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Biomedical subjects

H Alder

Publications and source records attributed to H Alder.

At least 55 records · Page 3Linked to original sources
PubMed1992

A conserved region in intron 1 negatively regulates the expression of the PCNA gene.

The Proliferating Cell Nuclear Antigen (PCNA) gene is a growth-regulated gene, whose expression is under the control of both transcriptional and posttranscriptional mechanisms. In previous work, it was shown that the 73 bp immediately upstream of the CAP site and intron 4 are major regulatory elements. We show here that intron 1 also plays a role in determining the levels of PCNA mRNA. Specifically, we show: 1) deletion of intron 1 increases the expression of PCNA mRNA in serum-deprived cells; 2) a 35 bp sequence in intron 1, containing a reverse CCAAT element specifically binds proteins from nuclear extracts; 3) this intron 1 sequence inhibits the expression of a co-tranfected human PCNA gene in transient expression assays suggesting that it competes for positive transcription factors; 4) mutations in the CCAAT region of the 35bp intron 1 probe abrogate both its protein-binding capacity and its ability to inhibit the expression of a co-transfected wt PCNA gene; and 5) the CCAAT region of human intron 1 is highly conserved in the mouse gene. We conclude that the reverse CCAAT region of intron 1 is a negative regulatory element of PCNA gene expression, and hypothesize that its inhibitory effect is abolished when certain protein(s) bind to it and that inhibition is restored if these proteins are competed out by an homologous sequence.

Antigens, Neoplasm↗
PubMed1992

The role of the promoter in the expression of the PCNA gene.

G1-specific temperature-sensitive (ts) mutants of the cell cycle arrest in G1 after serum stimulation at the restrictive temperature. Under these conditions, the RNA levels of late growth-regulated genes (such as DNA polymerase alpha, PCNA, thymidine kinase, and core histones) are markedly decreased or even undetectable, while early growth-regulated genes (for instance, c-myc) are normally expressed, and certain promoters are actually super-induced. We have used the human PCNA gene transfected into TK-ts13 cells (a G1-specific ts mutant) to investigate whether the inhibition of gene expression caused by this type of growth inhibition occurs at a transcriptional or post-transcriptional level. Constructs were made in which the 5' and 3' flanking sequences of the human PCNA gene were replaced by the corresponding elements of the SV40 T antigen coding gene. Using these constructs and data from run-on assays and RT-PCR, we conclude that the failure of expression of the PCNA gene in G1-arrested TK-ts13 cells occurs at the transcriptional level.

Animals↗
PubMed1991

Characterization of an enhancer-like structure in the promoter region of the proliferating cell nuclear antigen (PCNA) gene.

The steady-state mRNA levels of the proliferating cell nuclear antigen (PCNA) gene depend on the length of its promoter. A promoter extending from the HpaII restriction site at -210 from the cap site to the cap site itself is very active, while a -45 promoter (AatII restriction site) is very weak. We now show that the sequences between -73 and -45 of the human PCNA promoter contain an enhancer-like sequence that markedly increases the levels of PCNA mRNA. This sequence has characteristics of an enhancer, having an enhancing function also when placed away from the native position in the 5' flanking sequence. The increase in mRNA levels that occurs after serum stimulation, however, is independent of the enhancer. Synthetic promoters were also constructed containing mutations in the -73 to -45 sequence and these mutants completely lost their ability to drive the transcription of a heterologous cDNA. Nuclear proteins were shown to bind to this sequence, both by gel shift and by methylation interference analysis. We conclude that the levels of PCNA mRNA are controlled, in part, by a structure located in the 5' flanking sequence of the gene, but that this enhancer-like structure does not play a role in the serum regulation of the mRNA levels.

Animals↗
PubMed1991

A new growth-regulated complementary DNA with the sequence of a putative trans-activating factor.

A new complementary DNA (cDNA) clone has been isolated by differential screening of a cDNA library. The cognate RNA of this clone, called SC1, is growth regulated in human, mouse, and hamster cell lines. Its kinetics of growth regulation (time of increase in mRNA levels, sensitivity to cycloheximide, behavior in G1-specific temperature-sensitive mutants) classify the SC1 gene as a late growth-regulated gene, like the histone genes and the genes coding for the proteins of the DNA synthesis apparatus. By run-on assay, there is a modest increase in transcriptional rates after serum stimulation, which is not sufficient to explain the sharp increase in mRNA levels. The SC1 gene localizes to human chromosome 6p21-22. In bacteria, the SC1 cDNA clone makes a protein of Mr 39,000, in agreement with the putative reading frame. The amino acid sequence derived from the cDNA sequence indicates a previously unknown gene with a domain strongly suggestive of a trans-activating domain. The SC1 gene can be considered as coding for a possible new trans-activating factor that could play an important role in the transcription of genes required for the later stages of cell cycle progression.

Amino Acid Sequence↗
PubMed1989

Temporary complementation of temperature-sensitive mutants of the cell cycle by transfection with a wild-type or a mutant cDNA of ADP/ATP translocase.

