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J Becker

Publications and source records attributed to J Becker.

At least 19 recordsLinked to original sources
PubMed1999

The retinitis pigmentosa GTPase regulator, RPGR, interacts with the delta subunit of rod cyclic GMP phosphodiesterase.

Recently, the retinitis pigmentosa 3 (RP3) gene has been cloned and named retinitis pigmentosa GTPase regulator (RPGR). The amino-terminal half of RPGR is homologous to regulator of chromosome condensation (RCC1), the nucleotide exchange factor for the small GTP-binding protein Ran. In a yeast two-hybrid screen we identified the delta subunit of rod cyclic GMP phosphodiesterase (PDEdelta) as interacting with the RCC1-like domain (RLD) of RPGR (RPGR392). The interaction of RPGR with PDEdelta was confirmed by pull-down assays and plasmon surface resonance. The binding affinity was determined to be 90 nM. Six missense mutations at evolutionary conserved residues within the RLD, which were found in RP3 patients, were analyzed by using the two-hybrid system. All missense mutations showed reduced interaction with PDEdelta. A non-RP3-associated missense substitution outside the RLD, V36F, did not abolish the interaction with PDEdelta. PDEdelta is widely expressed and highly conserved across evolution and is proposed to regulate the membrane insertion or solubilization of prenylated proteins, including the catalytic subunits of the PDE holoenzyme involved in phototransduction and small GTP-binding proteins of the Rab family. These results suggest that RPGR mutations give rise to retinal degeneration by dysregulation of intracellular processes that determine protein localization and protein transport.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

Does local irradiation affect gastric emptying in humans?

The authors measured gastric emptying in 13 patients undergoing radiation therapy to the chest, abdomen, or pelvis for nongastrointestinal cancer to investigate whether gastric emptying (GE) was altered by this therapy. Symptoms and weight were monitored at regular intervals. Patients served as their own controls and were compared to a group of healthy subjects. When studied prior to radiation therapy (baseline), cancer patients had a gastric emptying rate that was similar to a healthy control group (t1/2 mean+/-SEM 92.0+/-15.3 vs. 80.4+/-8.2 min). Irradiation did not change the emptying rate, either after the first dose (early) of 180 cGy (t1/2 99.5+/-17.9) or after 2 weeks of therapy (late) with 3000 cGy (t1/2 75.5+/-7.3). There was no correlation of radiation field or tumor type with gastric emptying rate. Two of the 13 patients experienced nausea and vomiting during their course of radiation, but their gastric emptying was unchanged from baseline.

Abdomen↗
PubMed1998

Molecular and pharmacological characterization of recombinant rat/mice N-methyl-D-aspartate receptor subtypes in the yeast Saccharomyces cerevisiae.

The genes encoding the ionotropic N-methyl-D-aspartate (NMDA) receptor subunits NR1a, NR2B and NR2D were cloned in the multi-copy yeast-Escherichia coli shuttle vectors pMBO1 and pMB02. The protease-deficient yeast Saccharomyces cerevisiae c13-ABYS-86 (leu-, ura-, his-) was transformed with the recombinant plasmids pMBNR1a (leu+), pMBNR1a/pMBNR2D (ura+), pMBNR1a/pMBNR2D/ pMBNR2B (his+) or pMBNR1a/pMBNR2A/pMBNR2B, respectively, and was used to express the different NMDA receptor subunit genes. Western-blotting analysis with the specific NMDA receptor antibodies showed a clear but differently strong expression of the recombinant receptor proteins which were found to be only partially glycosylated in the cell membranes of the recombinant yeast strains. By immunofluorescence microscopy using the specific subunit antibodies and fluorescence-labeled secondary antibodies, the distinctly expressed NR1a and NR2D subunits could be located in the plasma membrane of the transformed yeast cells. Pharmacological characterization of crude membrane preparations of the recombinant yeast cells expressing 1-3 NMDA receptor subunits showed saturable binding of the glycine antagonist [3H]MDL105,519 with different Kd values of 56.88+/-5.38 nM (NR1a), 1365.11+/-76 nM (NR1a/NR2D), 22.97+/-3.37 nM for NR1a/NR2B/NR2D and 7.4+/-1.2 nM for NR1a/NR2A/NR2B. The bound capacities were 13.07+/-0.92 (NRla), 14.63+/-0.50 (NR1a/NR2D), 12.85+/-1.68 (NR1a/NR2B/NR2D) and 8.3+/-0.7 (NR1a/NR2A/NR2B) pmol/mg membrane protein. The [3H]MDL105,519 binding was inhibited by the glycine antagonist 5,7-dichlorokynurenate (DCKA), ethyl-2-carboxy-4.6-dichloro-3-indoleacetate (ECDI) and itself, but not by glycine, D-serine and 1-amino-cyclopropanecarboxylic acid (ACPC). Each of these recombinant receptor proteins consisting both of NR1 and NR2 subunits also showed a specific binding site for the NMDA agonist glutamate when using L-[3H]glutamate as a radioligand. Analysis of saturation experiments revealed that this ligand binds to a specific site with Kd values of 536+/-43, 688+/-60, and 856+/-48 nM for NR1a/NR2B, NR1a/NR2D, and NR1a/NR2B/NR2D respectively.

