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L Raeymaekers

Publications and source records attributed to L Raeymaekers.

At least 19 recordsLinked to original sources
PubMed2000

The Golgi PMR1 P-type ATPase of Caenorhabditis elegans. Identification of the gene and demonstration of calcium and manganese transport.

In recent years, it has been well established that the Ca(2+) concentration in the lumen of intracellular organelles is a key determinant of cell function. Despite the fact that essential functions of the Golgi apparatus depend on the Ca(2+) and Mn(2+) concentration in its lumen, little is known on the transport system responsible for ion accumulation. The Golgi ion pump PMR1 has been functionally studied only in yeast. In humans, mutations in the orthologous gene ATP2C1 cause Hailey-Hailey disease. We report here the identification of the PMR1 homologue in the model organism Caenorhabditis elegans and after ectopic expression the direct study of its ion transport in permeabilized COS-1 cells. The C. elegans genome is predicted to contain a single PMR1 orthologue on chromosome I. We found evidence for alternative splicing in the 5'-untranslated region, but no indication for the generation of different protein isoforms. C. elegans PMR1 overexpressed in COS-1 cells transports Ca(2+) and Mn(2+) with high affinity into the Golgi apparatus in a thapsigargin-insensitive manner. Part of the accumulated Ca(2+) can be released by inositol 1,4,5-trisphosphate, in agreement with the idea that the Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca(2+) store.

Adenosine Triphosphatases↗
PubMed2000

Abnormal intracellular ca(2+)homeostasis and disease.

A whole range of cell functions are regulated by the free cytosolic Ca(2+)concentration. Activator Ca(2+)from the extracellular space enters the cell through various types of Ca(2+)channels and sometimes the Na(+)/Ca(2+)-exchanger, and is actively extruded from the cell by Ca(2+)pumps and Na(+)/Ca(2+)-exchangers. Activator Ca(2+)can also be released from internal Ca(2+)stores through inositol trisphosphate or ryanodine receptors and is taken up into these organelles by means of Ca(2+)pumps. The resulting Ca(2+)signal is highly organized in space, frequency and amplitude because the localization and the integrated free cytosolic Ca(2+)concentration over time contain specific information. Mutations or functional abnormalities in the various Ca(2+)transporters, which in vitro seem to induce trivial functional alterations, therefore, often lead to a plethora of diseases. Skeletal-muscle pathology can be caused by mutations in ryanodine receptors (malignant hyperthermia, porcine stress syndrome, central-core disease), dihydropyridine receptors (familial hypokalemic periodic paralysis, malignant hyperthermia, muscular dysgenesis) or Ca(2+)pumps (Brody disease). Ca(2+)-pump mutations in cutaneous epidermal keratinocytes and cochlear hair cells lead to, skin diseases (Darier and Hailey-Hailey) and hearing/vestibular problems respectively. Mutated Ca(2+)channels in the photoreceptor plasma membrane cause vision problems. Hemiplegic migraine, spinocerebellar ataxia type-6, one form of episodic ataxia and some forms of epilepsy can be due to mutations in plasma-membrane Ca(2+)channels, while antibodies against these channels play a pathogenic role in all patients with the Lambert-Eaton myasthenic syndrome and may be of significance in sporadic amyotrophic lateral sclerosis. Brain inositol trisphosphate receptors have been hypothesized to contribute to the pathology in opisthotonos mice, manic-depressive illness and perhaps Alzheimer's disease. Various abnormalities in Ca(2+)-handling proteins have been described in heart during aging, hypertrophy, heart failure and during treatment with immunosuppressive drugs and in diabetes mellitus. In some instances, disease-causing mutations or abnormalities provide us with new insights into the cell biology of the various Ca(2+)transporters.

Animals↗
PubMed2000

Basic principles of quantitative PCR.

