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D Wöhrle

Publications and source records attributed to D Wöhrle.

At least 19 recordsLinked to original sources
PubMed2001

X chromosome-specific cDNA arrays: identification of genes that escape from X-inactivation and other applications.

Mutant alleles are frequently characterized by low expression levels. Therefore, cDNA array-based gene expression profiling may be a promising strategy for identifying gene defects underlying monogenic disorders. To study the potential of this approach, we have generated an X chromosome-specific microarray carrying 2423 cloned cDNA fragments, which represent up to 1317 different X-chromosomal genes. As a prelude to testing cell lines from patients with X-linked disorders, this array was used as a hybridization probe to compare gene expression profiles in lymphoblastoid cell lines from normal males, females and individuals with supernumerary X chromosomes. Measurable hybridization signals were obtained for more than half of the genes represented on the chip. A total of 53 genes showed elevated expression levels in cells with multiple X chromosomes and many of these were found to escape X-inactivation. Moreover, the detection of a male-viable deletion encompassing three genes illustrates the utility of this array for the identification of small unbalanced chromosome rearrangements.

Alleles↗
PubMed2000

Increase of FMRP expression, raised levels of FMR1 mRNA, and clonal selection in proliferating cells with unmethylated fragile X repeat expansions: a clue to the sex bias in the transmission of full mutations?

Fragile X syndrome is a triplet repeat disorder caused by expansions of a CGG repeat in the fragile X mental retardation gene (FMR1) to more than 220 triplets (full mutation) that usually coincide with hypermethylation and transcriptional silencing. The disease phenotype results from deficiency or loss of FMR1 protein (FMRP) and occurs in both sexes. The underlying full mutations arise exclusively on transmission from a mother who carries a premutation allele (60-200 CGGs). While the absolute requirement of female transmission could result from different mechanisms, current evidence favours selection or contraction processes acting at gametogenesis of pre- and full mutation males. To address these questions experimentally, we used a model system of cultured fibroblasts from a male who presented heterogeneous unmethylated expansions in the pre- and full mutation size range. On continual cell proliferation to 30 doublings we re-examined the behaviour of the expanded repeats on Southern blots and also determined the expression of the FMR1 gene by FMRP immunocytochemistry, western analysis, and RT-PCR. With increasing population doublings, expansion patterns changed and showed accumulation of shorter alleles. The FMRP levels were below normal but increased continuously while the cells that were immunoreactive for FMRP accumulated. The level of FMR1 mRNA was raised with even higher levels of mRNA measured at higher passages. Current results support the theory of a selection advantage of FMRP positive over FMRP deficient cells. During extensive proliferation of spermatogonia in fragile X males, this selection mechanism would eventually replace all full mutations by shorter alleles allowing more efficient FMRP translation. At the proliferation of oogonia of carrier females, the same mechanism would, in theory, favour transmission of any expanded FMR1 allele on inactive X chromosomes.

Base Sequence↗
PubMed1999

Effect of delivery system on the pharmacokinetic and phototherapeutic properties of bis(methyloxyethyleneoxy) silicon-phthalocyanine in tumor-bearing mice.

A Si(IV)-phthalocyanine bearing two methoxyethyleneglycol axial ligands bound to the central metal ion (SiPc) has been prepared by chemical synthesis and analyzed for its phototherapeutic activity after administration in a Cremophor or liposome formulation to C57B1/6 mice bearing a subcutaneously transplanted Lewis lung carcinoma (LLC). The maximum drug accumulation in the tumor is found at 24 h after intraperitoneal injection, independent of the delivery system. However, the tumor concentration of SiPc in the Cremophor formulation is about two-fold higher, while the drug concentration in liver and skin shows similar trends with the two delivery systems. The drug accumulation and retention in the brain is much larger when using Cremophor emulsion. Photodynamic therapy (672 nm, 370 mW m-2, 360 J cm-2) at 24 h after the injection of Cremophor emulsion- or DPPC liposome-formulated SiPc causes a very efficient and similar response for the LLC (approximately 8 versus 22 mm mean tumor diameter for the control groups at 21 days after phototreatment). These very promising effects, obtained both at higher and lower tumor drug concentrations, clearly demonstrate the potential phototherapeutical activity of the newly synthesized SiPc.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1998

Unusual mutations in high functioning fragile X males: apparent instability of expanded unmethylated CGG repeats.

