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Biomedical subjects

S Beck

Publications and source records attributed to S Beck.

At least 19 recordsLinked to original sources
PubMed1996

Evolutionary dynamics of non-coding sequences within the class II region of the human MHC.

About 40% (350 kb) of the human MHC class II region has been sequenced and a coordinated effort to sequence the entire MHC is underway. In addition to the coding information (22 genes/pseudogenes), the non-coding sequences reveal novel information on the organisation and evolution of the MHC as demonstrated here by the example of a 200 kb contig that has been analysed for local and global features. In conjunction with cross-species comparisons, our results present new evidence on the structure of isochores, the evolutionary dynamics of repeat-mediated recombination and its effect on certain MHC encoded genes, and a higher than average degree of natural polymorphism that has implications for sequencing the human genome. We also report the finding of a class I-related pseudogene (HLA-ZI) in the middle of the class II region, which provides the first direct evidence for DNA exchange between these two related regions in man.

Amino Acid Sequence↗
PubMed1996

Reevaluation of synaptic relationships of cerebellar terminals in the ventral lateral nucleus of the rhesus monkey thalamus based on serial section analysis and three-dimensional reconstruction.

Terminals of cerebellar afferents (CB) to different regions of the ventral lateral nucleus (VL) of the rhesus monkey thalamus were labeled with wheat germ agglutinin-horseradish peroxidase following injections into the dentate nucleus. Synaptic relationships of 17 CB with projection neuron dendrites (PNd) and local circuit neuron dendrites (LCNd) were analyzed in serial ultrathin sections from dorsal and ventral VL regions, which are known to differ cytoarchitecturally and functionally. Three terminals were reconstructed using three-dimensional (3D) computer image analysis techniques to obtain volumetric and planar measurements. CB in the ventral VL were often flat and elongated with synaptic vesicles arranged in clusters. Each CB was engaged with one PNd and one to four LCNd. A single bouton formed 8-50 synaptic contacts, with those on PNd outnumbering the ones on LCNd 4.1:1. Only some CB in the ventral VL were engaged in complex synaptic arrangements such as triads and serial synapses. Most CB in the dorsal VL displayed a roundish shape and numerous uniformly distributed synaptic vesicles. They formed 5-25 synaptic contacts with a 3:1 ratio of contacts on PNd compared with those on LCNd. CB in the dorsal VL participated in a variety of complex synaptic arrangements. Two types of triads were found: classic with CB, PNd and LCNd, and unconventional with CB and two LCNd. CB were also involved in serial synapses with two LCNd or LCNd and another PNd, and serial sequential synapses with two LCNd and a PNd. Three glomerulus-like structures were encountered in the dorsal VL. 3D reconstruction and volumetric measurements revealed that synaptic contacts formed by CB on PNd had varying shapes and sizes (0.022-0.274 microns2). Synapses formed on LCNd were larger (0.09-0.407 microns2). The total area of all active zones of a single CB on LCNd was either equal to or about 40% smaller than that of synapses on PNd. The entire active zone area comprised 1-1.6% of the total CB surface area and did not seem to correlate with the volume. Synaptic contacts formed by associated LCNd on PNd in complex arrangements were usually small (0.021-0.044 micron2). The results suggest that: synapses formed by CB on PNd and LCNd, and synapses formed by LCNd on PNd may differ in strength; a variety of different circuits participate in the processing of cerebellar afferent information in the primate VL; and these circuits differ in functionally different VL subdivisions.

Animals↗
PubMed1996

Six new polymorphic microsatellite markers used for the integration of genetic and physical maps of human chromosome 7.

We report the isolation and characterization of six new polymorphic dinucleotide repeat microsatellite markers (D7S1491, D7S1492, D7S1493, D7S1494, D7S1495, and D7S1496), their integration into the genetic map of human chromosome 7 by analysis of 40 CEPH (Centre d'Etude du Polymorphisme Humain) pedigrees, and their use for integration of physical and genetic maps of this chromosome.

Alleles↗
PubMed1996

Divergent intron arrangement in the MB1/LMP7 proteasome gene pair.