The polymerase chain reaction (PCR) is an extremely sensitive method owing to the repetitive multiplication of template molecules. This property is a drawback for quantitative measurements because small differences in the multiplication factor lead to large differences in the amount of product. Two methods can be used to solve the problem of quantification: kinetic methods based on the determination or comparison of the amplification factor; and coamplification methods, which compare the amount of product to that of a simultaneously amplified standard template. An overview of the theoretical background of both methods is presented. For selection of a suitable method, both theoretical and practical considerations are important. Kinetic methods are the most convenient if PCR can be performed without opening the tubes, as in some apparatus using fluorescence detection. Coamplification methods can be done without expensive equipment but requires the parallel running of several PCR tubes. When the number of initial template molecules is close to one, as in the limiting dilution technique, statistical considerations become important.

Gene Amplification↗
PubMed2000

[Intracellular calcium: physiology and physiopathology].

Many important aspects of our life are regulated by the free cytosolic Ca2+ concentration. The intracellular Ca2+ signal is regulated both in space, frequency and amplitude. Each cell chooses a unique set of Ca2+ signals to control its function. Ca2+ signal transduction is based on rises in free cytosolic Ca2+ concentration. Ca2+ can come from the extracellular space or be released from intracellular stores. Extracellular Ca2+ enters the cell through various types of plasma-membrane Ca2+ channels and leaves the cell using Ca2+ pumps and Na+/Ca(2+)-exchangers. Ca2+ is accumulated in intracellular stores by means of Ca2+ pumps and is released via inositol 1,4,5-trisphosphate (IP3) and ryanodine receptors. Mutations or abnormalities in one of the above mentioned Ca(2+)-transporting proteins can lead to disease. Skeletal-muscle pathology can be caused by abnormal ryanodine receptors (malignant hyperthermia, porcine stress syndrome, central core disease), plasma-membrane Ca2+ channels (hypokalemic periodic paralysis, muscular dysgenesis mice, paraneoplastic Lambert-Eaton myasthenia syndrome) or Ca2+ pumps (Brody disease). Neurologic disorders can be related to altered function of plasma-membrane Ca2+ channels (episodic ataxia type 2, spinocerebellar ataxia type 6, familial hemiplegic migraine, glutamate excitotoxicity, tottering, leaner, lethargic and stargazer mice), IP3 receptors (Lowe's oculocerebrorenal syndrome, manic depression, Alzheimer's disease, opisthotonos mice) and Ca2+ pumps (deafwaddler mouse and wriggle mouse sagami). Two skin diseases are caused by Ca(2+)-pump mutations (Darier disease and Hailey-Hailey disease). Incomplete X-linked congenital stationary night blindness is caused by a mutation in the plasma-membrane Ca2+ channels in rods and cones.

Animals↗
PubMed1999

Properties of heterologously expressed hTRP3 channels in bovine pulmonary artery endothelial cells.

1. We combined patch clamp and fura-2 fluorescence methods to characterize human TRP3 (hTRP3) channels heterologously expressed in cultured bovine pulmonary artery endothelial (CPAE) cells, which do not express the bovine trp3 isoform (btrp3) but express btrp1 and btrp4. 2. ATP, bradykinin and intracellular InsP3 activated a non-selective cation current (IhTRP3) in htrp3-transfected CPAE cells but not in non-transfected wild-type cells. During agonist stimulation, the sustained rise in [Ca2+]i was significantly higher in htrp3-transfected cells than in control CPAE cells. 3. The permeability for monovalent cations was PNa > PCs approximately PK >> PNMDG and the ratio PCa/PNa was 1.62 +/- 0.27 (n = 11). Removal of extracellular Ca2+ enhanced the amplitude of the agonist-activated IhTRP3 as well as that of the basal current The trivalent cations La3+ and Gd3+ were potent blockers of IhTRP3 (the IC50 for La3+ was 24.4 +/- 0.7 microM). 4. The single-channel conductance of the channels activated by ATP, assessed by noise analysis, was 23 pS. 5. Thapsigargin and 2,5-di-tert-butyl-1, 4-benzohydroquinone (BHQ), inhibitors of the organellar Ca2+-ATPase, failed to activate IhTRP3. U-73122, a phospholipase C blocker, inhibited IhTRP3 that had been activated by ATP and bradykinin. Thimerosal, an InsP3 receptor-sensitizing compound, enhanced IhTRP3, but calmidazolium, a calmodulin antagonist, did not affect IhTRP3. 6. It is concluded that hTRP3 forms non-selective plasmalemmal cation channels that function as a pathway for agonist-induced Ca2+ influx.

