Search PubMed⌕ Search

Biomedical subjects

T Ludwig

Publications and source records attributed to T Ludwig.

29 records · Page 2Linked to original sources
PubMed1994

Cloning and sequencing of cDNAs encoding the full-length mouse mannose 6-phosphate/insulin-like growth factor II receptor.

Overlapping clones encoding the complete coding sequence of the mouse mannose 6-phosphate/insulin-like growth factor II receptor have been isolated from a liver cDNA library and the nucleotide sequence has been determined. The open reading frame encodes a protein of 2482 amino acids that is 89 and 87% identical to the human and bovine receptors, respectively.

Amino Acid Sequence↗
PubMed1993

Targeted disruption of the mouse cation-dependent mannose 6-phosphate receptor results in partial missorting of multiple lysosomal enzymes.

In mammalian cells two mannose 6-phosphate receptors (MPRs) are involved in lysosomal enzyme transport. To understand the precise function of the cation-dependent mannose 6-phosphate receptor (CD-MPR), one allele of the corresponding gene has been disrupted in mouse embryonic stem cells and homozygous mice lacking this receptor have been generated. The homozygous mice appear normal, suggesting that other targeting mechanisms can partially compensate for the loss of the CD-MPR in vivo. However, homozygous receptor-deficient cells and animals clearly exhibit defects in targeting of multiple lysosomal enzymes when compared with wild-types. Increased levels of phosphorylated lysosomal enzymes were present in body fluids of homozygous animals. In thymocytes from homozygous mice or in primary cultures of fibroblasts from homozygous embryos, there is a marked increase in the amount of phosphorylated lysosomal enzymes that are secreted into the extracellular medium. The cultured fibroblasts have decreased intracellular levels of multiple lysosomal enzymes and accumulate macromolecules within their endosomal/lysosomal system. Taken together, these results clearly indicate that the CD-MPR is required for efficient intracellular targeting of multiple lysosomal enzymes.

Animals↗
PubMed1992

Gene and pseudogene of the mouse cation-dependent mannose 6-phosphate receptor. Genomic organization, expression, and chromosomal localization.

The cation-dependent mannose 6-phosphate receptor (CD-MPR) is one of the two transmembrane proteins involved in transport of lysosomal enzymes. We have cloned the mouse CD-MPR gene and also a very unusual processed-type CD-MPR pseudogene. They are both present at one copy per haploid genome and map to chromosomes 6 and 3, respectively. Comparison of the complete 10-kilobase (kb) sequence of the functional gene with the cDNA indicates that it contains seven exons. Exon 1 encodes the 5'-untranslated region of the mRNA, the others (exons 2-7) encode the luminal, transmembrane, and cytoplasmic domains of the CD-MPR. Exon 7 also contains a 1.2-kb-long 3'-untranslated region of the mRNA. A unique transcription-initiation site was determined by primer extension of mouse liver mRNA. The promoter elements in the 5' upstream region of this site resemble those contained in genes constitutively transcribed. However, Northern blot analysis demonstrates that the CD-MPR is variably expressed in adult mouse tissues and during mouse development. The pseudogene, which is flanked by direct repeats, is almost colinear with the cDNA indicating that it presumably arose by reverse transcription of an mRNA. However, the pseudogene differs from the cDNA. It contains at its 5' end, an additional 340-nucleotide (nt) sequence homologous to the promoter region of the functional gene. This sequence exhibits some promoter activity in vitro. Furthermore, a 24-nt insertion interrupts the region homologous to the 5'-noncoding region of the cDNA. In the functional gene, this 24-nt sequence occurs between exon 1 and 2, where it is flanked by typical consensus sequences of exon/intron boundaries. Therefore, it may represent an additional exon of the functional gene. These two features of the pseudogene suggest that expression of the CD-MPR gene may be regulated by use of different promoters and/or alternative splicing.

Amino Acid Sequence↗
PubMed1992

Hemispheric interactions: the bilateral advantage and task difficulty.

Twenty-five normal subjects made "same-different" responses to dot patterns presented in the LVF, RVF or bilaterally. Task difficulty was manipulated in each condition by varying the number of dots in the two patterns presented from two to four to six. The pairs of patterns always had the same number of dots on a given trial. Response latency and accuracy worsened as the number of dots increased for all three presentation conditions and for both "same" and "different" judgements. Overall, responding was faster and the number of errors lower on Bilateral presentations. For response latencies to identical patterns of dots, the size of the bilateral advantage increased relative to RVF responding as task difficulty increased but did not change significantly relative to LVF responding. When the two patterns were not identical the size of the advantage did not change as task difficulty increased. "Same" judgements were faster but less accurate than "different" judgements. A model of hemispheric interactions is proposed to account for the findings.

Adult↗
PubMed1991

Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells.

