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M W Berchtold

Publications and source records attributed to M W Berchtold.

At least 37 records · Page 2Linked to original sources
PubMed1996

V(D)J recombination is regulated similarly in RAG-transfected fibroblasts and pre-B cells.

Here we compare the regulation of V(D)J recombination in the fibroblast cell line L4 and the pre-B cell line 38B9. The former has been rendered recombination-competent by stable transfection of a genomic fragment comprising the recombination activating genes, RAG-1 and RAG-2, along with some of their flanking sequences. We show that V(D)J recombination is similarly regulated in these two cell lines. Activating signals are transmitted through the protein kinase A (PKA) pathway, and inhibitory signals through the protein kinase C (PKC) and the calcium signalling pathways. In both cell lines, recombinational activity reflects steady state levels of mRNA transcribed from the RAG-1 and RAG-2 genes. This suggests that transcription of the RAG genes is a major determinant regulating V(D)J recombination. A comparison of RAG-1 and RAG-2 mRNA levels within each cell line reveals almost identical response patterns indicating that RAG-1 and RAG-2 transcription is coordinately regulated. Together, these results imply that the RAG-containing fragment in L4 fibroblasts carries most, if not all, control regions regulating the transcription of the RAG-1 and RAG-2 genes.

Animals↗
PubMed1996

Down-regulation of the protein kinase A pathway by activators of protein kinase C and intracellular Ca2+ in fibroblast cells.

Many genes are regulated by the intracellular calcium, protein kinase C (PKC) and protein kinase A (PKA) pathways and it has been shown that these pathways synergize in some cell types, whereas they antagonize in others. Here we show that the calcium and PKC pathways suppress the effects mediated by the PKA pathway in a fibroblast cell line. The suppressing effect of elevated intracellular Ca2+ levels, but not of the PKC pathway, can be abrogated by the addition of cyclosporin A (CsA), indicating that the effect of Ca2+ is mediated by phosphatase-2B (PP-2B/calcineurin). Suppression by the PKC pathway is not mediated by the proto-oncogenes c-fos, c-jun and junB, as the co-transfection of these genes does not block the effects of the PKA stimulator 8-Br-cAMP. In addition, cotransfection with the catalytic subunit of PKA shows that the inhibitory effect of PKC occurs upstream of PKA activation.

3T3 Cells↗
PubMed1996

[PCR: basics and new developments].

Since its discovery 10 years ago PCR has been introduced for a variety of practical applications. PCR has opened new dimensions particularly in laboratory diagnostics because of its sensitivity, accuracy and speed. In spite of the availability of user friendly kits, basic knowledge is of great importance for the user especially if PCR has to be optimized for special needs or when specific problems arise. The general mechanism of the reaction and the significance of the reaction components and the PCR conditions are discussed initially. Several recent developments in PCR (new enzymes, RNA-PCR, improvements of the specificity, prevention of contamination and development of new equipment) that are critical for the user are shortly introduced. Finally, "long PCR" is discussed in order to demonstrate that even 10 years after the invention of PCR significant new breakthroughs in the PCR technology are still possible.

Animals↗
PubMed1995

Increase of skeletal muscle relaxation speed by direct injection of parvalbumin cDNA.

Parvalbumin (PV) is a high affinity Ca(2+)-binding protein found at high concentration in fast-contracting/relaxing skeletal muscle fibers of vertebrates. It has been proposed that PV acts in the process of muscle relaxation by facilitating Ca2+ transport from the myofibrils to the sarcoplasmic reticulum. However, on the basis of metal-binding kinetics of PV in vitro, this hypothesis has been challenged. To investigate the function of PV in skeletal muscle fibers, direct gene transfer was applied in normal and regenerating rat soleus muscles which do not synthesize detectable amounts of PV. Two weeks after in vivo transfection with PV cDNA, considerable levels of PV mRNA and protein were detected in normal muscle, and even higher amounts were detected in regenerating muscle. Twitch half-relaxation time was significantly shortened in a dose-dependent way in transfected muscles, while contraction time remained unaltered. The observed shortening of half-relaxation time is due to PV and its ability to bind Ca2+, because a mutant protein lacking Ca(2+)-binding capacity did not promote any change in physiology. These results directly demonstrate the physiological function of PV as a relaxing factor in mammalian skeletal muscle.

Animals↗
PubMed1995

Production and analysis of transgenic mice with ectopic expression of parvalbumin.