Adenosine Triphosphate↗
PubMed1999

Distribution of plasma membrane Ca2+ pump (PMCA) isoforms in the gerbil brain: effect of ischemia-reperfusion injury.

Non-species isoform-specific antibodies against three isoforms of the plasma membrane Ca2+ pump (PMCA) were used for immuno-localization of PMCA by Western blot analysis in membrane preparations isolated from different regions of gerbil brain. All three gene products were detected in the membranes from hippocampus, cerebral cortex and cerebellum. However, they showed a distinct distribution pattern. Two proteins were revealed in the case of PMCA1 with molecular masses 129 and 135 kDa. The antibody against PMCA2 recognized three proteins of about 130-137 kDa. Only one protein was detected with the anti-PMCA3 antibody. Levels of immuno-signal for the PMCA isoforms varied significantly among the different brain regions. The PMCA1 is the most abundant in the cerebro-cortical and hippocampal membrane preparations. The PMCA2 was detected in a lesser amount comparing to PMCA1 and was highest in the membrane preparations from cerebellum and in a slightly lesser amount from cerebral cortex. Anti-PMCA3 antibody stained weakly and was localized in the cerebellar and hippocampal membrane preparations. Transient forebrain ischemia (10 min) and reperfusion (for a prolonged period up to 10 d) leads to a significant decrease of PMCA immuno-signal. This decrease could be ascribed to the loss of PMCA1 signal, especially in hippocampal membrane preparations.

Animals↗
PubMed1998

Regulation of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) 2 gene transcript in neuronal cells.

The sarco/endoplasmic reticulum Ca(2+)-transport ATPase (SERCA2) pre-mRNA is alternatively processed in a tissue-specific manner. At its 3' end, two 5' splice donor sites compete for the same 3' acceptor splice site (3'A). While the upstream 5' donor splice site (5'D1) is used in muscle cells giving rise to the class 1 mRNA, the downstream one (5'D2) is exclusively used in neuronal cells generating the class 4 mRNA. Using a neuroblastoma cell line and a minigene containing the 3' end of the SERCA2 gene, we have investigated the regulation of the neuronal-type of splicing. We have shown that a strong 3'A is required for splicing because exchanging it for a weaker one abolishes splicing. A second region spanning the entire exon 25 downstream of the 3'A is also necessary for the repression of the muscle-specific splicing in neuronal cells. In addition the tissue-specific (muscle/neuron) selection of the appropriate 5' donor splice site seems to be determined by at least two distinct but adjacent negative cis-active elements located in the last 237 nt of the optional exon 24. The upstream negative element controls the neuronal splicing while the downstream one represses the muscle-specific splicing in neuronal cells. It is suggested that the cis-active elements in the gene transcript are the target of trans-acting factors that are responsible for the repression of neuronal- or muscle-specific splicing in a tissue-specific manner.

Animals↗
PubMed1998

Distribution and isoform diversity of the organellar Ca2+ pumps in the brain.

The gene family of organellar-type Ca2+ transport ATPases consists of three members. SERCA1 is expressed exclusively in fast skeletal muscle; SERCA2 is ubiquitously expressed, whereas SERCA3 is considered to be mainly expressed in cells of the hematopoietic lineage and in some epithelial cells. In the brain, the organellar-type Ca2+ transport ATPases are almost exclusively transcribed from the SERCA2 gene. Four different SERCA2 mRNAs have been described (classes 1-4). However, unlike in nonneuronal cells, which express the class 1, 2, and 3 splice variants, the main SERCA2 mRNA in the brain is the class 4 messenger. Similar to classes 2 and 3, the class 4 codes for the ubiquitously expressed SERCA2b protein. Recently, we have reported the distribution of the SERCA isoforms in the brain (Baba-Aissa et al., 1996a,b). SERCA2b was present in most neurons of all investigated brain regions. The highest levels were found in the Purkinje neurons of the cerebellum and in the pyramidal cells of the hippocampus. Interestingly, SERCA3 and SERCA2a are coexpressed along with SERCA2b in the Purkinje neurons, but are weakly expressed in the other brain regions if present at all. Since these three protein isoforms have a different affinity for Ca2+, their possible roles in relation to Ca2+ stores in neurons are discussed.

