Developmental expression of the cell adhesion molecule-like protein tyrosine phosphatases LAR, RPTPdelta and RPTPsigma in the mouse.

Using RNA in situ hybridization we compared the expression patterns of the cell adhesion molecule-like receptor-type protein tyrosine phosphatases LAR, RPTP sigma and RPTP sigma during mouse development. We found that LAR is expressed in basal lamina-associated epithelial tissues of (neuro)ectodermal, neural crest/ectomesenchyme and endodermal origin. RPTP sigma is found in (neuro)ectodermal, neural crest-derived systems and in mesoderm-derived tissues. The expression pattern of RPTP sigma largely parallels that of RPTP sigma, in concordance with their proposed evolutionary history

Protein-tyrosine phosphatases expressed in mouse epidermal keratinocytes.

The importance of growth factors acting via receptor-type protein-tyrosine kinases in the continuous renewal of the epidermis from the keratinocyte stem cell population has been well established. Protein-tyrosine phosphatases (PTPases), which dephosphorylate phosphotyrosine-containing proteins, may therefore be expected to play an equally important role in the control of epidermal growth and differentiation. In this study, we have made an inventory of the various PTPases that are expressed during mouse keratinocyte proliferation and maturation. A panel of 13 different PTPases probes was obtained by combining a set of PTPase cDNAs previously cloned from mouse brain and a set of PTPase probes obtained from a normalized keratinocyte PTPase cDNA library. This PTPase cDNA panel, spanning probes for receptor-type as well as cytoplasmic-type family members, was used to monitor RNA expression levels in keratinocyte fractions isolated from murine epidermis and in keratinocyte cell cultures. No overt changes were observed in PTPase mRNA levels in all strata of mouse epidermis, but comparison of cultured cells with freshly isolated keratinocytes revealed several conspicuous differences. In the cultured Balb/MK cell line, absence of PTP delta expression and upregulation of PTP kappa and, to a lesser extent, PTP gamma mRNA ratios were observed compared to the freshly isolated cells. These results provide a basis for further research on the impact of PTPase activity on epidermal growth control.

The neuronal nitric oxide synthase PDZ motif binds to -G(D,E)XV* carboxyterminal sequences.

PDZ motifs are small protein-protein interaction modules that are thought to play a role in the clustering of submembranous signalling molecules. The specificity and functional consequences of their associative actions is still largely unknown. Using two-hybrid methodology we here demonstrate that the PDZ motif of neuronal nitric oxide synthase (nNOS) can mediate the binding to several other proteins in brain. Peptide library screening showed that proteins bearing a carboxy-terminal G(D,E)XV* sequence are preferred targets for the nNOS amino-terminal PDZ motif. Potential nNOS targets include a melanoma-associated antigen, cyclophilins and the alpha1C-adrenergic receptor.

Effect of clofibrate on branched-chain amino acid metabolism.

Clofibric acid inhibited the oxidative decarboxylation of 4-methyl-2-oxopentanoate and 3-methyl-2-oxobutanoate in mitochondria and homogenates of rat liver and quadriceps muscle. In rat hemidiaphragms clofibric acid inhibited the oxidative decarboxylation of 4-methyl-2-oxopentanoate and had no effect on that of 3-methyl-2-oxobutanoate. Clofibric acid displaced branched-chain 2-oxo acids from bovine serum albumin. Clofibrate-treatment of rats decreased the actual activity and activity state of the branched-chain 2-oxo acid dehydrogenase complex in quadriceps muscle, and increased the total activity in heart and liver without a change of the activity state. All interactions of clofibric acid with the metabolism of branched-chain amino acids appear to relate to its structural resemblance to the branched-chain 2-oxo acids. Both reduced plasma and muscle concentrations of branched-chain amino acids and reduced muscle oxidation may play a role in the myopathic side-effects of clofibrate-treatment.

Studies on malaria and responses of Anopheles balabacensis balabacensis and Anopheles minimus to DDT residual spraying in Thailand.