We report on further cases of high functioning fragile X males showing decreased expression of FMR1 protein, absence of detectable methylation at the EagI site in the FMR1 gene promoter, and highly unusual patterns of fragile X mutations defined as smear of expansions extending from premutation to full mutation range. Very diffuse and therefore not easily detectable patterns of full mutations were also observed on prenatal testing using DNA from chorionic villi sampled at a time of development when full mutations were still unmethylated in this particular tissue. In the search for possible determinants of such unusual patterns, repeat expansions in the premutation and in the lower full mutation range were identified on genomic PstI blots previously prepared for fragile X DNA testing. Cases with 130 or more triplets, and a number of shorter repeats, were reinvestigated on EcoRI plus EagI digests. Among the 119 expansions, there were 22 in our sample showing either blurred bands or smears on PstI blots. This particular characteristic was strongly associated with the coincidence of a repeat size of more than 130 triplets and absence of EagI site methylation. Our data set also includes cases of mosaic patterns consisting of smears of unmethylated expansions to more than 130 CGGs and of clear bands of methylated expansions. We therefore suggest that in fragile X syndrome unusual smeared patterns of mutations result from somatic instability of larger repeats under circumstantial absence of repeat methylation.

Blotting, Southern↗
PubMed1997

Si(IV)-methoxyethylene-glycol-naphthalocyanine: synthesis and pharmacokinetic and photosensitizing properties in different tumour models.

A Si(IV)-naphthalocyanine bearing two methoxyethylenglycol axial ligands to the centrally coordinated metal ion (SiNc) was prepared by chemical synthesis and assayed for the phototherapeutic activity after administration in a Cremophor formulation to C57BI/6 mice bearing a subcutaneously transplanted Lewis lung carcinoma or B16 pigmented melanoma. Pharmacokinetic studies indicate that the maximal accumulation in the tumour occurs at 24 h after intraperitoneal injection of 0.5 mg kg-1 of SiNc, although the naphthalocyanine concentration in the Lewis lung carcinoma (0.70 microgram g-1) is significantly larger than that in the B16 pigmented melanoma (0.15 microgram g-1). This results in a higher selectivity of tumour targeting in the case of the lung carcinoma. Photodynamic therapy (782 nm, 370 mW cm-2, 360 J cm-2) at 24 h after SiNc injection causes an efficient tumour response for Lewis lung carcinoma (50% lower tumour diameter on day 19 post-treatment as compared to untreated controls) while the pigmented melanoma shows only a minor response regarding the rate of tumour growth.

Animals↗
PubMed1997

Characterization of FMR1 promoter elements by in vivo-footprinting analysis.

Fragile X syndrome is associated with silencing of the FMR1 gene. We studied the transcriptional regulation, by analysis of the FMR1 promoter region for the presence of in vivo protein/DNA interactions and for cytosine methylation at the single-nucleotide level. Four protein-binding sites were present in the unmethylated promoter of the active FMR1 gene. In the methylated promoter of inactive genes no footprints were detected, and no evidence of active repression was found in the region investigated. We propose that the silencing of FMR1 gene transcription results from a lack of transcription-factor binding.

Adult↗
PubMed1996

Tetraamido-substituted 2,3-naphthalocyanine zinc(II) complexes as phototherapeutic agents: synthesis, comparative photochemical and photobiological studies.

The aim of this study is to report the synthesis and photochemical and phototherapeutic activities of tetraamido-substituted 2,3-naphthalocyanine zinc(II) complexes (ZnNcs 5-8). Four naphthalocyanine complexes, tetrabenzamido- (ZnNc 5), tetramethoxybenzamido- (ZnNc 6), tetrahexylamido- (ZnNc 7) and tetradodecylamido- (ZnNc 8) naphthalocyanine zinc complexes absorbing at around 770 nm were synthesized. The dye-sensitized photo-oxidation of 1,3-diphenylisobenzofuran via 1O2 was studied in more detail in order to compare the quantum yields of these different sensitizers. Pharmacokinetic and photodynamic therapy studies of Lewis lung carcinoma in mice were carried out after administration of liposome incorporated ZnNcs 5-8. The phototherapeutic efficiency was evaluated by changes in the mean tumour diameter with time, regrowth delay (days), average survival time (days) and electron microscopy observations. According to all assessment criteria used, the most promising photosensitizer seems to be tetrabenzamido-substituted ZnNc 5. We suggest that mainly structural properties are the reason for the better results observed. The morphological analysis confirms the mechanism of photonecrosis, which was observed in our previous work with unsubstituted and substituted ZnNcs 1-4. Direct photodamage to the membrane, mitochondria and rough endoplasmic reticulum in the neoplastic cells and delayed photodestruction of the endothelial cells surrounding the tumour tissue were detected. Also, changes in lysosomes were observed. The data presented through different parameters are compared with such obtained for other photodynamic therapy sensitizers.