Transgenic mice expressing rat parvalbumin under the control of the human metallothionein IIA (MTII A), SV-40 early, and neuron-specific enolase (NSE) promoters were produced. Ectopic expression was analyzed by RNA polymerase chain reaction and RNase protection in combination with immunohistochemistry. From a total of 25 transgenic lines 18 were found to express the transgene. Expression strength and tissue specificity were dependent upon the promoter used and varied considerably among animal lines produced with the same construct. Highest constitutive MT IIA-driven expression was found in lung, liver, heart, and kidney, as well as in brain, and lower amounts of transgene expression were found in spleen, testis, and muscle. Immunohistochemistry of tissue sections of metallothionein-parvalbumin transgenic strain 29 in the non-induced state revealed that ectopic PV mRNA is translated into protein. Short-term induction of the MT IIA promoter by CdSO4 or CdCl2 leads to a shift in tissue specificity and does not increase ectopic expression in tissues where the transgene is active in the noninduced state. As expected the NSE promoter showed highest activity in brain. However, NSE-driven expression could also be detected to various degrees in all investigated tissues. SV-40-dependent PV expression showed no tissue preference and varied considerably among different strains. Except for the observation that the SV-40-PV construct showed lower yields in transgenic production and reduced numbers of positive offspring no obvious impairment of growth or behavior as a consequence of transgenic PV expression could be detected.

Animals↗
PubMed1995

Changes in shape and motility of cells transfected with parvalbumin cDNA.

Parvalbumin is thought to act as a Ca2+ buffer in skeletal muscle fibers, but its physiological role in brain, kidney, and testis remains unclear. We have transfected parvalbumin cDNA into a human ovarian adenocarcinoma cell line, which normally does not express this protein. The induced expression of parvalbumin under the control of three different promoters causes: (1) changes in the morphology of the cells from epitheloid to fusiform, (2) an increase in motility of whole cell clusters, and (3) a decrease in the mitotic rate. Transfection with a mutated cDNA of rat parvalbumin incapable of binding Ca2+ had no effect on these three parameters. Our results indicate that ectopic expression of parvalbumin influences not only cell division [Rasmussen and Means (1989) Mol. Endocrinol. 3, 588-596], but also cell shape and motility by modulating intracellular Ca2+ handling. This may be a basic function of parvalbumin when it is intrinsically expressed in differentiated nonmuscle cells.

Cell Line, Transformed↗
PubMed1995

Structure and chromosomal localization of the mouse oncomodulin gene.

The rat gene encoding oncomodulin (OM), a small calcium-binding protein, is under the control of a solo LTR derived from an endogenous intracisternal A-particle. The latter sequence is the only OM promoter analyzed so far. In order to study cell type-specific OM expression in a species lacking LTR sequences in the OM locus, we initially synthesized an OM cDNA from mouse placenta. By sequencing, we found a 137-bp-long 5'leader region that differed markedly from its rat counterpart but had high similarity to several mouse genomic sequences. Primers specific to this sequence in addition with primers specific for an exon 2/intron 2 sequence were used to screen a mouse ES cell line genomic P1 library. One positive clone contained the whole OM gene, including intron 1 of 25kb and a 5' flanking region of 27 kb lacking an LTR. The region upstream of exon 1 contains no TATA or CCAAT boxes but has a homopurine/homopyrimidine stretch of 102 bp as well as a (CA)22 repeat. The latter sequence is polymorphic and was therefore, used to map the OM gene to the distal end of the long arm of mouse Chromosome (Chr) 5 by interspecific backcross analysis. Additionally we localized the OM gene by in situ hybridization to the region G1-3 on Chr 5, confirming the genetic linkage results. Finally, the OM gene was found to be structurally conserved and to exist in a single copy in mammals.

Amino Acid Sequence↗
PubMed1994

Inactivation of individual Ca(2+)-binding sites in the paired EF-hand sites of parvalbumin reveals asymmetrical metal-binding properties.

