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Biomedical subjects

J Walter

Publications and source records attributed to J Walter.

At least 109 records · Page 6Linked to original sources
PubMed1998

Protein phosphatase 2A is required for the initiation of chromosomal DNA replication.

Protein phosphatase 2A (PP2A) is an abundant, multifunctional serine/threonine-specific phosphatase that stimulates simian virus 40 DNA replication. The question as to whether chromosomal DNA replication also depends on PP2A was addressed by using a cell-free replication system derived from Xenopus laevis eggs. Immunodepletion of PP2A from Xenopus egg extract resulted in strong inhibition of DNA replication. PP2A was required for the initiation of replication but not for the elongation of previously engaged replication forks. Therefore, the initiation of chromosomal DNA replication depends not only on phosphorylation by protein kinases but also on dephosphorylation by PP2A.

Animals↗
PubMed1998

Truncated presenilin 2 derived from differentially spliced mRNA does not affect the ratio of amyloid beta-peptide 1-42/1-40.

Numerous mutations in the presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD). Here we characterize the expression of two naturally occurring alternative PS2 transcripts which lack either exons 3 and 4 (PS2 deltaexon3,4) or exons 3, 4, and 8 (PS2 deltaexon3,4,8). These transcripts do not contain the natural initiation codon within exon 3. The transcripts are efficiently translated as N-terminal truncated proteins. These deleted proteins are still able to regulate formation of endogenous PS fragments, indicating that the C-terminal half of the PS2 protein is sufficient for this phenomenon. Although approximately 50% of the PS1 and both PS2 mutations occur within the N-terminal region lacking in the PS2 deltaexon3,4 and PS2 deltaexon3,4,8 proteins, expression of these truncated proteins does not affect pathological generation of amyloid beta-peptide (Abeta). This suggests that point mutations causing AD are gain of function mutations.

Alzheimer Disease↗
PubMed1998

Delta-aminolevulinic acid transport by intestinal and renal peptide transporters and its physiological and clinical implications.

Delta-aminolevulinic acid (ALA) is the precursor of porphyrin synthesis and has been recently used in vitro and in clinical studies as an endogenous photosensitizer for photodynamic therapy in the treatment of various tumors. For this purpose, ALA is given topically, systemically, or orally. When administered by the oral route, it shows excellent intestinal absorption. ALA is also efficiently reabsorbed in the renal proximal tubule after glomerular filtration. However, the pathways and mechanisms for its transmembrane transport into epithelial cells of intestine and kidney are unknown. Here we demonstrate that ALA uses the intestinal and renal apical peptide transporters for entering into epithelial cells. Kinetics and characteristics of ALA transport were determined in Xenopus laevis ooyctes and Pichia pastoris yeast cells expressing either the cloned intestinal peptide transporter PEPT1 or the renal form PEPT2. By using radiolabeled ALA and electrophysiological techniques in these heterologous expression systems, we established that: (a) PEPT1 and PEPT2 translocate 3H-ALA by saturable and pH-dependent transport mechanisms, (b) that ALA and di-/tripeptides, but not GABA or related amino acids, compete at the same substrate-binding site of the carriers, and (c) that ALA transport is electrogenic in nature as a consequence of H+/ALA cotransport. Reverse transcriptase-PCR analysis performed with specific primers for PEPT1 and PEPT2 in rabbit tissues demonstrates that, in particular, the PEPT2 mRNA is expressed in a variety of other tissues including lung, brain, and mammary gland, which have been shown to accumulate ALA. This suggests that these tissues could take up the porphyrin precusor via expressed peptide transporters, providing the endogenous photosensitizers for efficient photodynamic therapy.

Aminolevulinic Acid↗
PubMed1998

Adeno-associated viral vector-mediated gene transfer of human blood coagulation factor IX into mouse liver.