We sequenced the human MB1 gene from a cosmid clone mapping to chromosome 14q11.2-12. The gene spans about 6 kilobases and contains three exons and two introns. There was no evidence of an alternative leader exon, which is a characteristic of the major histocompatibility complex (MHC)-encoded LMP7 gene, the closest relative of MB1, with which it shares 67% amino acid identity. Conceptual translation of the 5' end of the gene calls for a cleaved leader sequence of 59 amino acids, consistent with western blot data. None of the MB1 gene's three exons were coincident with any of the six exons in LMP7. In contrast, in the delta-encoding gene and its counterpart, the MHC-encoded LMP2 gene (59% amino acid identity), all six exons are arranged at equivalent positions in respect to the coding frame. The unique structure of MB1 implies a separate origin or different selection pressures acting at this particular locus. DNA repeat analysis provides information on the minimum time of separation of the MB1/LMP7 pair of genes.

Base Sequence↗
PubMed1996

Cloning of contiguous genomic fragments from human chromosome 21 harbouring three trefoil peptide genes.

A group of small peptides with a typical cysteine-rich domain (termed trefoil motif or P-domain) is abundantly expressed at mucosal surfaces of specific normal and neoplastic tissues. Their association with the maintenance of surface integrity was suggested. The first known human trefoil peptide (pS2) was isolated from breast cancer cells (MCF7). Its oestrogen-inducible gene, and the human homologue to the porcine spasmolytic peptide gene (hSP/SML1) appear synchronously expressed in healthy stomach mucosa and several carcinomas of the gastrointestinal tract. Both genes were shown to be localised at 21q22.3. A new trefoil peptide from human intestinal mucosa (hITF/hP1.B) and its gene were described recently. By using suitable oligonucleotide primers and PCR and isolating large (110-250 kb) genomic recombinants cloned in the bacterial artificial chromosome (BAC) system, we present a genomic region from chromosome band 21q22.3 cloned in contiguous sequences and encoding all three members of human P-domain/trefoil peptides proving a physical linkage of all three trefoil peptide genes. Such genomic sequences will provide useful experimental material for analysis of gene regulation, for gene modification experiments and for establishing transgenic cells or animals.

Base Sequence↗
PubMed1996

Androgen-like and anti-androgen-like effects of antiprogestins in human mammary cancer cells.

In addition to their antiprogestational activity, the antiprogestins RU486, ZK98.299 and ZK98.734 possess varying antiglucocorticoid as well as androgen-like or antiandrogen-like properties in human mammary cancer cells. The human mammary cancer cell line MFM-223, which contains only androgen receptors, was used as a model to investigate androgen receptor mediated effects of these antiprogestins. Proliferation of MFM-223 cells is inhibited by androgens and does not respond to oestrogens, progestins and glucocorticoids. As shown in proliferation assays, ZK98.734 was a strong inhibitor of cell proliferation. This effect was antagonised by the antiandrogen hydroxyflutamide. ZK98.734 was found to displace [3H]R1881 from the androgen receptor in MFM-223 cells, substantiating the involvement of the androgen receptor. The antiprogestin ZK98.299 failed to influence the proliferation of MFM-223 cells. ZK98.299 did not bind to the androgen receptor and was devoid of androgenic or antiandrogenic activity. RU486 bound to the androgen receptor. It was a weak inhibitor of MFM-223 cell proliferation, but the inhibition of proliferation by RU486 was not antagonised by hydroxyflutamide. This effect was probably not mediated by the androgen receptor. RU486 had antiandrogenic activity in this cell line, as it antagonised the inhibitory effect of dihydrotestosterone at a 100-molar excess. These results were confirmed by transfection experiments with an MMTV-CAT construct in the same cell line, demonstrating the biological function of the ZK98.734-androgen receptor complex. ZK98.299 and RU486 were not able to induce CAT activity. The different androgenic or antiandrogenic properties of the antiprogestins investigated should be considered when selecting antiprogestational properties of the antiprogestins investigated should be considered when selecting antiprogestational compounds for clinical applications, as a partial androgenic activity may be of benefit in breast cancer but can have undesired side-effects in other diseases.

