PubMed Health⌕ Search

Biomedical subjects

D Meijer

Publications and source records attributed to D Meijer.

At least 19 recordsLinked to original sources
PubMed2004

Construction of stabilized proteins by combinatorial consensus mutagenesis.

We constructed stabilized variants of beta-lactamase (BLA) from Enterobacter cloacae by combinatorial recruitment of consensus mutations. By aligning the sequences of 38 BLA homologs, we identified 29 positions where the E.cloacae gene differs from the consensus sequence of lactamases and constructed combinatorial libraries using mixtures of mutagenic oligonucleotides encompassing all 29 positions. Screening of 90 random isolates from these libraries identified 15 variants with significantly increased thermostability. The stability of these isolates suggest that all tested mutations make additive contributions to protein stability. A statistical analysis of sequence and stability data identified 11 mutations that made stabilizing contributions and eight mutations that destabilized the protein. A second-generation library recombining these 11 stabilizing mutations led to the identification of BLA variants that showed further stabilization. The most stable variant had a mid-point of thermal denaturation (Tm) that was 9.1 degrees C higher than the starting molecule and contained eight consensus mutations. Incubation of three stabilized BLA variants with several proteases showed that all tested isolates have significantly increased resistance to proteolysis. Our data demonstrate that combinatorial consensus mutagenesis (CCM) allows the rapid generation of protein variants with improved thermal and proteolytic stability.

Amino Acid Sequence↗
PubMed2001

Cloning and expression of an endocellulase gene from a novel streptomycete isolated from an East African soda lake.

Alkaline cellulase-producing actinomycete strains were isolated from mud samples collected from East African soda lakes. The strains were identified as novel Streptomyces spp. by 16S rDNA sequence analysis. A cellulase gene (cel12A) from Streptomyces sp. strain 11AG8 was cloned by expression screening of a genomic DNA library in Escherichia coli. From the nucleotide sequence of a 1.5-kb DNA fragment, an open reading frame of 1,113 nucleotides was identified encoding a protein of 371 amino acids. From computer analysis of the sequence, it was deduced that the Cel12A mature enzyme is a protein of 340 amino acids. The protein contained a catalytic domain, a glycine-rich linker region, and a cellulose-binding domain of 221, 12, and 107 amino acids, respectively. FASTA analysis of the catalytic domain of Cel12A classified the enzyme as a family 12 endoglucanase and the cellulose-binding domain as a family IIa CBD. Streptomyces rochei EglS was determined as nearest neighbor with a similarity of 75.2% and 61.0% to the catalytic domain and the cellulose-binding domain, respectively. The cell2A gene was subcloned in a Bacillus high-expression vector carrying the Bacillus amyloliquefaciens amylase regulatory sequences, and the construct was transformed to a Bacillus subtilis host strain. Crude enzyme preparations were obtained by ultrafiltration of cultures of the Bacillus subtilis recombinant strain containing the 11AG8 cell2A gene. The enzyme showed carboxymethylcellulase (CMCase) activities over a broad pH range (5-10) with an optimum activity at pH 8 and 50 degrees C. The enzyme retained more than 95% of its activity after incubation for 30 min under these conditions.

Africa, Eastern↗
PubMed2000

A distal Schwann cell-specific enhancer mediates axonal regulation of the Oct-6 transcription factor during peripheral nerve development and regeneration.

The POU domain transcription factor Oct-6 is a major regulator of Schwann cell differentiation and myelination. During nerve development and regeneration, expression of Oct-6 is under the control of axonal signals. Identification of the cis-acting elements necessary for Oct-6 gene regulation is an important step in deciphering the complex signalling between Schwann cells and axons governing myelination. Here we show that a fragment distal to the Oct-6 gene, containing two DNase I-hypersensitive sites, acts as the Oct-6 Schwann cell-specific enhancer (SCE). The SCE is sufficient to drive spatially and temporally correct expression, during both normal peripheral nerve development and regeneration. We further demonstrate that a tagged version of Oct-6, driven by the SCE, rescues the peripheral nerve phenotype of Oct-6-deficient mice. Thus, our isolation and characterization of the Oct-6 SCE provides the first description of a cis-acting genetic element that responds to converging signalling pathways to drive myelination in the peripheral nervous system.

