Promoter of the gene encoding the 16 kDa DNA-binding and apoptosis-inducing C1D protein.

The 5' region of the gene encoding the human 16 kDa DNA-binding and apoptosis-inducing C1D protein was analysed for promoter activity. Sections of this region were cloned into a promoterless vector containing the enhanced green fluorescent protein (EGFP) as reporter gene. Expressed EGFP was estimated in transfected cells by quantitative fluorescence microscopy. The sequence between mRNA positions ATG -868 and ATG -12 results in relatively highest EGFP expression in transiently transfected human and murine cells. The upstream segment immediately adjacent to the 5' end of the most active fragment was identified as an inverted LINE-1 repeat element. Transient transfection experiments point to the presence of cis-acting repressing sequences on this LINE-1 element which reduce the transcriptional activity of the basal C1D promoter in human and murine cells by more than 95%. This result supports previous evidence suggesting that LINE-1 sequences may function as regulatory elements to control the expression of nearby genes.

Stability of DNA repeats in Escherichia coli dam mutant strains indicates a Dam methylation-dependent DNA deletion process.

In this study we report on the stabilization of short direct repetitive DNA elements. We arranged a 20 bp SK-primer element in a direct repeat manner within the cloning vector pBluescript KS (+). This resulted in an array of 27 direct repeats consisting of 24 bp units. We show that this plasmid could only be propagated without deletion of repeats in dam mutant Escherichia coli hosts, whereas all efforts to use strains that were defective in the methylation-dependent restriction system and the recA- or the mismatch repair-dependent deletion system failed. The deletions always affected whole repeat units and not parts of them, leading to an unpredictable reduction of the unit number down to a range of between 12 and two during propagation. We conclude that a Dam methylation-dependent, but recA- and mismatch repair-independent, deletion mechanism caused the DNA rearrangements without an obvious involvement of the known methylated-DNA restriction systems.

Targeting of the 22 kDa integral peroxisomal membrane protein.

Investigating targeting of the 22 kDa peroxisomal membrane protein (Pmp22p) to the peroxisomal membrane we have confined the targeting signal to amino acid residues 16-37 located in the N-terminal cytoplasmic tail. Comparison of Pmp22p orthologous sequences revealed a conserved motif Y3xL3xP3x(KQN) which might represent the core of this targeting signal not found so far in other Pmps. Fusion of the Pmp22p N-terminal tail to the C-terminal portion of Pmp22p which per se is not targeted to peroxisomes, conveys peroxisomal targeting. These data suggest that Pmp22p is targeted to peroxisomes by a new membrane targeting signal which is necessary and sufficient to target a polypeptide containing two transmembrane spans to peroxisomes.

Ether lipid biosynthesis: isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase.

In this paper we describe isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase (DAP-AT). The enzyme was extracted from rabbit Harderian gland peroxisomes and isolated as a trimeric complex by sucrose density gradient centrifugation. From peptide sequences matching EST-clones were obtained which allowed cloning and sequencing of the cDNA from a human cDNA library. The nucleotide-derived amino acid sequence revealed a protein consisting of 680 amino acid residues of molecular mass 77187 containing a C-terminal type 1 peroxisomal targeting signal. Monospecific antibodies raised against this polypeptide efficiently immunoprecipitated DAP-AT activity from solubilized peroxisomal preparations, thus demonstrating that the cloned cDNA codes for DAP-AT.

Synthesis of plasmalogens in eye lens epithelial cells.

The present paper describes cloning and sequencing of the mouse cDNA encoding dihydroxyacetonephosphate acyltransferase (DAPAT), the peroxisomal key enzyme of plasmalogen (PM) biosynthesis. Using monospecific antibodies, we localized DAPAT and alkyl dihydroxyacetonephosphate synthase to peroxisomes of mouse lens epithelial cells (LECs) and determined their enzymatic activity. By electrospray ionization mass spectrometry of mouse lens lipid extracts, we identified phosphatidyl ethanolamine including plasmenyl ethanolamine species as major constituents. Our data demonstrate the capacity of LECs to synthesize PMs and the high coincidence between deficiency of PM and early manifestation of cataract in patients with peroxisomal disorders suggests that ether-bonded lipids may play an important role in maintaining lens transparency.