Androgen Antagonists↗
PubMed1996

A third P-domain peptide gene (TFF3), human intestinal trefoil factor, maps to 21q22.3.

Small peptides displaying a cysteine-rich module (termed P-domain or trefoil motif) form a recently increasing group of peptides abundantly expressed at mucosal surfaces of specific tissues and are associated with the maintenance of surface integrity. The estrogen-inducible pS2 gene (BCEI) and the human homolog to the porcine spasmolytic peptide (hsP) gene (SML1) appear synchronously expressed in healthy stomach mucosa and several carcinomas of the gastrointestinal tract. Both genes were shown to be located at 21q22.3. A new trefoil peptide from human intestinal mucosa (hITF/hP1.B) and its gene (TFF3) were described recently. By PCR analysis of a somatic cell hybrid panel and FISH using two large genomic recombinants (110 kb, 210 kb) cloned in the Bacterial Artificial Chromosome (BAC) system, we show that this gene coding for the new member of human P-domain/trefoil peptides also maps to chromosome region 21q22.3 suggesting a physical linkage of all three trefoil peptide genes.

Amino Acid Sequence↗
PubMed1995

Construction of genetic maps using distance geometry.

The techniques of distance geometry, which generate coordinates from observed interpoint distances, have been applied to the problem of determining the relative positions of linked genetic loci from observed interlocus distances. Only the most precise data needed to join the loci are used, with missing distances substituted by sums of precise intermediate distances. Good initial positions (and therefore the order) of loci on a linear map are obtained in an operation of complexity O(N3). The method can therefore be used to generate good initial framework maps for the large numbers of markers encountered in current mapping projects. The locus positions can be subsequently refined to maximize the agreement with the originally observed distances, taking account of the weights of individual interlocus distances. By choosing only small distances from which to construct the map, the method reduces any error due to an incorrect choice of mapping function. It also prevents undue expansion of the map due to error-prone markers, since such markers are accommodated in higher dimensions. The method estimates the error in the positions of individual markers on the final map and identifies well- and ill-defined regions of the map.

Algorithms↗
PubMed1995

Identification and analysis of multigene families by comparison of exon fingerprints.

Gene families are often recognised by sequence homology using similarity searching to find relationships, however, genomic sequence data provides gene architectural information not used by conventional search methods. In particular, intron positions and phases are expected to be relatively conserved features, because mis-splicing and reading frame shifts should be selected against. A fast search technique capable of detecting possible weak sequence homologies apparent at the intron/exon level of gene organization is presented for comparing spliceosomal genes and gene fragments. FINEX compares strings of exons delimited by intron/exon boundary positions and intron phases (exon fingerprint) using a global dynamic programming algorithm with a combined intron phase identity and exon size dissimilarity score. Exon fingerprints are typically two orders of magnitude smaller than their nucleic acid sequence counterparts giving rise to fast search times: a ranked search against a library of 6755 fingerprints for a typical three exon fingerprint completes in under 30 seconds on an ordinary workstation, while a worst case largest fingerprint of 52 exons completes in just over one minute. The short "sequence" length of exon fingerprints in comparisons is compensated for by the large exon alphabet compounded of intron phase types and a wide range of exon sizes, the latter contributing the most information to alignments. FINEX performs better in some searches than conventional methods, finding matches with similar exon organization, but low sequence homology. A search using a human serum albumin finds all members of the multigene family in the FINEX database at the top of the search ranking, despite very low amino acid percentage identities between family members. The method should complement conventional sequence searching and alignment techniques, offering a means of identifying otherwise hard to detect homologies where genomic data are available.

Animals↗
PubMed1995

A procedure for selective DNA alkylation and detection by mass spectrometry.

A method which improves the detectability of DNA by mass spectrometry is presented. By quantitatively alkylating the backbone of phosphorothioate oligonucleotides the problems of gas phase ion generation by matrix assisted laser desorption ionization can be controlled. We have developed a selective alkylating protocol for phosphorothioate oligonucleotides which is a facile way of generating non-ionic nucleic acids. A variety of alkylating agents was studied and their kinetics were monitored in a gel electrophoretic assay and by mass spectrometry.