Studies on malaria and on A. b. balabacensis and A. minimus responses to DDT spraying were conducted in a forested hilly area in northern Thailand. In a first phase, base-line data were collected from July 1970 to March 1972. In a second phase, the study area received five round of DDT spraying over a period of two years and at the same time all malaria infections received radical treatment. During this two-year period of field operations, entomological and epidemiological observations were continued. The studies carried out in the second phase, showed that malaria transmission decreased under the applied optimum anti-malarial measures but was not interrupted. Human ecology and population movement inside the forest, especially during the dry season, contributed to a great extent to this result. The transmission occurring in the early part of the monsoon season clearly indicates the importance of the timing of DDT spraying. A. b. balabacensis appeared to be transmitting malaria all the year round in the deep forest but only in the monsoon season in the forest fringe. The vectorial capacity of both vectors was estimated separately for indoor and outdoor populations. The pre-spraying values obtained for A. b. balabacensis were much higher thaan for A. minimus. After DDT spraying A. b. balabacensis showed a decrease in vectorial capacity estimated at 31.5 times for the indoor population and 18 times for the outdoor population. A. minimus, on the other hand, showed a much smaller decrease, estimated at 6.8 and 1.9 times for the indoor and outdoor populations respectively.

Rapid assessment of protein-tyrosine phosphatase expression levels by RT-PCR with degenerate primers.

Using degenerate oligodeoxynucleotide primers we previously obtained cDNA fragments from ten different murine protein-tyrosine phosphatases (PTPases). Employing this same primer set, a method was developed to assess the expression levels of these PTPase family members in a fast and simple way. RT-PCR products of several cell types and tissue samples were used as probes on dot-blots containing the ten different PTPase fragments in equimolar amounts. Hybridization intensities at the various dots reflect the relative expression levels of the corresponding PTPases in the starting material. In this way expression of PTPases during mouse brain development could be monitored. Expression of PTP delta was found to be absent in embryonic stem cells but high in fetal and adult brain. PTP epsilon expression is shown to gradually increase in brain during maturation. Our method is generally applicable to gene families of which the transcripts can be detected with a single degenerate primer pair and is especially useful in situations where only limited amounts of RNA can be obtained.

Identification and typing of members of the protein-tyrosine phosphatase gene family expressed in mouse brain.

Protein-tyrosine phosphatases (PTPases) form a novel and important class of cell regulatory proteins. We evaluated the expression of PTPases in mouse brain by polymerase chain amplification of cDNA segments that encode the catalytic domains of these enzymes. Degenerate primer pairs devised on the basis of conserved protein motifs were used to generate a series of distinct PCR-derived clones. In this way, murine homologues of the human PTPases LRP, PTP beta, PTP delta, PTP epsilon and LAR were obtained. Corresponding regions in their catalytic domains were used to reveal the evolutionary relationships between all currently known mammalian PTPase protein family members. Phylogenetic reconstruction displayed considerable differences in mutation rates for closely related PTPases.

Increase of the activity state and loss of total activity of the branched-chain 2-oxo acid dehydrogenase in rat diaphragm during incubation.

Actual and total branched-chain 2-oxo acid dehydrogenase activities were determined in homogenates of incubated diaphragms from fed and starved rats. Incubation in Krebs-Ringer buffer increased the activity state, but caused considerable loss of total activity. Palmitate oxidation rates and citrate synthase activities did not significantly change on incubation. Starved muscles showed a higher extent of activation after 15 min of incubation (not after 30 and 60 min) and a smaller loss of total activity. Experiments with the transaminase inhibitor amino-oxyacetate confirm that the contribution of endogenous amino acids to the oxidation precursor pool is also smaller in diaphragms from starved rats on incubation in vitro. These phenomena together cause the higher 14CO2 production from 14C-labelled branched-chain amino acids and 2-oxo acids in muscles from starved than from fed rats. High concentrations of branched-chain 2-oxo acids, and the presence of 2-chloro-4-methyl-pentanoate, octanoate or ketone bodies, increase the extent of activation of the dehydrogenase complex; glucose and pyruvate had no effect. The observed changes of the activity state by these metabolites are discussed in relation to their interaction with branched-chain 2-oxo acid oxidation in incubated hemidiaphragms.