Recombinant adeno-associated virus vectors (AAV) were prepared in high titer (10(12) to 10(13) particles/mL) for the expression of human factor IX after in vivo transduction of murine hepatocytes. Injection of AAV-CMV-F.IX (expression from the human cytomegalovirus IE enhancer/promoter) into the portal vein of adult mice resulted in no detectable human factor IX in plasma, but in mice injected intravenously as newborns with the same vector, expression was initially 55 to 110 ng/mL. The expression in the liver was mostly transient, and plasma levels decreased to undetectable levels within 5 weeks. However, long-term expression of human F.IX was detected by immunofluorescence staining in 0.25% of hepatocytes 8 to 10 months postinjection. The loss of expression was likely caused by suppression of the CMV promoter, because polymerase chain reaction data showed no substantial loss of vector DNA in mouse liver. A second vector in which F.IX expression was controlled by the human EF1alpha promoter was constructed and injected into the portal vein of adult C57BL/6 mice at a dose of 6.3 x 10(10) particles. This resulted in therapeutic plasma levels (200 to 320 ng/mL) for a period of at least 6 months, whereas no human F.IX was detected in plasma of mice injected with AAV-CMV-F.IX. Doses of AAV-EF1alpha-F. IX of 2.7 x 10(11) particles resulted in plasma levels of 700 to 3, 200 ng/mL. Liver-derived expression of human F.IX from the AAV-EF1alpha-F.IX vector was confirmed by immunofluorescence staining. We conclude that recombinant AAV can efficiently transduce hepatocytes and direct stable expression of an F.IX transgene in mouse liver, but sustained expression is critically dependent on the choice of promoter.

Adult↗
PubMed1998

Proteolytic fragments of the Alzheimer's disease associated presenilins-1 and -2 are phosphorylated in vivo by distinct cellular mechanisms.

The majority of familial Alzheimer's disease mutations are linked to the recently cloned presenilin (PS) genes, which encode two highly homologous proteins (PS-1 and PS-2). Full-length PS proteins undergo endoproteolytic cleavage within their hydrophilic loop domain resulting in the formation of C-terminal (CTF) and N-terminal fragments (NTF). PS-2 was found to be phosphorylated as a full-length protein within its N-terminal domain. In contrast, PS-1 is phosphorylated selectively after proteolytic processing within its approximately 20 kDa CTF involving protein kinase C (PKC) and/or protein kinase A (PKA). We now have found that the CTF of the highly homologous PS-2 is also phosphorylated. Surprisingly, the PS-2 CTF is not phosphorylated by PKC or PKA. Instead, the PS-2 CTF is constitutively phosphorylated in vivo by serine/threonine protein kinases, which are independent of phorbol ester and intracellular cAMP. In vitro the large hydrophilic loop of PS-2 between transmembrane domains 6 and 7 can be phosphorylated by casein kinase-1 (CK-1) and CK-2, but not by PKA or PKC. Quantitative analysis of in vitro phosphorylation demonstrates the presence of two phosphorylation sites for CK-1 and a single site for CK-2. A deletion analysis revealed that the CTF of PS-2 is phosphorylated in vivo within an acidic sequence containing three potential phosphorylation sites for CKs (serines 327, 330, and 335). These data suggest that CK type protein kinases phosphorylate the CTF of PS-2 within its hydrophilic loop domain in vivo. Interestingly, the potential phosphorylation sites are located directly adjacent to the recently identified caspase cleavage sites.

Alzheimer Disease↗
PubMed1998

Alzheimer's disease associated presenilin-1 holoprotein and its 18-20 kDa C-terminal fragment are death substrates for proteases of the caspase family.

Mutations in the presenilin (PS) genes are linked to early onset familial Alzheimer's disease (FAD). PS-1 proteins are proteolytically processed by an unknown protease leading to the formation of two stable fragments of approximately 30 and approximately 20 kDa [Thinakaran, G., et al. (1996) Neuron 17, 181-190]. In addition to the conventional fragments, alternative cleavage products were observed as well. Here we characterize an alternative proteolytic pathway of PS-1 which involves proteases of the caspase superfamily. Caspase mediated cleavage occurs between aspartate 345 and serine 346 C-terminal to the conventional cleavage determined previously [Podlisny, M., et al., (1997) Neurobiol. Dis. 3, 325-337]. Full-length PS-1 can serve as a substrate for caspase-like proteases, as demonstrated by the generation of the alternative C-terminal fragment in cells expressing PS-1 containing the Deltaexon 10 deletion which is known to accumulate as a full-length molecule [Thinakaran, G., et al. (1996)]. By inhibition of de novo protein synthesis in untransfected cells we demonstrate that the conventional C-terminal fragment of PS-1 is a substrate for caspase-like proteases as well. Therefore full-length and the conventional C-terminal fragment of PS-1 can serve as potential death substrates. Due to the fact that very little full-length PS-1 is expressed in vivo, the much more abundant C-terminal fragment and not the full-length precursor is the major in vivo substrate for the alternative cleavage of PS-1 by proteases of the caspase superfamily.

Alzheimer Disease↗
PubMed1998

Familial segregation of hemangiomas and vascular malformations as an autosomal dominant trait.