Alkylation↗
PubMed1995

Coordinate regulation of the human TAP1 and LMP2 genes from a shared bidirectional promoter.

Recently, four genes (TAP1, TAP2, LMP2, LMP7) involved or potentially involved in the processing and transport of major histocompatibility complex class I-associated antigen to the endoplasmic reticulum have been identified. We now report the initial characterization of the bidirectional promoter for the human transporter associated with antigen processing 1 (TAP1) and low molecular mass polypeptide 2 (LMP2) genes. These genes are divergently transcribed from a central promoter region of only 593 bp. Functional analysis using a bidirectional reporter system demonstrates the minimal 593-bp promoter is sufficient for concurrent expression in both directions. There is no TATA box homology at either end but there is a prevalence of GC boxes. Transcription is initiated at multiple sites for each gene without any of the TAP1 transcripts overlapping with the LMP2 transcripts. The region proximal to the TAP1 gene is required for maximal basal level expression of not only TAP1 but also LMP2. Furthermore, this region is necessary for tumor necrosis factor alpha (TNF-alpha) induction of both genes. Site-specific mutations of an NF-kappa B element in the TAP1 proximal region blocked induction by TNF-alpha in both the TAP1 and LMP2 directions. An adjacent GC box was required for basal expression of both genes as well as augmenting the TNF-alpha induction of the distal LMP2 gene. In vivo genomic foot-printing of this region revealed strong protein/DNA interactions at the NF-kappa B and GC box consensus sequences. In vitro binding studies confirmed the capacity of the NF-kappa B site to bind p50/p65 and p52/p65 heterodimers and of the GC box to bind Sp1. Thus, the promoter elements proximal to the TAP1 gene play a significant role in regulating basal and induced expression of both TAP1 and LMP2. The findings presented in this report clearly link LMP2 expression with TAP1 expression and provide additional suggestive evidence linking LMP2 to class I antigen presentation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Perlecan in human bone marrow: a growth-factor-presenting, but anti-adhesive, extracellular matrix component for hematopoietic cells.

Human perlecan is a heparan sulfate proteoglycan with a large core protein of 467 kDa to which three glycosaminoglycan side chains are attached. It belongs to the heparan sulfate proteoglycan family which has been implicated in strong interactions between developing hematopoietic cells and their microenvironment in the bone marrow. Here we report that perlecan is highly expressed in the human bone marrow, as well as in long-term bone marrow cultures which are thought to mimic hematopoiesis in vitro. Expression of perlecan in this tissue was shown by Northern blotting of the 14-kb mRNA of the core protein and by immunofluorescence stainings. Functionally, perlecan shows a strong anti-adhesive effect on unfractionated bone marrow cells and on various hematopoietic cell lines, repelling the cells from the perlecan-coated area. In contrast, perlecan is adhesive for fibroblasts and endothelial cells. It is suggested that the anti-adhesive site is located within the core protein of perlecan since heparitinase-treated perlecan still shows the repellent effect. Although anti-adhesive for hematopoietic cells, perlecan is able to bind growth factors like granulocyte/macrophage-colony stimulating factor and present them to hematopoietic progenitor cells in a semi-solid colony assay. The functional role of a growth-factor-binding extracellular matrix component in the bone marrow microenvironment with anti-adhesive properties is uncertain but may be related to compartmentalization.

Adult↗
PubMed1995

Comparative sequence analysis of the human and pufferfish Huntington's disease genes.

The Huntington's disease (HD) gene encodes a novel protein with as yet no known function. In order to identify the functionally important domains of this protein, we have cloned and sequenced the homologue of the HD gene in the pufferfish, Fugu rubripes. The Fugu HD gene spans only 23 kb of genomic DNA, compared to the 170 kb human gene, and yet all 67 exons are conserved. The first coding exon, the site of the disease-causing triplet repeat, is highly conserved. However, the glutamine repeat in Fugu consists of just four residues. We also show that gene order may be conserved over longer stretches of the two genomes. Our work describes a detailed example of sequence comparison between human and Fugu, and illustrates the power of the pufferfish genome as a model system in the analysis of human genes.