We have investigated the distribution of newly synthesized lysosomal enzymes in endocytic compartments of normal rat kidney (NRK) cells. The mannose-6-phosphate (Man6-P) containing lysosomal enzymes could be iodinated in situ after internalization of lactoperoxidase (LPO) by fluid phase endocytosis and isolated on CI-MPR affinity columns. For EM studies, the ectodomain of the CI-MPR conjugated to colloidal gold was used as a probe specific for the phosphomannosyl marker of the newly synthesized hydrolases. In NRK cells, approximately 20-40% of the phosphorylated hydrolases present in the entire pathway were found in early endocytic structures proximal to the 18 degrees C temperature block including early endosomes. These structures were characterized by a low content of endogenous CI-MPR and were accessible to fluid phase markers internalized for 5-15 min at 37 degrees C. The bulk of the phosphorylated lysosomal enzymes was found in late endocytic structures distal to the 18 degrees C block, rich in endogenous CI-MPR and accessible to endocytic markers internalized for 30-60 min at 37 degrees C. The CI-MPR negative lysosomes were devoid of phosphorylated hydrolases. This distribution was unchanged in cells treated with Man6-P to block recapture of secreted lysosomal enzymes. However, lysosomal enzymes were no longer detected in the early endosomal elements of cells treated with cycloheximide. Immunoprecipitation of cathepsin D from early endosomes of pulse-labeled cells showed that this hydrolase is a transient component of this compartment. These data indicate that in NRK cells, the earliest point of convergence of the lysosomal biosynthetic and the endocytic pathways is the early endosome.

Animals↗
PubMed1990

Phosphorylation of the cytoplasmic domain of the bovine cation-independent mannose 6-phosphate receptor. Serines 2421 and 2492 are the targets of a casein kinase II associated to the Golgi-derived HAI adaptor complex.

A kinase activity of purified bovine brain clathrin-coated vesicles phosphorylates the bovine cation-independent mannose 6-phosphate receptor (CI-MPR) with high efficiency (Km approximately 50-100 nM). The kinase copurifies in gel filtration, adsorption on hydroxylapatite, and ion exchange chromatography with the HAI assembly proteins which are part of the coat of Golgi-derived clathrin-coated vesicles. The kinase is associated to the 47-kDa subunit of the complex and exhibits properties similar to a casein kinase II: it uses either ATP or GTP as substrate and its activity is stimulated by poly-L-lysine and inhibited by heparin. Using different domains of the CI-MPR as potential substrates, we show that the phosphorylation is restricted to its cytoplasmic domain. Inhibition studies using synthetic peptides and two-dimensional mapping of the tryptic phosphopeptides indicate that this posttranslational modification occurs on serines 2421 and 2492 of the full-length bovine CI-MPR precursor, residues which are located in typical casein-kinase II recognition sequences. Labeling of Madin-Darby bovine kidney cells followed by immunoprecipitation of the CI-MPR and analysis of the corresponding tryptic phosphopeptides shows that the same serines are phosphorylated in vivo.

Amino Acid Sequence↗
PubMed1990

In exocrine pancreas, the basolateral endocytic pathway converges with the autophagic pathway immediately after the early endosome.

Intracisternal granules (ICGs) are insoluble aggregates of pancreatic digestive enzymes and proenzymes that develop within the lumen of the rough endoplasmic reticulum of exocrine pancreatic cells, especially in guinea pigs. These ICGs are eliminated by autophagy. By morphological criteria, we identified three distinct and sequential classes of autophagic compartments, which we refer to as phagophores, Type I autophagic vacuoles, and Type II autophagic vacuoles. Lobules of guinea pig pancreas were incubated in media containing HRP for periods of 5-120 min to determine the relationship between the endocytic and autophagic pathways. Incubations with HRP of 15 min or less labeled early endosomes at the cell periphery that were not involved in autophagy of ICGs, but after these short incubations none of the autophagic compartments were HRP positive. After 30-min incubation with HRP, early endosomes at the cell periphery, late endosomes in the pericentriolar region, and, in addition, Type I autophagic vacuoles containing ICGs were all labeled by the tracer. Type II autophagic vacuoles were not labeled after 30-min incubation with HRP but were labeled after incubations of 60-120 min. Phagophores did not receive HRP even after 120 min incubations. We concluded that the autophagic and endocytic pathways converge immediately after the early endosome level and that Type I autophagic vacuoles precede Type II autophagic vacuoles on the endocytic pathway. We studied the distribution of acid phosphatase, lysosomal proteases and cation-independent-mannose-6-phosphate receptor (CI-M6PR) in the three classes of autophagic compartments by histochemical and immunocytochemical methods. Phagophores, the earliest autophagic compartment, contained none of these markers. Type I autophagic vacuoles contained acid phosphatase but, at most, only very low levels of cathepsin D and CI-M6PR. Type II autophagic vacuoles, by contrast, are enriched for acid phosphatase, cathepsin D, and other lysosomal enzymes, and they are also enriched for CI-M6PR. Moreover, soluble fragments of bovine CI-M6PR conjugated to colloidal gold particles heavily labeled Type II but not Type I autophagic vacuoles, and this labeling was specifically blocked by mannose-6-phosphate. This indicates that the lysosomal enzymes present in Type II autophagic vacuoles carry mannose-6-phosphate monoester residues. Using 3-C2, 4-dinitroanilino-3'-amino-N-methyldipropylamine (DAMP), we showed that Type II autophagic vacuoles are acidic. We interpret these findings as indicating that Type II autophagic vacuoles are a prelysosomal compartment in which the already combined endocytic and autophagic pathways meet the delivery pathway of lysosomal enzymes.

