria Erythrobacter as well as Erythromicrobium and more distantly to Sphingomonas spp. Supplementary to those genotypic classifications the chemotaxonomic signatures including the major respiratory lipoquinone systems, the cellular fatty acid compositions as well as the polyamine contents of the bacteria were investigated. The isolated organisms displayed differences in their physiological and biochemical properties to already described strains belonging to the same genera or families, as revealed by the comparative 16S rRNA analysis. Despite the fact that these bacteria were isolated from a 0.2 microm filtrate, the cultured organisms which were all rod-shaped, displayed width dimensions ranging from 0.4 up to 0.7 microm, indicating that these bacteria were starvation forms at the time of isolation and not ultramicrobacteria as defined by Torella and Morita (1981) or by Schut et al. (1993). Because our isolated strains represent potentially new taxa, this first investigation on 0.2 pm filterable bacteria from the Western Mediterranean Sea supports the hypothesis that this bacterial fraction contributes to the diversity of marine bacteria.

Journal Article↗
PubMed1999

Gene transfer to mammalian cells using genetically targeted filamentous bacteriophage.

We have genetically modified filamentous bacteriophage to deliver genes to mammalian cells. In previous studies we showed that noncovalently attached fibroblast growth factor (FGF2) can target bacteriophage to COS-1 cells, resulting in receptor-mediated transduction with a reporter gene. Thus, bacteriophage, which normally lack tropism for mammalian cells, can be adapted for mammalian cell gene transfer. To determine the potential of using phage-mediated gene transfer as a novel display phage screening strategy, we transfected COS-1 cells with phage that were engineered to display FGF2 on their surface coat as a fusion to the minor coat protein, pIII. Immunoblot and ELISA analysis confirmed the presence of FGF2 on the phage coat. Significant transduction was obtained in COS-1 cells with the targeted FGF2-phage compared with the nontargeted parent phage. Specificity was demonstrated by successful inhibition of transduction in the presence of excess free FGF2. Having demonstrated mammalian cell transduction by phage displaying a known gene targeting ligand, it is now feasible to apply phage-mediated transduction as a screen for discovering novel ligands.

Animals↗