Animals↗
PubMed1996

Unsymmetrically substituted benzonaphthoporphyrazines: a new class of cationic photosensitizers for the photodynamic therapy of cancer.

Unsymmetrical zinc(II) complexes of benzonaphthoporphyrazines 5a-12a bearing between one and eight pyridyloxy substituents are synthesized by statistical tetramerization of 6-(1,1-dimethylethyl)-2,3-naphthalenedicarbonitrile (1) with 4-(3-pyridyloxy)- or 4,5-bis-(3-pyridyloxy)-1,2-benzenedicarbonitrile (2, 3). Methylation of 5a-12a leads to the catianic pyridyloxybenzonaphthoporphyrazines 5b-12b having between one to eight positive charges. The Q-band transition in the visible spectra exhibits a bathochromic shift from 680 to 760 nm dependent upon the number of annelated naphthalene rings. The singlet oxygen quantum yields of the benzonaphthoporphyrazines determined by the dye-sensitized photooxidation of 1,3-diphenylisobenzofurane is surprisingly high (in the range of zinc phthalocyanine). The photooxidative stabilities of the photosensitizers described quantitatively by first-order kinetics decrease with the number of annelated naphtho groups. A linear correlation between the logarithm of the decomposition rate constant and the position of the highest occupied molecular orbital (HOMO)-level of the photosensitizers is found. Destabilization of the HOMO leads to a decrease of the photostability. Due to their adjustable long wavelength absorption, their intramolecular polarity axis and their different hydrophilic/hydrophobic character, these novel compounds may be suitable photosensitizers for the photodynamic therapy of cancer.

Cations↗
PubMed1996

FRAXE testing.

Explore the source record for details and available documents.

Female↗
PubMed1995

The X-chromosomal human biglycan gene BGN is subject to X inactivation but is transcribed like an X-Y homologous gene.

We report the mRNA and protein expression levels of human biglycan (BGN) in patients with different numbers of sex chromosomes. BGN maps to the distal long arm of the X chromosome, band Xq28, near the second pseudoautosomal region. BGN expression levels are reduced in 45,X Turner patients and increased in patients with additional sex chromosomes. This is suggestive of a pseudoautosomal gene or a gene that escapes X inactivation and that has an active Y chromosomal copy. However, we also provide evidence from hybrid cell lines that BGN is subject to X inactivation and that there is no homolog on the Y chromosome. This evidence excludes an escape from X inactivation. Moreover, additional Y chromosomes increase BGN expression levels, despite the absence of a Y chromosomal BGN gene. Therefore, another explanation has to be invoked. The "pseudoautosomal expression" of BGN may be attributed to a gene or genes that escape X inactivation and that regulate the transcriptional activity of BGN. This is the first report concerning an X chromosomal gene that does not show the conventional correlation between gene dosage and expression rate known from other X chromosomal genes.

Adult↗
PubMed1995

Heterogeneity of DM kinase repeat expansion in different fetal tissues and further expansion during cell proliferation in vitro: evidence for a casual involvement of methyl-directed DNA mismatch repair in triplet repeat stability.

We have analysed the mitotic behaviour of expanded CTG repeats in somatic tissues and cultured somatic cells from myotonic dystrophy (DM) fetuses using indirect and direct methods. Heterogeneity of expansions between fetal tissues was demonstrated in a 16 week old fetus whereas there was no evidence for such a somatic heterogeneity in a 13 week old fetus. Dilution plating of cultured cells from an adult patient and a fetus resulted in isolation of clones showing single expanded restriction fragments when the donor showed a heterogeneous smear of expansions or a single expanded fragment. During proliferation in vitro to 45 doublings, DM cells experienced highly synchronous further repeat expansion which first became evident at approximately 15 cell generations and reached a plateau of maximum expansion at approximately 200 days. When mathematically expressed as a function of culture time the dynamics of expansion of restriction fragments followed a sigmoid curve. This unstable behaviour of CTG repeat expansions in DM was compared to the mitotically stable patterns of full mutation in fragile X fetal tissues and led to the hypothesis that methylation of CpGs within the repeat sequence is a stabilizing factor of largely expanded CGG and GCC repeats allowing for efficient methyl-directed strand-specific DNA mismatch repair.