Animals↗
PubMed1998

Structure determination of the small ubiquitin-related modifier SUMO-1.

The recently discovered small ubiquitin-related modifier SUMO-1 belongs to the growing family of ubiquitin-related proteins involved in postranslational protein modification. Unlike ubiquitin, SUMO-1 does not appear to target proteins for degradation but seems to be involved in the modulation of protein-protein interactions. Independent studies demonstrate an essential function of SUMO-1 in the regulation of nucleo-cytoplasmic transport, and suggest a role in cell-cycle regulation and apoptosis. Here, we present the first three-dimensional structure of SUMO-1 solved by NMR. Although having only 18% amino acid sequence identity with ubiquitin, the overall structure closely resembles that of ubiquitin, featuring the betabetaalphabetabetaalphabeta fold of the ubiquitin protein family. In addition, the position of the two C-terminal Gly residues required for isopeptide bond formation is conserved between ubiquitin and SUMO-1. The most prominent feature of SUMO-1 is a long and highly flexible N terminus, which protrudes from the core of the protein and which is absent in ubiquitin. Furthermore, ubiquitin Lys48, required to generate ubiquitin polymers, is substituted in SUMO-1 by Gln69 at the same position, which provides an explanation of why SUMO-1 has not been observed to form polymers. Moreover, the hydrophobic core of SUMO-1 and ubiquitin is maintained by conserved hydrophobic residues, whereas the overall charge topology of SUMO-1 and ubiquitin differs significantly, suggesting specific modifying enzymes and target proteins for both proteins.

Amino Acid Sequence↗
PubMed1998

Functional expression of recombinant N-methyl-D-aspartate receptors in the yeast Saccharomyces cerevisiae--localization and pharmacological characterization.

The yeast Saccharomyces cerevisiae was used for expressing the genes encoding the ionotropic N-methyl-D-aspartate (NMDA) receptor subunits from rats (NR1a, NR2A, NR2C) and mice (NR2B). Four plasmids were constructed by cloning the different NMDA receptor genes in the two multi-copy yeast-Escherichia coli shuttle vectors pMB01 (--> NR1a gene) and pMB02 (--> NR2A-2C genes). The protease-deficient S. cerevisiae strain cI3-ABYS-86 (leu-, ura-) was transformed or co-transformed with the resulting plasmids pMBNR1a (leu+) or pMBNR1a/pMBNR2A-C (ura+) respectively. Western blotting analysis with antibodies raised against amino acid sequences at the C-termini of the respective subunits revealed that the recombinant receptor proteins were differently expressed and only partially glycosylated in the cell membranes of the recombinant yeast strains. The expression and localization of the recombinant NMDA receptor proteins were also proved by immunofluorescence microscopy which indicated a distinct expression of the different NMDA receptor subunits in the plasma membrane of the transformed yeast cells. Pharmacological characterization of crude membrane preparations of the recombinant yeast cells showed saturable binding of the glycine antagonist [3H]MDL105,519 with Kd values of 56.9 +/- 6.19 nM (NR1a/NR2A), 26.72 +/- 2.13 nM (NR1a/NR2B), and 21.22 +/- 1.64 nM (NR1a/NR2C), and bound capacities of 17.94 +/- 1.24 pmol/mg membrane protein (NR1a/NR2A), 11.45 +/- 0.67 pmol/mg (NR1a/NR2B), and 16.15 +/- 0.86 (NR1a/NR2C) pmol/mg. The [3H]MDL105,519 binding was inhibited by the glycine antagonist 5,7-dichlorokynurenate, ethyl-2-carboxy-4,6-dichloro-3-indoleacetate, and itself, but not by glycine, D-serine or 1-amino-cyclopropanecarboxylic acid. Specific binding of [3H]glycine or the NMDA channel blocker [3H]dizolcipine were not observed.

Animals↗
PubMed1998

The 1.7 A crystal structure of the regulator of chromosome condensation (RCC1) reveals a seven-bladed propeller.