BACKGROUND: The pathogenesis of infantile hemangiomas is not yet understood. Growth factors and hormonal and mechanical influences have been thought to affect the focal abnormal growth of endothelial cells in these lesions. However, these influences may represent secondary responses to an underlying primary molecular event leading to the development of hemangiomas. OBSERVATIONS: We report the rare familial occurrence of hemangiomas and/or vascular malformations in 6 kindreds, suggesting autosomal dominant inheritance. In these families, multiple generations (2-4) were affected by hemangiomas or vascular malformations. In contrast to the generally accepted female-male ratio of 3:1 to 4:1 associated with sporadic hemangiomas, the families with hemangiomas in our study demonstrated a 2:1 ratio. Additionally, vascular malformations and hemangiomas were present in different members of the same family. The vascular lesions appeared to be transmitted in an autosomal dominant fashion with moderate to high penetrance. CONCLUSIONS: We have identified 6 families demonstrating autosomal dominant segregation of childhood hemangiomas. Additionally, family members with vascular malformations were identified in these kindreds. Physicians caring for children with hemangiomas and vascular malformations should include in their medical histories inquiries about vascular lesions in other family members, even when obvious lesions are not present in the parents. The identification of the mutation(s) underlying vascular lesions will provide insight into the pathogenesis of these familial hemangiomas and, potentially, common sporadic hemangiomas. In addition, such research would shed light on the regulation of angiogenic processes during development.

Arteriovenous Malformations↗
PubMed1998

Proteolytic processing of Alzheimer's disease associated proteins.

Amyloid beta-peptide (A beta), the major component of senile plaques, is generated by proteolytic processing from the beta-amyloid precursor protein (beta APP). Mutations within the beta APP gene cause early onset familial AD (FAD) by affecting A beta generation. Interestingly, the much more abundant mutations within the presenilin (PS) genes also result in the abnormal generation of a 42 residue A beta (A beta 42), thus clearly supporting a pivotal role of A beta for the pathology of AD. PS proteins are proteolytically processed into stable 30 kDa N-terminal fragments (NTF) and 20 kDa C-terminal fragments (CTF). Beside the conventional proteolytic pathway. PS proteins can also be cleaved further C-terminal by proteases of the caspase superfamily. PS proteins were localized within the endoplasmic reticulum (ER) and early Golgi, compartments which we have demonstrated to be involved in A beta 42 generation and intracellular accumulation. Using Caenorhabditis elegans as a simple animal model, we demonstrate that PS proteins are involved in NOTCH signaling FAD causing mutations interfere with the biological function of PS proteins in NOTCH signaling.

Alzheimer Disease↗
PubMed1998

H19 and Igf2 are expressed and differentially imprinted in neuroectoderm-derived cells in the mouse brain.

Igf2 and H19 are reciprocally imprinted genes that are closely linked and coexpressed in tissues of mesodermal and endodermal origin. Here we report that coexpression of these genes is also found in specific fetal tissues of neuroectodermal origin, that is in the ventral midline region of both the hindbrain and spinal cord. For cells of neuroectodermal origin, complete absence of Igf2 and H19 transcription was previously described. Analysis of allele-specific expression of both Igf2 and H19 in the ventral midline region of the hindbrain shows that H19 is expressed monoallelically, with the paternal allele being silent, whereas Igf2 is expressed biallelically. Furthermore, we observed a strong influence of the parental species background, in that the Mus musculus allele was always expressed at higher levels than the M. spretus allele. This was observed when the M. spretus allele was contributed by the mother or by the father. An analysis of Igf2 methylation by bisulphite genomic sequencing provided no clear answer as to whether Igf2 expression and methylation are linked in a tissue of neuroectodermal origin. Taken together, our results provide novel information on H19 and Igf2 expression and imprinting patterns in the fetal mouse brain. In addition, they indicate that some aspects of Igf2 regulation in cells of neuroectodermal origin do not follow the pattern that exists in mesoderm- and endoderm-derived tissues. Apart from the ventral midline region, H19 and Igf2 were found to be coexpressed in the ectodermally derived Rathke's pouch and in some circumventricular organs of the brain, such as the organum vasculosum of the lamina terminalis (OVLT) and the pineal gland.

Animals↗
PubMed1998

Prevalence of the factor V leiden mutation in children and neonates with thromboembolic disease.