Animals↗
PubMed2000

Halomonas magadii sp. nov., a new member of the genus Halomonas, isolated from a soda lake of the East African Rift Valley.

A number of novel alkaliphilic organotrophic bacteria have been isolated from several saline and alkaline East African soda lakes. The new isolates grow at pH values between 7.0 and 11.0, with pH optima for growth between 9.0 and 10.0. Growth occurs at total salts concentration between 0% and 20% (w/v) with optimum at 0%-7% (w/v). Phylogenetic analyses based on 16S rDNA sequence comparison indicate that these isolates are related (>96% similarity) to members of the Halomonadaceae within the gamma-3 subdivision of the Proteobacteria. These analyses indicate that existing species within the Halomonadaceae fell within three main groups, one group comprising the type species of Halomonas, Halomonas elongata, and a number of other known species including one soda lake isolate. A second group constituting most of the remaining known species of Halomonas and related Chromohalobacter spp. includes 3 soda lake isolates with high DNA-DNA homologies. The third group included Halomonas halodenitrificans, Halomonas desiderata, Halomonas cupida, and 13 soda lake isolates. Phenotypic comparisons indicated that the majority of soda lake strains shared similar morphological, phenotypic, and chemotaxonomic properties to known strains of Halomonas but grew under alkaline conditions. The 3 soda lake isolates with high DNA-DNA homologies were, however, significantly different in antibiotic sensitivity pattern and in the utilization of several substrates, were unable to reduce nitrite, and showed low DNA-DNA homologies with known halomonads in the same group. We propose that these isolates comprise a new species of the genus Halomonas that we name Halomonas magadii sp. nov. The type strain is strain 21 MI (NCIMB 13595).

Africa↗
PubMed2000

Erythroid defects and increased retrovirally-induced tumor formation in Evi1 transgenic mice.

Aberrant expression of the Evi1 (ecotropic virus integration site 1) proto-oncogene has been associated with hematopoietic malignancies in both mice and man. To determine the effect of enforced expression of Evi1 in vivo, we developed a transgenic mouse model utilizing the murine Sca-1 (Ly-6E.1) promoter. Here, we describe the generation and analysis of three independent lines of Evi1 transgenic mice. Transgenic animals of two founder lines developed normally. These mice did not show any obvious hematological abnormalities but showed a significant reduction in the number of bone marrow colony-forming unit erythroid (CFU-E)-derived colonies. This implies a defect of normal erythroid hematopoiesis affecting relatively late erythroid progenitor cells. We also show that when newborn Evi1 transgenic mice of these two lines were infected with Cas-Br-M MuLV, tumor incidence was greatly enhanced in comparison with nontransgenic littermates, indicating an increased susceptibility for leukemia development. Interestingly, analysis of a third founder line revealed that all male progeny consistently displayed severely impaired erythropoiesis with major defects in the bone marrow, spleen and peripheral blood. Taken together, our results present the first evidence of Evi1 disturbing normal erythropoiesis in vivo and provides evidence for cooperative potential of Evi1 in tumor progression.

Animals↗
PubMed1999

Transcriptional regulation of the POU gene Oct-6 in Schwann cells.

Genetic evidence suggests that the POU transcription factor Oct-6 plays a pivotal role as an intracellular regulator of Schwann cell differentiation. In the absence of Oct-6 function Schwann cells are generated in appropriate numbers and these cells differentiate normally up to the promyelin stage at which they transiently arrest. During peripheral nerve development Oct-6 expression is initiated in Schwann cell precursors and is strongly upregulated in promyelin cells. Oct-6 expression is subsequently extinguished in terminally differentiating Schwann cells. Thus, identification and characterisation of the DNA elements involved in this stage specific regulation may lead us to the signaling cascade and the axon-derived signals that drive Schwann cell differentiation and initiate myelination. Here we present experiments that aim at identifying such regulatory sequences.