The gene encoding the regulator of chromosome condensation (RCC1) was cloned by virtue of its ability to complement the temperature-sensitive phenotype of the hamster cell line tsBN2, which undergoes premature chromosome condensation or arrest in the G1 phase of the cell cycle at non-permissive temperatures. RCC1 homologues have been identified in many eukaryotes, including budding and fission yeast. Mutations in the gene affect pre-messenger RNA processing and transport, mating, initiation of mitosis and chromatin decondensation, suggesting that RCC1 is important in the control of nucleo-cytoplasmic transport and the cell cycle. Biochemically, RCC1 is a guanine-nucleotide-exchange factor for the nuclear Ras homologue Ran; it increases the dissociation of Ran-bound GDP by 10(5)-fold. It may also bind to DNAvia a protein-protein complex. Here we show that the structure of human RCC1, solved to 1.7-A resolution by X-ray crystallography, consists of a seven-bladed propeller formed from internal repeats of 51-68 residues per blade. The sequence and structure of the repeats differ from those of WD40-domain proteins, which also form seven-bladed propellers and include the beta-subunits of G proteins. The nature of the structure explains the consequences of a wide range of known mutations. The region of the protein that is involved in guanine-nucleotide exchange is located opposite the region that is thought to be involved in chromosome binding.

Amino Acid Sequence↗
PubMed1998

Prenatal diagnosis of autosomal recessive polycystic kidney disease (ARPKD): molecular genetics, clinical experience, and fetal morphology.

Autosomal recessive polycystic kidney disease (ARPKD) is one of the most common hereditary renal cystic diseases and has a high infant mortality. Prenatal diagnosis using fetal sonography can be unreliable, especially in early pregnancy. The ARPKD locus has been mapped to proximal chromosome 6p allowing haplotype-based prenatal diagnosis in "at-risk" families. From December 1994 to March 1997, we received 258 inquiries regarding prenatal evaluation and we have completed analyses in 212 families. To date, 65 prenatal analyses have been performed in 57 families. In the majority of the requesting families (45/57), the index children are deceased and their DNA was extracted from paraffin-embedded tissue. Eighteen fetuses were homozygous for the disease-associated haplotypes. In 12 of these fetuses, pathoanatomical examination demonstrated typical ARPKD changes consisting of dilated collecting ducts and the characteristic hepatic ductal plate malformation. These changes were detected in two fetuses as early as 13 weeks gestational age. These cases represent the earliest demonstration of ARPKD-associated histopathology reported to date. One high risk fetus was carried to term and turned out to be unaffected. However, the diagnosis of ARPKD remained doubtful in the index patient. Forty-three fetuses were either heterozygous or homozygous for a nondisease-associated haplotype and all infants born were phenotypically unaffected at birth. In four cases, a recombination event occurred between the flanking markers and no genotypic prediction was possible. Three of these pregnancies were terminated and necropsy of the fetuses confirmed ARPKD, while one fetus was carried to term and showed no abnormalities at birth. These results show that haplotype-based prenatal testing is feasible and reliable in pregnancies "at risk" for ARPKD. An absolute prerequisite for these studies is an accurate diagnosis of ARPKD in previously affected sib(s).

Adult↗
PubMed1998

Fine mapping of the autosomal recessive polycystic kidney disease locus (PKHD1) and the genes MUT, RDS, CSNK2 beta, and GSTA1 at 6p21.1-p12.

A total of 33 polymorphic markers were analyzed to generate a high-resolution genetic linkage map of the locus PKHD1 (polycystic kidney and hepatic disease 1) for the autosomal recessive polycystic kidney disease (ARPKD), using a combination of recombination mapping and linkage analysis in 164 families. Recombinants narrowed the PKHD1 region from 3.8 cM to a 1-cM interval flanked by the markers D6S1024 and D6S1714. Linkage disequilibrium analysis in 13 Finnish ARPKD families identified two different highly conserved haplotypes with four distal flanking markers, suggesting the existence of at least two major mutations of Finnish origin. The genes MUT (methylmalonyl coenzyme A-mutase), RDS (retinal degeneration, slow), CSNK2 beta (casein kinase II, beta subunit), and GSTA1 (glutathione S-transferase alpha, type 1) were excluded as PKHD1 genes using both established and novel intragenic polymorphisms in families with key recombinants. These genetic data, combined with our YAC-based physical map of the 6p21-p12 region, will facilitate efforts to positionally clone the PKHD1 gene.

Casein Kinase II↗
PubMed1998

Autosomal recessive polycystic kidney disease.