Effect of starvation and exercise on actual and total activity of the branched-chain 2-oxo acid dehydrogenase complex in rat tissues.

Starvation does not change the actual activity per g of tissue of the branched-chain 2-oxo acid dehydrogenase in skeletal muscles, but affects the total activity to a different extent, depending on the muscle type. The activity state (proportion of the enzyme present in the active state) does not change in diaphragm and decreases in quadriceps muscle. Liver and kidney show an increase of both activities, without a change of the activity state. In heart and brain no changes were observed. Related to organ wet weights, the actual activity present in the whole-body muscle mass decreases on starvation, whereas the activities present in liver and kidney do not change, or increase slightly. Exercise (treadmill-running) of untrained rats for 15 and 60 min causes a small increase of the actual activity and the activity state of the branched-chain 2-oxo acid dehydrogenase complex in heart and skeletal muscle. Exercise for 1 h, furthermore, increased the actual and the total activity in liver and kidney, without a change of the activity state. In brain no changes were observed. The actual activity per g of tissue in skeletal muscle was less than 2% of that in liver and kidney, both before and after exercise and starvation. Our data indicate that the degradation of branched-chain 2-oxo acids predominantly occurs in liver and to a smaller extent in kidney and skeletal muscle in fed, starved and exercised rats.

[Biological effect of laser-photo-coagulation on the retina (author's transl)]. / Biologische Wirkung des Lasers auf die Netzhaut

The effects of laser-photo-coagulation on retina, pigment epithelium and choroid has been studied in animals (rabbit and mini-pig). During the first two hours after photocoagulation the permeability of the blood-retinal barrier and of the pigment epithelium is temporarily increased. Later on (2-4 days) pigment epithelial cells proliferate within the coagulated areas and "tighten" the epithelial leaks. Photoreceptor outer segments undergo during the same period reversible breakdown. Normal retinal blood vessels are hardly altered by photocoagulation whereas pathological or neoformed vessels may be obliterated. Functional deficiences are observed after extensive photocoagulations only and even then rarely implicated by the patients.

A novel receptor-type protein tyrosine phosphatase with a single catalytic domain is specifically expressed in mouse brain.

Protein tyrosine phosphatases (PTPases) are important regulatory proteins that, together with protein tyrosine kinases, determine the phosphotyrosine levels in cell signalling proteins. By PCR amplification of mouse brain cDNA fragments encoding the catalytic domains of these enzymes, we identified three novel members of the PTPase gene family. Northern-blot analysis showed that two of these novel clones represent brain-specific PTPases, whereas the third originates from a large-sized mRNA that is more ubiquitously expressed. A full-length cDNA encoding one of the brain-specific PTPases, PTP-SL, was isolated. Sequence analysis revealed a transmembrane PTPase containing a single catalytic phosphatase domain that has 45% homology to a rat cytoplasmic brain-specific PTPase named STEP. This suggests a role for PTP-SL in cell-cell signalling processes in the brain.

Modulation of gene activity by consecutive gene targeting of one creatine kinase M allele in mouse embryonic stem cells.

The cytosolic creatine kinases (CK's; EC 2.7.3.2) BB, BM and MM are dimeric isoenzymes which have an important role in energy metabolism and display characteristic tissue- and stage-specific patterns of expression in mammals. To study the functional role of the distribution of the CK isoenzymes we have focussed on the modulation of expression of the genes encoding the individual B and M subunits, starting at the muscle creatine kinase (CKM) gene which is transcriptionally inactive during early embryogenesis. Using repeated rounds of gene targeting in mouse embryonic stem (ES) cells, two types of mutant cell lines were obtained. First, we generated a cell line in which insertion of a neomycin resistance (neor) gene had disrupted one of the CKM alleles. Subsequently, from this cell line, following introduction of an insertion type vector designed for replacement of the muscle specific CKM-enhancer by the constitutively acting polyoma virus enhancer PyF441, several independent doubly targeted clones were isolated which all had insertions in the previously neo-disrupted CKM allele. In some of these ES clones, the targeted enhancer replacement resulted in gene correction and functional activation of the silent CKM gene. Dimerisation between the ectopically expressed CKM subunits and CKB subunits which are normally present at high levels in ES cells, led to the formation of the BM isoform of CK in these clones.