Animals↗
PubMed1999

Ultrasonic dissection for endoscopic surgery. The E.A.E.S. Technology Group.

With the development of endoscopic surgery, new hazards of high-frequency (HF) electrosurgery have been recognized. The potential risks of monopolar electrosurgery, the limitations of bipolar technique, and the need to reduce instrument interchange have favored the use of ultrasonic technology, which becomes more and more popular. This work aims at presenting the main features of the currently available ultrasonically activated scalpels, as well as their advantages, limitations, and indications.

Dissection↗
PubMed1998

Role of Oct-6 in Schwann cell differentiation.

Research into the POU transcription factor Oct-6 has been the focus of much current attention, in particular its role in Schwann cell development and differentiation. Based on published data and data presented here, we propose a model for Oct-6 function at two distinct stages of Schwann cell maturation. First, Oct-6 function is required in promyelin cells for their timely differentiation into myelinating cells. Second, Oct-6 functions during myelination and is required for the proper downregulation of its own gene. While the first function of Oct-6 is firmly established, the second function is still highly hypothetical. Experiments to establish a distinct role for Oct-6 in late Schwann cell differentiation are discussed.

Animals↗
PubMed1998

Dietzia natronolimnaios sp. nov., a new member of the genus Dietzia isolated from an east African soda lake.

Two novel alkaliphilic aerobic organotrophic bacteria have been isolated from a moderately saline and alkaline East African soda lake. The new isolates grow at pH values between 6 and 10, with a pH optimum for growth of 9.0, and at a salt concentration between 0% and 10% (w/v). Phylogenetic analysis based on 16S rDNA sequence shows that these isolates are very closely related (99.6% similarity) and are members of the monospecific genus Dietzia (98.8% and 98.7% similarity). DNA/DNA hybridization revealed a relatedness of 83% between the two isolates, but only 8% between them and the type strain Dietzia maris. The G + C content as measured by thermal denaturation is 66.1 mol%. Phenotypic comparisons between D. maris and one isolate showed that they share very similar morphological and chemotaxonomic properties, but differ significantly in carbon source utilization profiles and halotolerance in alkaline medium. We propose a second species of this genus which we name Dietzia natronolimnaios (type strain 15LN1 = CBS 107.95).

Bacteria, Aerobic↗
PubMed1998

Comparison of sequence and function of the Oct-6 genes in zebrafish, chicken and mouse.

To examine the role of the Oct-6 gene in Schwann cell differentiation we have cloned and characterized the chicken and zebrafish homologues of the mouse Oct-6 gene. While highly homologous in the Pit1-Oct1/2-Unc86 (POU) domain, sequence similarities are limited outside this domain. Both genes are intronless and both proteins lack the amino acid repeats that are a characteristic feature of the mammalian Oct-6 proteins. However as in mammals, the aminoterminal parts of the chicken and zebrafish Oct-6 proteins are essential for transactivation of octamer containing promoters. By immunohistochemistry we have found that the chicken Oct-6 protein is expressed in late embryonic ensheathing Schwann cells of the sciatic nerve and is rapidly downregulated when myelination proceeds. This expression profile in glial cells is identical to that in the mouse and rat. Furthermore the zebrafish Oct-6 homolog is expressed in the posterior lateral nerve at a time when it contains actively myelinating Schwann cells. Thus despite extensive primary sequence divergence among the vertebrate Oct-6 proteins, the expression of the chicken and zebrafish Oct-6 proteins is consistent with the notion that Oct-6 functions as a 'competence factor' in promyelin cells to execute the myelination program.