Differential display PCR reveals induction of immediate early genes by vasoactive intestinal peptide in PC12 cells.

In order to identify genes regulated by vasoactive intestinal peptide, we performed differential display PCR as originally described by Liang and Pardee. Messenger RNA of PC12 cells treated with vasoactive intestinal peptide or nerve growth factor for one hour was reverse transcribed and amplified using different sets of oligo-dT and random primers. Radioactively labeled PCR products were displayed on polyacrylamide gels and candidate cDNAs extracted from the gel, re-amplified by PCR, cloned, and sequenced. Differential expression was verified by RT-PCR applying sets of specific primers obtained from the sequence. The specificity of the PCR product was confirmed by Southern blotting using a radioactively labeled internal primer and semi-quantitative densitometric analysis. This rapid and sensitive protocol led to the isolation of two immediate early genes, pip92 and PC4, known to be increased on mRNA level by nerve growth factor in PC 12 cells.

lacZ transgenic mice to monitor gene expression in embryo and adult.

In transgenic experiments, lacZ can be used as a reporter gene for activity of a given promoter. Its main advantage is the ease of visualization in situ, on sections or in whole mount preparations, and the availability of simple protocols. In the following, we describe our procedure for detecting promoter activity in transgenic mice, including choice of lacZ vectors, generation of the transgenic mice, and analysis of expression. We had recently used this protocol to detect tyrosinase gene promoter activity in embryonic and adult brain.

Phosphate Transport across the Plasma Membrane of Wheat Leaf Protoplasts: Characteristics and Inhibitor Specificities.

The kinetics and inhibitor specificities of phosphate transport across the plasma membrane of wheat leaf mesophyll protoplasts have been examined. Studies were also carried out on the effects of light and pH on phosphate transport and the plasma membrane electropotential. At pH 5.8 (30 degrees C), protoplasts accumulated phosphate at the rate of 3.9 +/- 0.2 nanomoles per milligram protein per hour. Phosphate uptake rates and inhibitor specificities for the leaf cell plasma membrane phosphate transporter were qualitatively similar to those observed with root protoplasts. Neither picrylsulfonic acid, or p-chloromercuribenzene sulfonate affected phosphate uptake significantly at 0.1 millimolar. Of all compounds tested, carbonyl cyanide-p-trifluoromethoxy phenylhydrazone was the most effective inhibitor of phosphate uptake (60% at 0.1 millimolar). Tribenzylphosphate inhibited uptake by 34% while dibenzylphosphate had no effect. The plasma membrane electropotential was found to be -37 +/- 3 millivolts. Initiation of photosynthesis lowered the membrane potential to -39 +/- 3 millivolts. Inhibition of phosphate uptake by 34% with the substrate analog tribenzylphosphate resulted in a measured membrane potential of -33 +/- 3 millivolts. These changes in potential were not significant at the 5% probability level. Phosphate uptake rates remained constant under photosynthetic and nonphotosynthetic conditions. The utility of tribenzylphosphate as an inhibitor in plant systems is demonstrated.

Transgenic mouse models for tumors of melanocytes and retinal pigment epithelium.

Cutaneous and ocular melanomas are due to malignant transformation of neural crest-derived melanocytes. The rising incidence of this tumor in humans has stimulated experiments to devise suitable mouse models. In the past years, transgenic mouse lines have been generated using different oncogenes - Ha-ras, SV40 T antigen (Tag), ret - which develop benign lesions of melanocytes, melanoma, and/or eye tumors. Pigment cell tumors in humans, although rather rare, can also develop from the retinal pigment epithelium (RPE), a cell layer of neuroectodermal origin. We, therefore, established transgenic models for this ocular tumor. Regulated by the promoter of tyrosinase-related protein-1 (TRP-1), two oncogenes, ret and SV40 Tag, were targeted to the developing RPE in transgenic mice. The TRP-1/ret transgenic mice displayed microphthalmia and benign tumors of the RPE. Expression of SV40 T antigen (TRP-1/Tag) led to malignant tumors, which were invasive and metastasized to inguinal lymph node and spleen.