Autosomal recessive polycystic kidney disease (ARPKD) is a rare inherited disorder which usually becomes clinically manifest in early childhood, although the spectrum of ARPKD is much more variable than generally known. Presentation of ARPKD at later ages and survival into adulthood have been observed in many cases. The responsible gene has been mapped to chromosome 6p. Thus there is no evidence of genetic heterogeneity. The most important indication for DNA diagnosis is the prenatal diagnosis in families with at least one affected child. The critical region has been narrowed with the use of recombinant families of about 4 cM. Several possible candidate genes have been excluded.

Chromosome Mapping↗
PubMed1998

[p53 mutations and HPV infections in squamous epithelial carcinomas of the head-neck region. Long-term follow-up].

Mutations of the p53 gene are the most commonly observed genetic alterations in malignant tumors and are often associated with a loss of the tumor suppressor function of the p53 protein. We have analyzed specimens of head and neck squamous cell carcinomas (HNSCC) from 110 patients for p53 gene mutations and 92 of them additionally for human papillomavirus (HPV) infection in order to evaluate the prognostic significance of these factors by comparison with clinical follow-up data. Using the method of polymerase chain reaction (PCR)/temperature gradient gel electrophoresis (TGGE), mutations within the exons 5 to 8 of the p53 gene were found in 48 tumors (44%). Sequencing revealed missense mutations in most cases (15/20). Frequency of p53 gene mutations was not related to the tumor stage, the grade of differentiation, the presence of lymph node metastases, or the smoking history of the patients. With the help of a highly sensitive PCR/hybridization assay, an infection with the high-risk HPV types 16 and 18 could be detected in 39/92 tumor specimens (42%). Follow-up data were obtained from 99 patients with a range of 2-112 months. No correlation of overall survival on the presence of p53 gene mutations or HPV infection could be observed. The absence of statistically significant correlations between p53 gene mutations and progressive disease, however, does not exclude its putative relevance in early phases of tumor development.

Adult↗
PubMed1998

Lung volume histograms after computed tomography of the chest with three-dimensional imaging as a method to substantiate successful surgical expansion of the rib cage in achondroplasia.

The authors describe a radiographic method to quantify a surgical procedure of thoracic expansion in a 2-year-old patient with achondroplasia, small chest cage, and severe restrictive lung disease. The patient had undergone three surgical procedures of thoracic expansion since birth. The authors utilized computer-generated lung volume histograms after spiral computed tomographic scanning and three-dimensional imaging of the lungs to calculate his lung volumes before and after the third surgical thoracic expansion. The lung volumes, calculated by the histograms, were 363 mL and 406 mL before and after surgery, respectively. This 40-mL difference in the patient's lung volumes (4 mL/kg) accounted for a significant clinical improvement. Lung volume histograms obtained by this radiographic method are very helpful in substantiating a successful surgical chest expansion or provide an explanation for an unsuccessful repair.

Achondroplasia↗
PubMed1998

A family of Drosophila genes encoding quaking-related maxi-KH domains.

We recently identified a Drosophila gene, wings held out (who), that specifies a STAR (signal transduction and RNA activation) protein expressed within mesoderm and muscles. Genetic evidence suggests that WHO regulates muscle development and function in response to steroid hormone titer. who is related to the mouse quacking gene, essential for embryogenesis and neural myelination, and gld-1, a nematode tumor suppressor gene necessary for oocyte differentiation, both of which contain RNA binding "maxi-KH" domains presumed to link RNA metabolism to cell signaling. To initiate a broader study of Drosophila WHO-related proteins we used degenerate primers encoding peptides unique to maxi-KH domains to amplify the corresponding genes. We recovered nine genes, all specifying single maxi-KH domain proteins having tripartite regions of similarity that extend over 200 amino acids. One is located within the 54D chromosome subdivision, and one within 58C, while the remaining seven are within the 58E subdivision. At least four of these STAR proteins are expressed in a general manner, suggesting that maxi-KH domains are employed widely in Drosophila.

Amino Acid Sequence↗
PubMed1998

Dosing of oral sumatriptan: a review of our first 104 patients.