A FERM domain governs apical confinement of PTP-BL in epithelial cells.

PTP-BL is a cytosolic multidomain protein tyrosine phosphatase that shares homologies with several submembranous and tumor suppressor proteins. Here we show, by transient expression of modular protein domains of PTP-BL in epithelial MDCK cells, that the presence of a FERM domain in the protein is both necessary and sufficient for its targeting to the apical side of epithelial cells. Furthermore, immuno-electron microscopy on stable expressing MDCK pools, that were obtained using an EGFP-based cell sorting protocol, revealed that FERM domain containing fusion proteins are enriched in microvilli and have a typical submembranous location at about 10-15 nm from the plasma membrane. Immunofluorescence microscopy suggested colocalization of the FERM domain moiety with the membrane-cytoskeleton linker ezrin. However, at the electron microscopy level this colocalization cannot be confirmed nor can we detect a direct interaction by immunoprecipitation assays. Fluorescence recovery after photobleaching (FRAP) experiments show that PTP-BL confinement is based on a dynamic steady state and that complete redistribution of the protein may occur within 20 minutes. Our observations suggest that relocation is mediated via a cytosolic pool, rather than by lateral movement. Finally, we show that PTP-BL phosphatase domains are involved in homotypic interactions, as demonstrated by yeast two-hybrid assays. Both the highly restricted subcellular compartmentalization and its specific associative properties may provide the appropriate conditions for regulating substrate specificity and catalytic activity of this member of the PTP family.

The activity state of the branched-chain 2-oxo acid dehydrogenase complex in rat tissues.

An assay is described to define the proportion of the branched-chain 2-oxo acid dehydrogenase complex that is present in the active state in rat tissues. Activities are measured in homogenates in two ways: actual activities, present in tissues, by blocking both the kinase and phosphatase of the enzyme complex during homogenization, preincubation, and incubation with 1-14C-labelled branched-chain 2-oxo acid, and total activities by blocking only the kinase during the 5 min preincubation (necessary for activation). The kinase is blocked by 5 mM-ADP and absence of Mg2+ and the phosphatase by the simultaneous presence of 50 mM-NaF. About 6% of the enzyme is active in skeletal muscle of fed rats, 7% in heart, 20% in diaphragm, 47% in kidney, 60% in brain and 98% in liver. An entirely different assay, which measures activities in crude tissue extracts before and after treatment with a broad-specificity protein phosphatase, gave similar results for heart, liver and kidney. Advantages of our assay with homogenates are the presence of intact mitochondria, the simplicity, the short duration and the high sensitivity. The actual activities measured indicate that the degradation of branched-chain 2-oxo acids predominantly occurs in liver and kidney and is limited in skeletal muscle in the fed state.

Toward early diagnosis of myotonic dystrophy: construction and characterization of a somatic cell hybrid with a single human der(19) chromosome.

We have constructed a somatic cell hybrid line, designated 908K1, with a single human der(19) chromosome on a Chinese hamster background by employing conventional as well as microcell-mediated cell fusion techniques. The der(19) chromosome comprises the 19p13.1----q13.2 segment, as well as the distal (Xq24----qter) portion of the X chromosome long arm, and is stably retained by HAT selection. Extensive characterization of this hybrid line and comparison with other somatic cell hybrids has enabled us to regionally assign PGK2 to the distal short arm of chromosome 19 and to narrow down the assignments of CYP1, TGFB, and ERCC1 on 19q. Moreover, a cosmid library has been constructed from this microcell hybrid. By screening this library, as well as a chromosome 19-enriched library obtained elsewhere, 14 single-copy probes have been isolated that map on the 19p13.1----q13.2 segment, and 5 probes were assigned to the distal Xq. It is anticipated that these probes will be useful for the diagnosis of myotonic dystrophy and fra(X) mental retardation.

Genetic variability of the murine creatine kinase B gene locus and related pseudogenes in different inbred strains of mice.