A number of cell-cycle-specific temperature-sensitive (ts) mutants have been isolated from animal cells, especially Syrian hamster cells. These ts mutants, like cell cycle ts mutants of yeast, can be complemented by specific genes, some of which have been molecularly cloned. We have isolated a cDNA clone that complements TK-ts13 cells, but only temporarily. This clone, called B1, differs from a previously isolated clone (Sekiguchi et al.: EMBO Journal 7:1683-1687, 1988) that specifically complements ts13 cells. In addition, B1 also complemented temporarily three other ts mutants of the cell cycle, tsAF8, ts694, and ts550C cells. These mutants have different mutations since, in cell fusion experiments, they complement each other. Sequencing of the B1 cDNA clone revealed that it was a mutant of human ADP/ATP translocase in which some human sequences at the 5' end have been replaced by SV40 sequences. The wild-type translocase was less effective but could still increase the survival time of cell cycle ts mutants at the restrictive temperature. Using the polymerase chain reaction, it was possible to demonstrate that the B1 plasmid is expressed in TK-ts13 cells undergoing temporary complementation.

Amino Acid Sequence↗
PubMed1988

Transdifferentiation from striated muscle of medusae in vitro.

We have established an in vitro transdifferentiation and regeneration system which is based entirely on mononucleated striated muscle cells. The muscle tissue is isolated from anthomedusae and activated by various means to undergo cell cycles and transdifferentiation to several new cell types. In all cases DNA-replication is initiated and the division products are smooth muscle cells, characterized by their ultrastructure and monoclonal antibodies, and nerve/sensory cells, characterized by their ultrastructure and FMRFamide-staining. Both cell types are found at a 1:1 ratio after the first division. The nerve cells stop to replicate, whereas the smooth muscle cells continue and keep producing in each successive division a smooth muscle cell and a nerve cell. The observed data indicate that smooth muscle cells behave like stem cells. Depending on the destabilization and culturing methods, some isolated muscle tissue will form a bilayered fragment and within only two cell cycles manubria (the feeding and sexual organ) or tentacles will regenerate. In this case six to eight new non-muscle cell types have been formed by transdifferentiation.

Animals↗
PubMed1987

Cell cycles and in vitro transdifferentiation and regeneration of isolated, striated muscle of jellyfish.

Isolated, mononucleated, cross-striated muscle cells of a medusa can transdifferentiate in vitro to various new cell types and even form a complex regenerate. The transdifferentiation events follow a strict pattern. The first new cell type resembles smooth muscle and is formed without a preceding DNA replication. This cell type behaves like a stem cell and by quantal cell cycles produces all other new cell types. Some preparations develop an inner and an outer layer separated by a basal lamella. Formation of these layers does not depend on DNA replication. When layers do not form, each division results in nerve cells and smooth muscle cells. If separation into layers occurs, then a regenerate will be formed, and in the course of only two cell cycles all necessary cell types to form a functional regenerate will differentiate.

Animals↗
PubMed1987

In vitro transdifferentiation of striated muscle to smooth muscle cells of a medusa.

Mononucleated striated muscle cells can be isolated from anthomedusae and cultivated in artificial seawater. In the cultivated muscle the differentiated state is maintained and DNA synthesis is not observed. The isolated striated muscle can be activated by collagenase treatment to transdifferentiate into various new cell types. Between the second and third day following collagenase treatment DNA synthesis is initiated, and mitosis and de novo flagellum formation occur in the isolated muscle. Under these circumstances all isolated striated muscle fragments produce both smooth muscle cells and y-cells (Schmid and Alder, 1984). In experiments, in which either transcription (actinomycin D) or translation (cycloheximide) is inhibited, the activated striated muscle cells do not transdifferentiate but maintain their differentiated state. Inhibition of DNA replication (aphidicolin), however, results in uniform transdifferentiation of striated muscle to smooth muscle cells in the absence of y-cell types (Schmid and Alder, 1984). The fluorescence stain NBD-phallacidin is used to monitor the characteristic change of F-actin pattern of these isolates.

Actins↗
PubMed1984

Isolated, mononucleated, striated muscle can undergo pluripotent transdifferentiation and form a complex regenerate.

Isolated, mononucleated, cross-striated muscle of a medusa can be activated by collagenase treatment to transdifferentiate completely to various new cell types and to regenerate autonomously the sexual (without gametes) and feeding organ of the animal. Under these circumstances all isolated muscle fragments produce smooth muscle cells and a glandular cell type (y-cells). When culture conditions are appropriate, endoderm is also formed, followed by regeneration of a complex organ of seven or eight new non-muscle cell types, including nematocytes, digestive, secretory, gland, interstitial, and presumably nerve cells.

Animals↗
PubMedDate not supplied

A highly specific functional test for factor V leiden: A modified tissue factor assay for activated protein C resistance.

We compared the sensitivity and specificity of a tissue factor-based assay (FVR) with the addition of a phospholipid/silica preparation, to the commercially available aPTT-based method, APCR (CoatestTM), and a modified aPTT-based method (APCM) which utilized factor V-depleted plasma, for the detection of the factor V Leiden mutation. A total of 110 patients were included in this study. This included 32 patients on coumadin therapy, 7 patients on heparin therapy, 5 patients on both anticoagulants therapy, and 24 patients who were positive for anticardiolipin antibody (ACL) and/or lupus inhibitor (LI). Our data demonstrate that the FVR is not affected by anticoagulation treatment or ACL/LI antibodies, whereas in the APCR method, 33 patients cannot be determined either due to the anticoagulant therapy or presence of the ACL and/or LI. With the APCM method, the clotting endpoint could not be determined in 1 patient due to the presence of a strong LI. The additional phospholipid/silica material utilized in the FVR enhanced the APC degradation of factor Va and therefore sharpened the demarcation between the factor V Leiden-positive and -negative patients. The sensitivity for the APCR, APCM and FVR was 42, 97 and 100% respectively. The specificity for the APCR, APCM and FVR was 94, 96 and 100% respectively.

Activated Protein C Resistance↗