Amino Acid Sequence↗
PubMed1996

Oct-6 (SCIP/Tst-1) is expressed in Schwann cell precursors, embryonic Schwann cells, and postnatal myelinating Schwann cells: comparison with Oct-1, Krox-20, and Pax-3.

The POU domain transcription factor Oct-6 (SCIP/Tst-1) is likely to control important stages of Schwann cell development, including the initiation of myelination around birth. Here, we use immunocytochemical and reverse transcriptase-polymerase chain reaction techniques to examine Oct-6 earlier in nerve development, to test the idea that Oct-6 has an additional role in Schwann cell precursors or early embryonic Schwann cells, a possibility raised by previous studies on transgenic mice. Consistent with this, we find low but unambiguous levels of Oct-6 mRNA and protein in Schwann cell precursors of mouse and rat (nerves from 12- and 14-day-old embryos, respectively), with expression levels gradually increasing during early Schwann cell development and towards birth. Unexpectedly, Oct-6 immunoreactivity is clearly present in nuclei of most myelinating cells at least as late as postnatal day 12. Furthermore, many nonmyelinating Schwann cells express Oct-6 in adult life. A comparison of Oct-6 mRNA with other Schwann cell transcription factors-namely, Oct-1, Krox-20, and Pax-3-reveals that each factor exhibits strong developmental regulation and a unique expression pattern in embryonic nerves. Therefore, they are likely to play distinct regulatory roles in early development of the Schwann cell lineage.

Animals↗
PubMed1996

The POU factor Oct-6 and Schwann cell differentiation.

The POU transcription factor Oct-6, also known as SCIP or Tst-1, has been implicated as a major transcriptional regulator in Schwann cell differentiation. Microscopic and immunochemical analysis of sciatic nerves of Oct-6(-/-) mice at different stages of postnatal development reveals a delay in Schwann cell differentiation, with a transient arrest at the promyelination stage. Thus, Oct-6 appears to be required for the transition of promyelin cells to myelinating cells. Once these cells progress past this point, Oct-6 is no longer required, and myelination occurs normally.

Animals↗
PubMed1996

The restricted expression pattern of the POU factor Oct-6 during early development of the mouse nervous system.

Oct-6 is a POU transcription factor that is thought to play a role in the differentiation of cells of neuroectodermal origin. To investigate whether the Oct-6 protein could play a role in the establishment of neuroectoderm in vivo we studied the expression of the Oct-6 protein during early mouse development. Expression is first observed in the primitive ectoderm of the egg cylinder stage embryo. In gastrulating embryos, Oct-6 protein is found in the extra-embryonic ectoderm of the chorion and the anterior ectoderm of the embryo proper. As development proceeds, Oct-6 expression becomes more restricted to the anterior medial part of the embryo until Oct-6 positive cells are observed only in the neural groove of the headfold stage embryo. In the late headfold stage embryo, Oct-6 expression is detected in the neuroepithelium of the entire brain and later is restricted to a more ventral and anterior position. As the anterior neuropore closes, Oct-6 protein is detected in a segment-like pattern in the mid-and forebrain. Thus, the expression pattern of the Oct-6 gene agrees with a role for the Oct-6 protein in the establishment and regional specification of the neuroectoderm in vivo. The two waves of widespread induction of the Oct-6 gene, one in the primitive ectoderm and another in the primitive brain, both followed by a progressive restriction in the expression patterns suggest a mechanism for the regulation of the gene.

Animals↗
PubMed1996

Beagle model used in a tissue tolerance study of the response of normal and surgically manipulated liver to single high-dose intraoperative radiotherapy.