Amino Acid Sequence↗
PubMed1995

Educational and consultative functions, topics, and methods of hospital general tumor conferences.

The educational and consultative functions, topics, and methods of 20 hospital general tumor conferences in Colorado were studied over a three-year period. Methods used included questionnaires completed by hospital admitting staff members, by tumor conference directors, and by conference attendees. Workshops were held in which a multidisciplinary panel of physicians, nurses, and cancer registrars discussed the educational and patient-management aspects of tumor conferences with tumor conference directors. The study design included evaluation of the effects of providing certain teaching aids, including newsletters, Physician Data Query printouts, and tumor registry data on the functions, topics, and methods of the conferences. Although certain temporal trends were suggested, the handouts as employed added little of perceived effectiveness for the busy clinicians attending. The characteristics of the tumor conferences were analyzed according to hospital size, presence of one or more residency programs, and location (metropolitan or otherwise). One observation was that relatively few primary care physicians attended these tumor conferences, especially those in large hospitals, and approaches to meeting the needs of that group are discussed. The importance of the interpersonal skills of the tumor conference director in the effectiveness of the conference was very evident.

Colorado↗
PubMed1995

Stark self-referral law.

Should Iowa physicians worry about the Stark law? Probably. In September, after a nearly four-year delay, the regulations for Stark I took effect. This article discusses how the Stark I and II self-referral laws might affect your practice.

Iowa↗
PubMed1994

Genomic organization of HLA-DMA and HLA-DMB. Comparison of the gene organization of all six class II families in the human major histocompatibility complex.

The genomic nucleotide sequences of HLA-DMA and HLA-DMB have been determined and their gene organizations have been compared with other human class II genes. The following features were found to be highly conserved throughout all human class II families. (i) All alpha genes are composed of 5 exons and all beta genes of 6 exons. (ii) The intron-exon boundary classes of exons 1-4 (alpha genes) and exons 1-5 (beta genes) are 100% conserved. Only the last boundary class which falls within the cytoplasmic domain appears to be variable. (ii) The size of exon 3 (membrane proximal domain) is also 100% conserved except for DMA (-1 codon) and DMB (+1 codon). The position and a possible functional implication of this deletion/insertion are discussed. Our findings confirm and extend the evidence based on sequence homology that DMA and DMB are different from typical class II genes suggesting that they may originate from a time prior to the divergence of the main class II genes. In addition we have identified various new repeat sequences within class II genes. Analysis of their classification and distribution reveal single and multiple repeat mediated recombination events. One of these events seems to have partially replaced exon 1 in DPA2. The possibility of this event causing DPA2 to become a pseudogene is discussed.

Amino Acid Sequence↗
PubMed1994

Androgen responsiveness of the new human endometrial cancer cell line MFE-296.

MFE-296 endometrial cancer cells express androgen receptors in vitro. These cells, which are tumorigenic in nude mice, are derived from a moderately differentiated human endometrial adenocarcinoma. They express vimentin and the cytokeratins 7, 8, 18, and 19. Karyotyping revealed near-tetraploidy for most of the cells. No marker chromosomes were observed. DNA analyses confirmed the genetic identity of the cell line and the patient from whom the cell line was derived. Proliferation of MFE-296 cells was inhibited by the progestin R5020 and the androgen dihydrotestosterone (DHT). The inhibition of proliferation by DHT was antagonized by the antiandrogen Casodex, demonstrating the involvement of the androgen receptor. Androgen binding was determined at 22,000 binding sites per cell using a whole-cell assay (KD = 0.05 nM) and 30 fmol/mg protein with the dextran charcoal method; 7 fmol/mg protein of progesterone receptors were found, whereas estrogen receptors were below 5 fmol/mg protein. The androgen receptor was functionally intact, as demonstrated by transfection experiments with a reporter-gene construct, containing an androgen-responsive element. In MFE-296 cells the content of the androgen receptor was up-regulated by its own ligand.

Androgens↗