Transport of hydrophobic ions in erythrocyte membrane: I. Zero membrane potential properties.

The permeability and partition coefficients of tetraphenylarsonium (TPA) and several other organic cations were studied in the human erythrocyte using an ion-selective electrode. The permeability constant for the different cations could be explained quite well by differences in oil/water partition coefficients. No evidence for facilitated transport could be found. Binding of the organic ions occurred to both the cell membrane and to intracellular contents. Partitioning to the membrane remained relatively constant despite variation from ion intracellular binding with blood samples from different donors. TPA flux is stimulated by substoichiometric amounts of tetraphenylboron and other organic anions, suggesting an ion-pairing mechanism.

Regionalized self-care hemodialysis. A solution to the increasing cost.

To cope with an ever-increasing number of patients with end-stage renal disease, a regionalized self-care hemodialysis program was set up and combined with the existing home and center hemodialysis, peritoneal dialysis, and renal transplantation. Five years later, of a total of 105 patients treated by hemodialysis, 66 were dialyzing themselves--31 at home and 35 in seven local self-care facilities. This proportion of patients engaging in autonomous treatment (69%) was obtained without restrictive selection criteria, since we treat 210 patients per million population with hemodialysis. The annual intake of new patients could be managed without increasing the number of center dialysis beds and by only slightly increasing the specialized staff. The cost reduction obtained with this program when compared with all center dialysis treatment represents 37%, or approximately 1,200,000 US dollars per year. These results were obtained without compromising the quality of treatment.

Detection and quantification of protein phosphatase inhibitor-1 gene expression in total rat liver and isolated hepatocytes.

The mRNA expression of protein phosphatase inhibitor-1 (inhibitor-1) in rat liver was demonstrated using highly sensitive semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Quantification by real-time RT-PCR (LightCycler technology) yielded the same copy number of inhibitor-1 mRNA in total rat liver and isolated hepatocytes (12 copies per cell). This novel finding shows that rat liver expresses indeed inhibitor-1 mRNA, albeit in low amounts. The low copy number explains why the mRNA had not been detected by Northern blotting so far. For comparison, about 425 copies/cell were detected in brain and 2500 copies/cell in skeletal muscle from rat. The full-length coding sequence of rat liver inhibitor-1 was cloned and sequenced, 100% homology with the muscle cDNA was obtained, indicating the expression of the same gene in liver and muscle. In vitro transcription and translation yielded a protein (Mr approximately 30 kDa) which could be detected with a specific antibody by immunoblotting. This indicates an intact open reading frame of inhibitor-1 in rat liver. Immunoblotting of liver extract yielded a very weak band which comigrated with the inhibitor-1 proteins from muscle and brain. It is concluded that mRNA expression of inhibitor-1 may have implications for the regulation of protein phosphatase-1 (PP1) in rat liver.

Impaired membrane traffic in defective ether lipid biosynthesis.

The first steps of ether lipid biosynthesis are exclusively localized to peroxisomes and hence some peroxisomal disorders are characterized by a severe deficiency of plasmalogens, the main ether lipids in humans. Here we report on gene defects of plasmalogen biosynthesis, chromosomal localization of the corresponding genes and, as a consequence of plasmalogen deficiency, on structural alterations of caveolae, clathrin-coated pits, endoplasmic reticulum and Golgi cisternae, as well as on the reduced rate of transferrin receptor cycling. The data suggest that plasmalogens, analogous to cholesterol, are essential for correct membrane functioning and their deficiency results in impaired membrane trafficking.