The role of genetic variation in isoenzyme gene families is often poorly appreciated. We report here on the determination of DNA sequences and typing of genetic variability in four creatine kinase B (CKB) gene loci in different inbred strains of mice. The unique functional murine CKB gene was found to be nearly identical to the previously characterised rat and human sequences in both size and exon-intron structure. In this gene, approximately 0.5% allelic nucleotide positions as well as the lengths of simple A-rich and [TG]n repetitive elements located at the 5' and 3' sides of the transcribed segment, differed between inbred strains of mice. Preliminary experiments suggest that this sequence divergence is of importance for design of gene targeting strategies involving homologous DNA recombination. The three additional CKB-like gene loci in mice all had the characteristics of processed pseudogenes. By Southern blot analysis we could demonstrate that both the type and number of pseudogenes differed between inbred strains. Analysis of the CKB gene sequences enabled us to speculate about the evolutionary history of this highly polymorphic subfamily of genes.

Isolation and characterization of alphoid DNA sequences specific for the pericentric regions of chromosomes 4, 5, 9, and 19.

We have cloned and characterized two distinct types of alphoid DNA elements. Probe pG-Xba 11/340 was obtained by random cloning of human satellite DNA and contains two basic units with overall 88% homology to the 171-bp consensus alphoid sequence. pG-Xba 11/340-like elements are represented about 2,000-4,000 times in the haploid genome and, by in situ hybridization, are found exclusively at the primary constrictions of chromosomes 4 and 9. Probe pG-A16 was cloned from a chromosome 19-specific cosmid library and represents a 2.25-kb higher-order DNA element which is present at roughly 75-150 copies per haploid genome and which hybridizes to the centromeres of chromosomes 5 and 19. Using the pG-A16 probe, further genetic and physical dissection of the central area of chromosome 19 can be envisaged.

Molecular cloning of a mouse epithelial protein-tyrosine phosphatase with similarities to submembranous proteins.

Protein-tyrosine phosphatases (PTPases) form an important class of cell regulatory proteins. We have isolated overlapping cDNA clones that together comprise an 8 kb transcript encoding a novel murine PTPase which is expressed in various organs. Sequence analysis revealed an open reading frame of 2,460 amino acid residues. The predicted protein, PTP-BL, is a large non-transmembrane PTPase that exhibits 80% homology with PTP-BAS, a recently described human PTPase. PTP-BL shares some intriguing sequence homologies with submembranous proteins. It contains a band 4.1-like motif also present in the tumor suppressors neurofibromatosis 2 and expanded, five 80 amino acid repeats also present in the discs-large tumor suppressor, and a single catalytic phosphatase domain. No obvious homologies to other proteins were found for the N-terminal region of the protein other than human PTP-BAS. RNA in situ hybridization experiments show that the PTP-BL gene is expressed in epithelial cells, predominantly in kidney, lung, and skin. These data suggest a cell cortical localization for PTP-BL in epithelial cells and a possible role in the morphology and motility of epithelial tissues.

The mouse gene Ptprf encoding the leukocyte common antigen-related molecule LAR: cloning, characterization, and chromosomal localization.

The human receptor-like protein tyrosine phosphatase leukocyte common antigen-related molecule (LAR; gene symbol PTPRF) closely resembles cell adhesion molecules, which suggests that it may be involved in the regulation of phosphotyrosine levels through cell-cell or cell-matrix interactions. To obtain a better understanding of LAR function, we have characterized the mouse Ptprf gene as a first step toward site-directed mutagenesis studies in vitro and in vivo. The cytoplasmic region of the mouse LAR (mLAR) protein is encoded by 11 exons that span only 4.5 kb of genomic DNA. Compared to the known exon-intron structures of other mammalian receptor-like protein tyrosine phosphatase genes, such as Ptpra (encoding LRP) and Ptprc (coding for Ly-5), the Ptprf gene part encoding the cytoplasmic region of mLAR contains not only smaller, but also fewer introns. Sequence analysis of both phosphatase domains of mLAR and its homologs MPTP delta and mRPTP sigma revealed a higher evolutionary conservation of the second, C-terminal domain in comparison to the first domain. Fluorescence in situ hybridization was used to map the Ptprf gene to region C6-D1 on mouse chromosome 4.