Adult↗
PubMed1993

Molecular analysis of mutations in the gene FMR-1 segregating in fragile X families.

Molecular genetic analysis of the transmission of mutations in 73 families with fragile X (one of the largest samples evaluated so far) has confirmed previous hypotheses that the fragile X syndrome results from two consecutive mutational steps, designated "premutation" and "full fragile X mutation". These mutations give rise to expansions of restriction fragments, most probably by amplification of the FMR-1 CGG repeat. Premutations are identified by small expansions that apparently have no effect on either the clinical or the cellular phenotype. Full mutations are reflected by large expansions and hypermethylation of the expanded gene region. All males showing large expansions were affected. Individuals with full mutations also expressed the fragile X, with only one exception. An affected "mosaic" male, showing a predominance of premutated fragments in his leukocytes, was shown to be fragile-X-negative on different occasions. About 50% of heterozygotes with full mutations were reported by clinicians to be mentally retarded. Conversion of the premutation to the full mutation may occur at oogenesis, as previously suggested, or after formation of a zygote at an early transitional stage in development when the CGG repeat behaves as a mitotically unstable element on maternally derived/imprinted X chromosomes carrying a premutation of sufficient repeat length.

Alternative Splicing↗
PubMed1993

Liposome-delivered Zn(II)-2,3-naphthalocyanines as potential sensitizers for PDT: synthesis, photochemical, pharmacokinetic and phototherapeutic studies.

The aim of this investigation is to report the synthesis and fundamental photochemical properties of naphthalocyanines with potential interest for photodynamic therapy (PDT), as well as their pharmacokinetics and phototherapeutic effects in a tumor model. Four zinc naphthalocyanines (ZnNc), unsubstituted ZnNc 1, tetraacetylamido-substituted ZnNc 2, tetraamino-substituted ZnNc 3 and tetramethoxy-substituted ZnNc 4 absorbing around 760-770 nm, were synthesized. The dye-sensitized photo-oxidation of 1,3-diphenylisobenzofuran via 1O2 was studied in dimethylsulfoxide (DMSO). Quantum yields for this photoreaction are 0.135-0.164 and are relatively independent of the kind of substituent. In addition, the photoinduced electron transfer studied in N,N-dimethylformamide-water in the presence of methylviologen and mercaptoethanol is only slightly influenced by the kind of substituent. The pharmacokinetic properties of ZnNc 1 in hamsters bearing a transplanted rhabdomyosarcoma were studied using dipalmitoylphosphatidylcholine liposomes. Experimental PDT of rhabdomyosarcoma was carried out using liposome-delivered ZnNc 1-4. The phototherapeutic effect was evaluated by tumor photonecrosis, the mean tumor diameter during the observation period and the percentage of cured animals. The best effect was found after PDT with ZnNc 2 (50% of the treated animals were cured). A slightly lower effect was observed after application of ZnNc 4 (40% cured animals). No effect at all was noted after PDT with ZnNc 3 and a very low efficiency was found after treatment with ZnNc 1 as photosensitizer. Obviously, the photodynamic effect depends on the biological characteristics as well as on the nature of the substituents.

Animals↗
PubMed1993

A human pseudoautosomal gene, ADP/ATP translocase, escapes X-inactivation whereas a homologue on Xq is subject to X-inactivation.

We report the cloning of a highly conserved pseudoautosomal gene on the human sex chromosomes. A cDNA clone was selected by crosshybridization with a microdissected clone from the chromosomal subregion Xp22.3. It encodes a previously characterized member of the ADP/ATP translocase family and plays a fundamental role in cellular energy metabolism. This gene, ANT3, is located approximately 1,300 kilobases from the telomere, proximal to the pseudoautosomal gene CSF2RA, and escapes X-inactivation. Interestingly, a homologue of ANT3, ANT2, maps to Xq and is subject to X-inactivation. These genes provide the first evidence of two closely related X-chromosomal genes, which show striking differences in their X-inactivation behaviour.

Animals↗