Animals↗
PubMed1998

Modelling of some potential effects of lumenal Ca2+ binding on the kinetics of Ca2+ release from the endoplasmic reticulum.

The time course of Ca2+ release from intracellular stores during a prolonged exposure to a constant concentration of inositol 1,4,5-trisphosphate does not follow a single exponential but presents a progressive slowing down of the relative rate of efflux. Among the many factors that may contribute to this phenomenon, the possible contribution of lumenal Ca2+ buffering has been largely neglected. However, since the efflux rate depends on the free Ca2+ concentration whereas the total Ca2+ content is mainly determined by bound Ca2+, simple Ca2+ efflux kinetics can be expected only if there is a linear relation between free Ca2+ and bound Ca2+. Although little is known on lumenal Ca2+ binding, reasonable assumptions predict that the ratio of (Ca2+ bound)/(Ca2+ free) may decrease with increasing free Ca2+ concentrations, implying a continuously decreasing rate coefficient, as is experimentally observed. Simulated Ca2+ release curves show significant deviations from a single exponential if high-affinity binding sites are present in addition to low-affinity sites. Although this simple model does not predict several of the experimentally observed properties, it is concluded that a full understanding of Ca2+ release kinetics requires more detailed information on lumenal Ca2+ buffering within Ca2+ stores.

Binding Sites↗
PubMed1997

Phosphorylation by protein kinases A and C of myofibrillar proteins in rabbit stunned and non-stunned myocardium.

We tested the hypothesis that altered phosphorylation of myofibrillar proteins is involved in post-ischemic myocardial stunning. Myofibrillar proteins were isolated from Langendorff perfused control rabbit hearts, hearts submitted to 15 min normothermic ischemia and hearts submitted to 15 min ischemia followed by 10 min of reperfusion (stunned hearts). The in vivo level of phosphorylation of specific contractile proteins by protein kinases A and C was indirectly detected by the amount of 32P incorporated in vitro in the presence of these protein kinases and saturating concentration of [gamma-32P]-ATP (back-phosphorylation method). In control experiments the back-phosphorylation technique was able to detect PKA- or PKC-induced protein phosphorylation in hearts treated with isoproterenol and phorbol ester, respectively. In stunned hearts, contractile function was significantly suppressed compared to the period before ischemia. We found no difference in myofibrillar protein profile (on densitometry of the Coomassie-stained gels after SDS-PAGE) and in PKA mediated 32P incorporation when comparing control, ischemic and stunned myocardium. Three different PKCs were used for phosphorylation: commercial purified rat brain PKC, partially purified rat brain PKC or rabbit partially purified cardiac PKC. Cardiac PKC mainly phosphorylated troponin I, whereas brain PKC phosphorylated both troponin T and troponin I. No significant difference in 32P incorporation mediated by either brain or cardiac PKC was found between control, ischemic and ischemic/reperfused myofibrils. These data indicate that myocardial stunning does not cause changes in PKC- or PKA-mediated Pi incorporation into myofibrillar proteins detectable by the back-phosphorylation method.

Animals↗
PubMed1997

Co-ordinated regulation of the plasma membrane calcium pump and the sarco(endo)plasmic reticular calcium pump gene expression by Ca2+.