Previously a rat parvalbumin mutant protein PVF102W was constructed with a reporter Trp at position 102 in the middle of the hydrophobic center [Pauls, T. L., et al. (1993) J. Biol. Chem. 268, 20897-20903]. In the present study three new parvalbumin mutant proteins, derived from PVF102W and containing alterations at positions essential for Ca2+ binding in either one of the two Ca(2+)-binding sites (PV-CD and PV-EF) or in both (PV-CD/-EF), were expressed and purified. With the flow dialysis method it was established that both PV-CD and PV-EF bind 1 Ca2+ with affinity constants KCa of 1.1 x 10(7) and 3.2 x 10(6) M-1, respectively. Mg2+ binding, monitored by equilibrium gel filtration in the absence of Ca2+, showed that both mutants bind 1 Mg2+ with KMg = 8 10(4) for PV-CD and 3 x 10(3) M-1 for PV-EF. Compared to the parameters of the parent mutant PVF102W (two sites with equal affinities of 2.7 x 10(7) and 3 x 10(4) M-1 for Ca2+ and Mg2+, respectively), these data indicate that inactivation of the EF site, much more than of the CD site, impairs divalent cation binding. The binding of Ca2+ and Mg2+ is mutually exclusive, indicative of a Ca2+/Mg2+ mixed site. However, as for PVF102W, the KMg values obtained from the competition equation are approximately 40-fold lower than the affinities measured by direct binding. PV-CD/-EF binds neither Ca2+ nor Mg2+. Trp fluorimetry revealed that in the three mutant PVs the residue Trp-102 is deeply buried in the hydrophobic core.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Calmodulin-like effect of oncomodulin on cell proliferation.

Oncomodulin (OM) is a Ca2+ binding protein (CABP) structurally closely related to parvalbumin. Expression of OM is restricted to early embryonic stages, the placental cytotrophoblasts, and neoplastic tissues. The function of OM as a calmodulin (CaM)-like enzyme modulator is controversial. Two types of experiments demonstrate that OM may act in an analogous fashion to CaM in T14 and T10 cancerous cell lines, which both express OM. First, both OM transcript and protein levels increased at the G1/S boundary in a similar manner to CaM, though not to the same extent, in the chemically transformed rat fibroblast cell line T14 synchronized at mitosis by nocodazole. Second, antisense oligonucleotides specific to the OM ATG region inhibited growth of T14 in a similar dose-dependent manner as observed with CaM-specific antisense probes.

Animals↗
PubMed1994

Intracellular Ca2+ and Ca(2+)-binding proteins in chemically transformed rat fibroblasts.

Disturbed Ca2+ handling and altered levels of Ca(2+)-binding proteins (CaBPs) have been found in many transformed cells. To investigate whether there exists a correlation between expression of CaBPs and intracellular Ca2+ we analyzed three transformed rat fibroblast cell lines (T14, T10, and T43) exhibiting different levels of CaBPs. All three cell lines express calmodulin (CaM) at elevated levels compared to normal tissues. In addition, oncomodulin (OM) is expressed at high levels in T14 cells and at much lower levels in T10 cells, while T43 cells do not express OM. We demonstrate that CaBP levels are affected by changes of extracellular [Ca2+] ([Ca2+]o). In reduced [Ca2+]o, CaM levels were increased up to 60%, while OM levels were decreased up to 65% in T14 and T10 cells. Increase of [Ca2+]o, on the other hand, led to a decrease of CaM levels and an increase of OM levels, suggesting that [Ca2+]o exerts its effect on OM and CaM differently. Comparison of the growth rates in reduced [Ca2+]o revealed that T14 cells with the most pronounced CaM increase induced by low [Ca2+]o grew best under these conditions. These results indicate that elevated CaM expression but not the presence of OM reduces the Ca2+ requirement for growth. Intracellular [Ca2+] transients are not buffered by the high concentration of CaBPs in T14 cells. In contrast, [Ca2+]i transients induced by increase of [Ca2+]o or addition of serum were most pronounced in T14 cells compared to T10 and T43 cells.

Animals↗
PubMed1993

Cloning of a mouse cDNA encoding DNA polymerase delta: refinement of the homology boxes.

A mouse DNA polymerase delta (Pol delta)-encoding cDNA (pol delta) was isolated by PCR amplification and cDNA library screening. The sequenced cDNA has a length of 3386 bp and the open reading frame (ORF) encodes a protein of 1105 amino acids (aa) with an M(r) of 123,743. The aa identity to the proteins encoded by the corresponding cDNA from Bos taurus (93%) and Homo sapiens (92%) is very high. The identity to the Pol delta from Schizosaccharomyces pombe, Saccharomyces cerevisiae and Plasmodium falciparum is around 50%. An aa comparison between all available Pol delta sequences reveals several common structural motifs. Polyclonal antibodies raised against a 31-aa synthetic peptide deduced from the ORF specifically recognize Pol delta polymerases from human cells and calf thymus in an immunoblot.

Amino Acid Sequence↗
PubMed1993

Metal binding properties of recombinant rat parvalbumin wild-type and F102W mutant.