To investigate the feasibility of delivering a single large dose of intraoperative electron beam radiotherapy (IORT) to the liver of clinically normal and partially hepatectomized beagles, an experimental study was designed. The purpose of the study was to obtain dose guidelines for the delivery of IORT to the liver of human patients with colorectal cancer metastases to the liver. After partial resection of the liver, IORT in doses up to 30 Gy was applied to the resection plane as well as to a nonsurgically manipulated part of the liver of 25 beagles. The temporal sequence of histologic changes of these irradiated parts of the liver tissue was investigated. There were no postoperative complications and no morbidity or mortality associated with a minimal follow-up of 3 years. Necropsy performed 3 months after IORT revealed only mild histopathologic changes. One year after IORT, more distinct histopathologic changes consisting of capsular thickening, diffuse parenchymal fibrosis, and subcapsular hepatocellular atrophy were found. Three years after IORT, the parenchymal architecture seemed to be restored, although loss of liver tissue was definitive at the irradiation site; liver function remained intact. These results indicate that IORT to part of the liver in the canine model can be safely applied and that, although doses up to 30 Gy can result in severe local tissue damage, wound healing and liver function are not disturbed.

Animals↗
PubMed1995

[Laparoscopic splenectomy revisited].

We present a new technique for laparoscopic splenectomy which allows manual control of endoscopic dissection. After induction of pneumoperitoneum and insertion of an endoscope to check the absence of any contraindications, an incision is made for insertion of the hand and forearm. An airtight system allowing insertion of the hand while maintaining the pneumoperitoneum is fixed around the incision. A second port is inserted in the left flank. The procedure is then performed with laparoscopic instruments while the left hand allows for spleen mobilisation and easier exposure of the structures to be dissected and divided. Eight patients with a mean age of 51.3 years have been operated on for Idiopathic Thrombocytopenia. The average duration of the procedure was 90 min (80-130 min). No postoperative complication occurred. The mean duration of stay was 3.8 days (3-7 days). These results of this technique are encouraging. The procedure is quicker and safer than an exclusive laparoscopic approach. The incision allows removal of the intact spleen. Other applications of this system can be envisaged.

Adult↗
PubMed1995

Induction of differentiation-regulated transcription factor Oct-6 specifically accompanies major histocompatibility complex class I down-regulation by E1A of oncogenic adenovirus type 12.

The E1A genes from adenovirus (Ad) types 5 and 12 share the capacity to cooperate with a second oncogene to transform primary rodent cells in vitro. However, only Ad12-transformed cells are oncogenic in immunocompetent rodents, an event that requires conserved region 3 (CR3) of E1A to be intact. Ad12-induced tumorigenicity correlates with the E1A-CR3-dependent down-modulation of MHC class I transcription, contributing to escape from CTL-mediated immune surveillance. Expression of MHC class I antigens is also lacking in undifferentiated embryonal carcinoma cells. In these cells, MHC class I expression increases during differentiation in a process possibly involving octamer-binding proteins. We found that both nononcogenic and oncogenic Ad-transformed cells contained the ubiquitously expressed factor Oct-1. In contrast, only oncogenic Ad12-transformed cells that are derived from primary cell cultures expressed an additional octamer-binding factor, which we identified as Oct-6. The induction of Oct-6 expression was at the RNA level and was found to require an intact CR3 domain in Ad12 E1A. Like MHC class I expression, Oct-6 expression was not affected in already established cell lines expressing Ad12 E1A. The presence of Oct-6 in Ad12-transformed cells correlated with an increase in octamer-dependent transcription of a reporter gene, relative to Ad5-transformed cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenovirus E1A Proteins↗
PubMed1993

The mouse low density lipoprotein receptor gene: cDNA sequence and exon-intron structure.

The low density lipoprotein (LDL) receptor plays a central role in the cholesterol metabolism. The cDNA sequence of the mouse low density lipoprotein receptor (Ldlr) gene has been determined and shows 76% homology with the human gene. The exon-intron structure has been determined for the 129/J mouse strain. The gene is composed of 18 exons and spans a region of 28 kb. In addition, the promoter regions of the mouse and human genes are homologous. Northern blot analysis revealed an mRNA of approximately 5 kb. The cloning of the Ldlr gene will enhance the usefulness of the mouse for the study of cholesterol metabolism and, in particular, for carrying out gene targeting experiments.

Amino Acid Sequence↗