The plasma membrane calcium pump (PMCA) and sarco(endo)plasmic reticular calcium pump (SERCA) are important components of the Ca2+ homeostasis system responsible for intracellular Ca2+ signaling, yet the factors which govern their expression remain unclear. Recently, we have found that overexpression of PMCA by a gene transfection approach caused a down-regulation of the SERCA pump [Liu B.F., Xu X., Fridman R., Muallem S., Kuo T.H. Consequences of functional expression of the plasma membrane calcium pump isoform 1a. J Biol Chem 1996; 271: 5536-5544]. The results suggest an interdependence between PMCA and SERCA gene expression which has prompted us to investigate further on the mechanisms that regulate the expression of these Ca2+ pump genes in various cultured cell lines. In the present study, we have analyzed the isoforms of the PMCA and SERCA in different cells and presented evidence in favor of a co-induction of the PMCA and SERCA gene expression by second messengers, such as protein kinase C, cAMP, and Ca2+. Ectopic expression of PMCA or SERCA led to downregulation of the endogenous forms of both pumps. Changes in the level of mRNAs were paralleled by corresponding altered pump protein contents. The Ca(2+)-mediated increase of gene expression is accompanied by increased transcription rates as indicated by nuclear run-on assay. The co-ordinated induction of the PMCA and SERCA gene expression by thapsigargin was not blocked by the cytosolic application of the Ca2+ chelator BAPTA. We conclude that genes encoding components of the major Ca2+ transport pathways, including pumps and IP3 receptor channels, are regulatorally linked and this link is provided by the Ca2+ load of the ER store. This study points to the importance of gene expression as an integral component in the regulation of cellular Ca2+ homeostasis.

Animals↗
PubMed1996

Distribution of the organellar Ca2+ transport ATPase SERCA2 isoforms in the cat brain.

Of the three genes encoding the Ca2+ transport ATPases of the endoplasmic reticulum, the SERCA2 gene is the major isoform expressed in the mammalian brain. The SERCA2 transcript is alternatively processed generating two protein isoforms: SERCA2a which is expressed in cardiac and slow-skeletal muscle, and SERCA2b, the house-keeping isoform which is ubiquitously expressed. We have studied the expression of SERCA2 in the cat brain, and at a less refined level also in the rat brain, using antibodies specific for either SERCA2a or SERCA2b. The SERCA2a staining was very restricted. The SERCA2a antibody clearly labeled the cell body of the Purkinje neurons and weakly stained the giant cells of the gigantocellular reticular nuclei. In contrast, the SERCA2b isoform was found in most regions of the brain. It appeared to be largely confined to neuronal cells. Neuroglial cells were negative. The antibody stained the cell body. In heavily labeled cells such as the pyramidal cells of the hippocampus and of the cerebral cortex, it also stained the proximal portion of the dendrites. The most intense labeling was observed in the Purkinje neurons, which were stained all over the cell including the distal ramifications of the dendritic tree. Remarkably the SERCA2b labeling in neuronal cells of the hypothalamic area and the substantia nigra was very weak. The possible physiological significance of these results is discussed.

Animals↗
PubMed1996

Purkinje neurons express the SERCA3 isoform of the organellar type Ca(2+)-transport ATPase.

We report the distribution of the sarco(endo)plasmic reticulum Ca2+ ATPase 3 (SERCA3) isoform in the rat brain. Compared to SERCA2 isoform, which is found in all brain regions, SERCA3 is specifically expressed in the Purkinje neurons. This conclusion is based on immunochemical observations using SERCA3- and SERCA2b-specific antibodies, in-situ hybridization using SERCA3-specific oligonucleotide probes and single-cell reverse transcription-polymerase chain reaction (RT-PCR). Immunocytochemistry clearly revealed the expression of SERCA3 in the cell body and in the dentritic processes of the Purkinje neurons. Single-cell ratio RT-PCR showed that Purkinje neurons expressed 3-fold lower levels of SERCA3 mRNA compared to SERCA2 mRNA. SERCA3 expression is very low or absent in the rat cerebrum and brainstem. It is known that the SERCA3 Ca2+ pump has an approximately 5-fold lower affinity for Ca2+ when expressed in COS cells as compared to other SERCA members [15]. If this property is also valid in a neuronal context, the expression of the SERCA3 Ca(2+)-pump isoform could have important functional implications for the regulation of the cytosolic Ca2+ concentration in Purkinje neurons.