Rat parvalbumin (PV), an EF-hand type Ca(2+)-binding protein, was expressed in Escherichia coli and mutated by replacing a Phe at position 102 with a unique Trp in order to introduce a distinct fluorescent label into the protein. Mass spectroscopy and NMR data indicate that the recombinant wild-type (PVWT) and F102W mutant (PVF102W) proteins have the expected molecular weight and retain the native structure. Both proteins contain two non-cooperative Ca2+/Mg(2+)-binding sites with intrinsic affinity constants, KCa and KMg, of 2.4 +/- 0.9 x 10(7) M-1 and of 2.9 +/- 0.2 x 10(4) M-1, respectively, for PVWT, and KCa and KMg, of 2.7 +/- 1.1 x 10(7) M-1 and of 4.4 +/- 0.3 x 10(4) M-1, respectively, for PVF102W. Based on the highly similar metal binding properties of PVWT and PVF102W the latter protein was used to study cation-dependent conformational changes. Trp fluorescence emission and UV difference spectra of PVF102W indicated that the Trp residue at position 102 is confined to a hydrophobic core and conformationally strongly restricted. Upon Ca2+ or Mg2+ binding the structural organization of the region around the Trp is hardly affected, but there are significant changes in its electrostatic environment. The conformational change upon binding of Ca2+ and Mg2+, as monitored by UV difference spectrophotometry, increases linearly from 0 to 2 cations bound, indicating that the binding of both ions contributes equally to the structural organization in this protein.

Animals↗
PubMed1993

Localization of the human bona fide calmodulin genes CALM1, CALM2, and CALM3 to chromosomes 14q24-q31, 2p21.1-p21.3, and 19q13.2-q13.3.

The three bona fide genes coding for calmodulin CALM1, CALM2, and CALM3, were assigned to human chromosomes 14, 2, and 19, respectively, by polymerase chain reaction-based amplification of calmodulin gene-specific sequences using DNA from human-hamster cell hybrids as template. Employing calmodulin gene-specific DNA probes of mainly intronic or flanking parts of the individual genes, regional sublocalization was performed by in situ hybridization on metaphase spreads of human lymphocytes. The three calmodulin genes map to chromosomes 14q24-q31, 2p21.1-p21.3, and 19q13.2-q13.3, respectively. These results represent the first complete chromosomal localization study on a mammalian calmodulin gene family and indicate that these structurally closely related genes were most likely dispersed throughout the genome concomitantly with their generation from an ancestral precursor gene.

Animals↗
PubMed1993

Localization of the intronless gene coding for calmodulin-like protein CLP to human chromosome 10p13-ter.

The functional intronless gene coding for a calmodulin-like protein (CLP) has been localized to human chromosome 10p13-ter. Chromosomal assignment was performed by Southern blot analysis of DNA from human-rodent somatic cell hybrids and amplification of a CLP gene-specific 1090-bp DNA fragment by the polymerase chain reaction (PCR) on DNA from human-hamster cell hybrids. Chromosomal sublocalization was carried out by in situ hybridization of human chromosome metaphase spreads. The CLP gene is the first member of the human calmodulin/calmodulin-like gene family to be chromosomally sublocalized. Its presence near the telomeric end of the short arm of chromosome 10 may be of significance with respect to its highly (epithelial) cell-type restricted expression in vivo and strong downregulation upon malignant transformation. The generation of a human CLP gene-specific sequence tag site specified by the two primers used for PCR should prove useful for future linkage studies.

Animals↗
PubMed1993

Evolution of EF-hand calcium-modulated proteins. V. The genes encoding EF-hand proteins are not clustered in mammalian genomes.

The chromosomal assignments of genes belonging to the EF-hand family which have a common origin are compiled in this article. So far data are available from 27 human gene loci belonging to 6 subfamilies and 8 murine loci belonging to 4 subfamilies. Chromosomal localization has been obtained by somatic-cell hybrid analysis using the Southern blot technique or PCR amplification, metaphase spread in situ hybridization, or isolation of the particular genes from chromosome-specific libraries. Except for genes of the S-100 alpha proteins which are grouped on human chromosome 1q12-25 and mouse chromosome 3, no linkage has been found for genes encoding EF-hand proteins, indicating absence of selective pressure for maintaining chromosomal clustering. Six of these genes map to known syntenic groups conserved in the human and mouse genomes. This suggests that chromosomal translocations occurred before divergence of these species. The possible significance of chromosomal positioning with respect to nearby located known genes and genetic disease loci is discussed.

Animals↗