Acid Phosphatase↗
PubMed1990

Microtubule depolymerization inhibits transport of cathepsin D from the Golgi apparatus to lysosomes.

Lysosomes as well as a prelysosomal compartment rich in the mannose 6-phosphate receptor are clustered close to the Golgi apparatus in the perinuclear region of the microtubule organizing center in interphase human skin fibroblasts. The spatial organization of these organelles depends on an intact microtubule network. Depolymerization of the microtubules by treatment of cells with nocodazole leads to random scattering of Golgi elements, the prelysosomal compartment, and lysosomes throughout the cytoplasm. To test whether microtubules and the spatial organization of these organelles are important for efficient transport of lysosomal enzymes, the effect of microtubule depolymerization on the maturation of newly synthesized cathepsin D was studied. An up to fivefold inhibition of proteolytic maturation of cathepsin D was observed in drug-treated cells. This effect was due to a decreased rate of transport of cathepsin D from the Golgi apparatus to lysosomes. Depolymerization of microtubules did not inhibit transport of cathepsin D from the endoplasmic reticulum to the trans-Golgi network. Furthermore, synthesis of the phosphomannosyl marker present on cathepsin D was not affected by nocodazole. These results suggest that efficient transport of cathepsin D from the Golgi apparatus to a prelysosomal compartment and lysosomes is facilitated by microtubules and the spatial organization of these organelles.

Biological Transport↗
PubMedDate not supplied

Aldosterone remodels human endothelium.

AIM: In response to aldosterone endothelial cells swell and stiffen. Although amiloride-sensitive sodium and water uptake is known to be involved, the underlying mechanisms are yet unclear. We tested the hypothesis whether the intracellular accumulation of water or organic matter is responsible for the structural and functional alterations. METHODS: Atomic force microscopy was used as an imaging tool and a mechanical nanosensor. Cell water, organic cell matter and cell pressure was measured at single cell level in human umbilical vein endothelial cells (HUVEC). Furthermore, we tested by means of a miniature perfusion chamber in vitro the physical robustness to blood flow of the aldosterone-treated endothelium. RESULTS: In response to a three-day treatment with 1 nM aldosterone HUVEC swell. To our surprise, cell water decreased from 82+/-6% to 71+/-5% while intracellular organic matter increased from 18+/-1.8% to 29+/-3.0%. These changes were paralleled by a rise in cell pressure of 114%, measured in living HUVEC in vitro. Blood flow across the endothelium was found significantly altered after aldosterone treatment. Imaging the endothelial monolayer after blood perfusion disclosed large gaps between cells treated with aldosterone. The mineralocorticoid receptor blockers, spironolactone and eplerenone could prevent the aldosterone actions. CONCLUSION: Mild aldosteronism causes intracellular accumulation of organic matter at the cost of cell water. This makes endothelium stiff and vulnerable to shear stress. The measurements could explain clinical observations that high blood pressure combined with high plasma aldosterone concentration may damage the endothelium of blood vessels.

Aldosterone↗
PubMedDate not supplied

Comparison of the activity profiles of two fixed combinations of regular/NPH human insulin (recombinant DNA) of different compositions with a fixed regular/NPH porcine insulin combination (PPI) in insulin-dependent diabetic individuals.

In a randomized double-blind crossover study, 10 insulin-dependent diabetic individuals were treated with a commercial brand of neutral, highly purified porcine insulin (PPI) with 30% regular and 70% NPH fractions (Mixtard, Nordisk, Denmark), and with human insulins (recombinant DNA) with 20% regular and 80% NPH fractions, and with 30% regular and 70% NPH fractions. Each of the preparations was given for 4 consecutive days. The human insulin combination with the 20% regular and 80% NPH components showed a similar activity profile as the PPI 30/70 combination. In contrast, the human insulin combination with the 30% regular and 70% NPH fractions produced substantially lower blood sugar concentrations with the associated risk of hypoglycemia.

Adult↗