Animals↗
PubMed1996

cDNA cloning, expression and chromosomal localization of the human sarco/endoplasmic reticulum Ca(2+)-ATPase 3 gene.

cDNA and genomic clones encoding human sarco/endoplasmic reticulum Ca(2+)-ATPase 3 (SERCA3) were isolated. The composite nucleotide sequence of the 4.6 kb cDNA, as well as the partial structure of 25 kb of genomic DNA encoding all but the 5' region of the gene, was determined. The nucleotide sequence coding for the last six amino acids of the pump and the 3'-untranslated region were identified within the sequence of the last exon. Northern blot hybridization analysis using cDNA probes derived from this exon detected a 4.8 kb transcript in several human tissues. Using a cDNA probe derived from the 5'-coding region an unexpected mRNA distribution pattern, consisting of two mRNA species of 4.8 and 4.0 kb, was detected in thyroid gland and bone marrow only. This is the first indication of an alternative splicing mechanism operating on the SERCA3 gene transcript, which most likely generates SERCA3 isoforms with altered C-termini. Human SERCA3 expressed in platelets and in COS cells transfected with the corresponding cDNA was detected with the previously described antibody N89 (directed against the N-terminal region of rat SERCA3) and with a new SERCA3-specific antiserum C91, directed against the extreme C-terminus of the human isoform. A monoclonal antibody PL/IM430, previously assumed to recognize SERCA3 in human platelets, does not react with the 97 kDa human SERCA3 transiently expressed in COS cells. Therefore the 97 kDa isoform detected by PL/IM430 more likely represents a novel SERCA pump, as recently suggested [Kovács, Corvazier, Papp, Magnier, Bredoux, Enyedi, Sarkadi and Enouf (1994) J. Biol. Chem. 269, 6177-6184]. Finally, by fluorescence in situ hybridization and chromosome G-banding analyses, the SERCA3 gene was assigned to human chromosome 17p13.3.

Amino Acid Sequence↗
PubMed1996

Kinetics of the non-specific calcium leak from non-mitochondrial calcium stores in permeabilized A7r5 cells.

We have investigated the detailed kinetics of the passive Ca2+ leak from non-mitochondrial Ca2+ stores in permeabilized A7r5 cells. The decrease in the content of stored Ca2+ in the presence of 2 microM thapsigargin deviated from a single-exponential curve in the initial phase of the efflux. The deviation persisted after correcting this efflux for passively bound Ca2+. The non-single-exponential nature of the spontaneous release also occurred when the initial store Ca2+ content was reduced to 40% of its original value by pretreatment with 200 nM inositol 1,4,5-trisphosphate (InsP3). The passive Ca2+ leak could be modelled by two exponential curves with discrete rate constants of 0.06 min-1 and 0.98 min-1, and not by any other type of non-exponential decay. We concluded that individual store units are heterogeneous with respect to their passive Ca2+ permeability. This non-exponential nature of the passive Ca2+ release is unrelated to the non-single-exponential InsP3-induced Ca2+ release.

Animals↗
PubMed1996

The ubiquitously expressed pICln protein forms homomeric complexes in vitro.

Endothelial cells contain a cell-volume sensitive chloride conductance with biophysical properties similar to those of a ubiquitously expressed chloride current that is activated by cell swelling. The latter current has been associated with the ICln protein (pICln) which may be the chloride channel itself or, alternatively, a channel regulator. We were therefore interested in whether pICln is involved in the endothelial volume-sensitive chloride current. As a first step, we have cloned human pICln and studied its expression at the protein level. Using a polyclonal antiserum raised against human pICln we found a widespread expression of pICln, both in endothelial cells and in other cell lines. A characteristic feature of pICln is its anomalous migration during denaturing polyacrylamide gel electrophoresis. We also demonstrate that bacterially expressed pICln forms homomeric complexes in vitro.

Base Sequence↗