OBJECTIVE: Resistance to activated protein C (APC) has been identified as a risk factor for thrombotic disease in adults. In over 90% of cases, the basis for the APC resistance is a mutation in the coagulation factor V gene (factor V Leiden) that renders the protein more resistant to inactivation by APC. We sought to determine the prevalence of the factor V Leiden (FVL) mutation in neonates and children who had experienced an arterial or venous thromboembolic event. STUDY DESIGN: We retrospectively analyzed the clinical records of 33 neonates and 52 children with thromboembolic disease. Screening for the FVL mutation was performed by DNA analysis, allowing for identification of patients as normal, heterozygous, or homozygous. RESULTS: Of the 85 patients studied, 12 (14.1%) were heterozygous for FVL; none were homozygous. Of the 47 patients who had arterial central nervous system events, 8 (17%) were positive for the FVL mutation, including 6 of 22 (27%) neonates. Of those patients who had a venous thrombosis, 4 of 32 (12.5%) were FVL positive. None of the 85 patients had protein C deficiency, 3.5% had protein S deficiency, 1.2% had antithrombin III deficiency, and 16.5% had anti-phospholipid antibodies. CONCLUSION: These data suggest that the FVL mutation plays a role in the development of arterial and venous thrombotic events in neonates and children.

Adolescent↗
PubMed1998

Imprinting mechanisms in mammals.

Imprinting is a genetic mechanism that determines expression or repression of genes according to their parental origin. Some imprinted genes occur in clusters in the genome. Recent work using transgenic mice shows that multiple cis-acting sequences are needed for correct imprinting. Mutation analysis in a normal chromosomal context reveals the importance of imprinting centres for regional establishment or maintenance of imprinting in a cluster. Elements that contribute to the function of imprinting centres and regional propagation of the imprints are CpG-rich differentially methylated regions (that during development retain germline imposed methylation or demethylation), direct repeat clusters, and unusual RNAs (antisense, non-translated etc.). The interaction of these cis elements with transacting factors such as methylase and chromatin factors establishes a hierarchical control system with local and regional effects.

Alleles↗
PubMed1998

Regulated chromosomal DNA replication in the absence of a nucleus.

Using Xenopus egg extracts, we have developed a completely soluble system for eukaryotic chromosomal DNA replication. In the absence of a nuclear envelope, a single, complete round of ORC-dependent DNA replication is catalyzed by cytosolic and nuclear extracts added sequentially to demembranated sperm chromatin or prokaryotic plasmid DNA. The absence of rereplication is explained by an activity present in the nucleus that prevents the binding of MCM to chromatin. Our results indicate that the role of the nuclear envelope in DNA replication is to concentrate activators and inhibitors of replication inside the nucleus. In addition, they provide direct evidence that metazoans use the same strategy as yeast to activate DNA replication and to restrict it to a single round per cell cycle.

Animals↗
PubMed1998

Intercellular adhesion molecule-1 (ICAM-1) in the mouse facial motor nucleus after axonal injury and during regeneration.

Intercellular adhesion molecule 1 (ICAM-1, CD54) is a widely expressed glycoprotein, which plays an important role in leukocyte extravasation and in the interaction of lymphocytes with antigen-presenting cells. In the current study we examined the regulation of ICAM-1 in the mouse facial motor nucleus after facial nerve transection, using immunohistochemistry, confocal laser microscopy and electron microscopy. In the normal facial nucleus ICAM-1 immunoreactivity was restricted to vascular endothelium. Transection of the facial nerve led to a strong and selective upregulation of ICAM-1 on activated microglia. Quantitation of microglial ICAM-1 immunoreactivity revealed a biphasic increase. The first peak 1-2 days post operation paralleling the early stage of microglial activation was followed by a decline at 4-7 days. The second induction of ICAM-1 occurred at day 14 accompanying the period of neuronal cell death and microglial phagocytosis of neuronal debris. Immunoelectron microscopy showed strong ICAM-1 reactivity on the cell membrane of activated microglia at day 2. During the second peak (day 14), ICAM-1 was also observed on lymphocytes adhering to phagocytotic microglia forming aggregates around neuronal debris. No immunolabelling was observed on neurons, astrocytes or oligodendroglia. These data suggest the involvement of ICAM-1 in the adhesion of activated microglia, in their phagocytosis of neuronal debris, and also in the interaction with infiltrating lymphocytes following this injury.

Animals↗
PubMed1998

The effect of electrical stimulation on colonic transit following spinal cord injury in cats.