BACKGROUND: The introduction of oral sumatriptan in the United States at doses of 25 and 50 mg, compared with 100-mg tablets worldwide, has created the need to develop a protocol for appropriate dosing. METHODS: We evaluated the first 104 patients in our practice to treat two migraine attacks with oral sumatriptan. For their first treatment with oral sumatriptan, patients were evaluated on their response to 25-mg tablets and the total number of tablets taken. For their second treatment, patients were evaluated on their response to sumatriptan, number of 25-mg tablets taken, and dosage prescribed for future migraines. RESULTS: [table see text] After the second treatment, 41 patients (40%) continued therapy with 25-mg tablets, 54 (53%) were prescribed 50-mg tablets, 2 patients (2%) were prescribed two 50-mg tablets, and 5 patients (5%) were prescribed injectable sumatriptan. Seventy patients had previously used injectable sumatriptan, while 34 had not previously used sumatriptan. There were no significant differences in their response to oral sumatriptan. CONCLUSION: Oral sumatriptan was effective in clinical practice at doses of 25 and 50 mg. The majority of patients required more than one 25-mg tablet for a migraine attack, reflecting both inadequacy of dosing for some migraines and recurrence of headache, yet 40% of patients continued on treatment with 25-mg tablets. There were no significant differences in response to therapy in patients being switched from injectable to oral sumatriptan compared with those initiating therapy with oral sumatriptan. Both tablet strengths of oral sumatriptan are useful in clinical practice.

Administration, Oral↗
PubMed1998

False-positive LSD testing in urine samples from intensive care patients.

Unexpected positive results for lysergic acid diethylamide (LSD) were found in urine samples from 12 patients in an intensive care unit in a routine screening using the CEDIA DAU assay. None of these test results could be confirmed by high-performance liquid chromatography analysis, but all samples contained the mucolytic drug ambroxol. Further studies demonstrated that ambroxol exhibits a significant cross-reactivity in the CEDIA DAU LSD assay. Therefore, positive LSD results obtained with the CEDIA DAU assay have to be critically evaluated, particularly during the cold season, when infections of the respiratory tract often result in more frequent use of mucolytic medications.

Adult↗
PubMed1998

Assessment of conjunctival epithelium after severe burns and surgical reconstruction with Tenon plasty by means of a modified impression cytology procedure.

PURPOSE: Tenon plasty has been used to reconstruct the conjunctival surface in severe burns in which ischemic sclera was exposed or undergoing ulceration. A modified impression cytology procedure was applied to investigate the conjunctival epithelium. The quality of the regenerated epithelium on the advanced Tenon sheets was assessed. METHODS: The 63 conjunctival samples of eye-burn patients were investigated. Among these, 41 patients had very severe bums. Conjunctival samples were collected from 6 weeks after surgery to 5 years after the accident. They were compared with conjunctival epithelia obtained from 53 normal eyes of healthy volunteers. A 25-mm2 Biopore membrane (Millipore Catalogue PICM 01250) was placed on the bulbar conjunctiva surface in the lower temporal quadrant, at a distance of 3-5 mm from the limbus, till it was soaked with fluid. The ablated cell sheets were stained with periodic acid-Schiff (PAS). RESULTS: In all cases, an intact conjunctival epithelium was observed. In healthy eyes, 2,338 epithelial cells/mm2 and 155 goblet cells/ mm2 were found. Eyes after a surgical reconstruction with Tenon plasty resulted only in 1,575 epithelial cells/mm2 and 72 goblet cells/mm2. The differences were highly significant. The ratio of epithelial to goblet cell counts revealed an increase of goblet cells during the postoperative period. CONCLUSION: Conjunctival epithelium as well as goblet cell densities were reduced after heat, lime, alkali, and acid burns. However, after concrete burns, cell densities were increased. Tenon plasty provided the regeneration of the fully intact conjunctival epithelium. Goblet cells were present from 6 weeks after the surgery on; their number increased gradually. The stimulation of the goblet cell mucous secretion is discussed.

Adult↗
PubMed1998

Dependency and self-criticism in bipolar and unipolar depressed women.

OBJECTIVES: The dependency and self-criticism scores of women with histories of either unipolar disorder (N = 74) or bipolar disorder (N = 20) or no psychiatric history (N = 24) were compared to determine the influence of state depression on these personality dimensions. DESIGN: All women completed the Depressive Experiences Questionnaire and the Beck Depression Inventory. Patients were divided into currently depressed and remitted groups. RESULTS: Currently depressed women with unipolar disorder were more self-critical and dependent than non-psychiatric controls; women with unipolar disorder whose depression remitted were more self-critical only. Women in both the depressed and remitted bipolar groups were more self-critical than controls. Depressed bipolar patients did not differ from the controls in their level of dependency; remitted bipolar patients were significantly less dependent than controls. CONCLUSIONS: Dependency needs in depressed women are heavily influenced by mood state. Self-criticism appears to be a-characterological trait in both major depression and bipolar disorder. Women with bipolar disorder in remission report fewer dependency needs than women with no history of psychiatric disorder. The data provide partial support for Blatt's (1974) hypothesis that dependency and self-criticism reflect relatively stable personality dimensions in patients with a mood disorder.

Adult↗