The effect of direct electrical stimulation on colinic transit and manometric recordings following spinal cord injury were assessed in five adult male cats. Intra-colonic catheters were surgically placed, stimulating electrodes were sutured to the colonic serosa and a laminectomy with spinal cord clamping at a T4 level was done to induce spinal cord injury (SCI). Twenty radiopaque markers were inserted through an intra-colonic catheter located 1 cm distal to the cecum and were monitored with daily fluoroscopy as a measure of colonic transit. Transit measurements were compared before SCI, after SCI and after SCI with electrical stimulation of 40 pps, 1 ms, and 0-50 mA. Colonic transit following SCI was significantly prolonged (P<0.05) when compared to the transit before SCI. Electrical stimulation following SCI improved colonic transit to values not significantly different from those before SCI. Spontaneous colonic phasic motor activity was similar both before and after SCI. Manometric defection patterns were also observed to be similar before SCI and after SCI with electrical stimulation. Based on our scoring criteria, the most frequent response to electrical stimulation was an abdominal contraction. These findings demonstrate that colonic transit is prolonged following SCI and that direct electrical stimulation of the colon following SCI improves colonic transit in an animal model.

Animals↗
PubMed1998

Mutant presenilin 2 transgenic mouse: effect on an age-dependent increase of amyloid beta-protein 42 in the brain.

The N141I missense mutation in presenilin (PS) 2 is tightly linked with a form of autosomal dominant familial Alzheimer's disease (AD) in the Volga German families. We have generated transgenic mouse lines overexpressing human wild-type or mutant PS2 under transcriptional control of the chicken beta-actin promoter. In the brains of transgenic mice, the levels of human PS2 mRNA were found to be five- to 15-fold higher than that of endogenous mouse PS2 mRNA. The amyloid beta-protein (Abeta) 42 levels in the brains of mutant PS2 transgenic mice were higher than those in wild-type PS2 transgenic mice at the age of 2, 5, or 8 months. In addition, the Abeta42 levels appeared to increase steadily in the mutant PS2 transgenic mouse brains from 2 to 8 months of age, whereas there was only a small increase in wild-type transgenic mice between the ages of 5 and 8 months. There was no definite difference in the levels of N-terminal and C-terminal fragments between wild-type and mutant PS2 transgenic mice at the age of 2, 5, or 8 months. These data show a definite effect of the PS2 mutation on an age-dependent increase of Abeta42 content in the brain.

Aged↗
PubMed1998

Methylation levels at selected CpG sites in the factor VIII and FGFR3 genes, in mature female and male germ cells: implications for male-driven evolution.

Transitional mutations at CpG dinucleotides account for approximately a third of all point mutations. These mutations probably arise through spontaneous deamination of 5-methylcytosine. Studies of CpG mutation rates in disease-linked genes, such as factor VIII and FGFR3, have indicated that they more frequently originate in male than in female germ cells. It has been speculated that these sex-biased mutation rates might be a consequence of sex-specific methylation differences between the female and the male germ lines. Using the bisulfite-based genomic-sequencing method, we investigated the methylation status of the human factor VIII and FGFR3 genes in mature male and female germ cells. With the exception of a single CpG, both genes were found to be equally and highly methylated in oocytes and spermatocytes. Whereas these observations strongly support the notion that DNA methylation is the major determining factor for recurrent CpG germ-line mutations in patients with hemophilia and achondroplasia, the higher mutation rate in the male germ line is apparently not a simple reflection of sex-specific methylation differences.

Achondroplasia↗
PubMed1998

Syntenic organization of the mouse distal chromosome 7 imprinting cluster and the Beckwith-Wiedemann syndrome region in chromosome 11p15.5.

In human and mouse, most imprinted genes are arranged in chromosomal clusters. Their linked organization suggests co-ordinated mechanisms controlling imprinting and gene expression. The identification of local and regional elements responsible for the epigenetic control of imprinted gene expression will be important in understanding the molecular basis of diseases associated with imprinting such as Beckwith-Wiedemann syndrome. We have established a complete contig of clones along the murine imprinting cluster on distal chromosome 7 syntenic with the human imprinting region at 11p15.5 associated with Beckwith-Wiedemann syndrome. The cluster comprises approximately 1 Mb of DNA, contains at least eight imprinted genes and is demarcated by the two maternally expressed genes Tssc3 (Ipl) and H19 which are directly flanked by the non-imprinted genes Nap1l4 (Nap2) and Rpl23l (L23mrp), respectively. We also localized Kcnq1 (Kvlqt1) and Cd81 (Tapa-1) between Cdkn1c (p57(Kip2)) and Mash2. The mouse Kcnq1 gene is maternally expressed in most fetal but biallelically transcribed in most neonatal tissues, suggesting relaxation of imprinting during development. Our findings indicate conserved control mechanisms between mouse and human, but also reveal some structural and functional differences. Our study opens the way for a systematic analysis of the cluster by genetic manipulation in the mouse which will lead to animal models of Beckwith-Wiedemann syndrome and childhood tumours.

Amino Acid Sequence↗