Interaction of Wnt-1 proteins with the binding protein BiP.

The mouse Wnt-1 gene, a target for insertional activation in mouse mammary tumor virus-induced mammary tumors, encodes poorly secreted, cysteine-rich glycoproteins required for proper central nervous system development. We have been analyzing the biosynthesis of Wnt-1 proteins in several cell lines that express Wnt-1 cDNA from heterologous promoters. A protein of 78 kDa was found to be associated with the intracellular forms of Wnt-1 proteins in mammalian and avian cells by using multiple antisera against Wnt-1 proteins. We have identified p78 as the binding protein BiP with anti-BiP antisera and by its release from Wnt-1 immunoprecipitates upon incubation with MgCl2 and ATP. Experiments with a Wnt-1 mutant that lacks the sequence encoding the signal peptide indicates that Wnt-1 proteins must enter the secretory pathway in order to interact with BiP. We demonstrate that Wnt-1 proteins are associated with BiP in cells in which active Wnt-1 proteins are produced, such as a cultured mammary epithelial cell line and Wnt-1 transgenic mouse mammary tumor cells. The association of Wnt-1 proteins with BiP may be a factor in determining the efficiency of secretion of Wnt-1 gene products.     

Molecular heterogeneity underlying the G6PD Mediterranean phenotype

Summary As part of a study aiming to define the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency, we analysed a sample from a Portugese boy with a family history of favism. Although the biochemical properties of red-cell G6PD from this subject were similar to those of the common variant G6PD Mediterranean, the corresponding mutation (563 CT) was not present. Instead, polymerase chain reaction (PCR) amplification and sequencing of the entire gene detected a CT transition at nucleotide 592 in exon VI, changing an arginine residue to a cysteine residue only 10 amino acids downstream from the Mediterranean mutation. Single-strand conformation polymorphism analysis of a PCR-amplified DNA fragment spanning exons VI and VII of the G6PD gene has detected the same mutation, confirmed by sequencing, in a G6PD-deficient patient from Southern Italy. We name this new variant G6PD Coimbra.     

Dolichol and N-linked oligosaccharide synthesis in the rat testis: interaction between Sertoli and spermatogenic cells, evidence for paracrine effects.

The relative contribution of the Sertoli cell and the pachytene spermatocyte to dolichol and N-linked oligosaccharide biosynthesis within the seminiferous tubule was investigated. Evidence is presented to show that the interaction between these two cell types affects dolichol and N-linked oligosaccharide biosynthesis. Analysis of the dolichol content of Sertoli cultures confirms earlier data suggesting that the Sertoli cell constitutes the major pool of dolichols within the seminiferous tubule. [14C]Acetate incorporation studies suggest that the Sertoli cell in culture synthesizes dolichol much more rapidly than does the isolated pachytene spermatocyte. This information, in addition to previous data in the literature, infers an interactive effect whereby the presence of the spermatogenic cell in the tubule stimulates dolichol synthesis in the Sertoli cell. The absence of normal Sertoli-spermatocyte interactions in in vitro incubations may also limit dolichol synthesis in the pachytene spermatocyte. The distribution of dolichol kinase between the Sertoli and the pachytene spermatocyte was also examined. The concentration of this enzyme in the Sertoli cell suggests the presence of an active salvage pathway within that cell. The correlation between the appearance of the pachytene spermatocyte and the previously described peak of dolichol kinase activity in the seminiferous tubules of the prepubertal animal implies cell-cell interactions. Radiolabelling studies of N-linked oligosaccharides were conducted using [3H]mannose and concanavalin A affinity chromatography to identify multiantennary, biantennary, and high-mannose oligosaccharide pools. An in vitro bicameral coculture system was used to demonstrate that pachytene spermatocytes stimulate incorporation of [3H]mannose into Sertoli cell oligosaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of radical adduct reduction and reoxidation of the corresponding hydroxylamines in in vivo spin trapping of carbon tetrachloride-derived radicals.

In vivo spin trapping of radical metabolites has become a promising tool in understanding and predicting toxicities caused by different xenobiotics. However, in biological systems radical adducts can be reduced to electron paramagnetic resonance (EPR)-silent hydroxylamines. To overcome this difficulty, different procedures for reoxidation of the reduced radical adducts were systematically investigated and some metabolic inhibitors of nitroxide reduction were tested. As a test system, carbon tetrachloride (CCl4), a known hepatotoxic substance, was used. CCl4 is metabolized by liver to .CCl3 and, in the presence of the spin trap phenyl N-t-butylnitrone (PBN), forms the PBN/.CCl3 and PBN/.CO2- radical adducts. These radical adducts were measured in the bile using electron paramagnetic resonance after administration of CCl4 and PBN to the rat. We have shown that these radical adducts were reduced to the corresponding hydroxylamines in vivo, since immediately after the collection of bile only traces of the radical adducts could be detected, but after oxidation by different procedures such as bubbling with oxygen, addition of mild oxidant potassium ferricyanide or autoxidation the EPR spectra intensity increases, indicating that the hydroxylamines had been re-oxidized back to nitroxides. The collection of bile into plastic Eppendorf tubes containing the sulfhydryl reagent N-ethylmaleimide (NEM) or the enzyme ascorbate oxidase did not increase the intensity of the spectra significantly, demonstrating that neither reduction by reduced glutathione (GSH) nor ascorbic acid occurred ex vivo. However in the presence of NEM faster re-oxidation was observed. A new radical adduct that was not observed previously in any in vivo experiment and which exhibited 13C hyperfine coupling was detected when the rats were injected with 13CCl4. We have proven that this is the same adduct detected previously in vitro in microsomal incubations of CCl4, PBN, GSH, and reduced nicotinamide adenine dinucleotide phosphate (NADPH). As a general rule, we have shown that a variety of oxidation procedures should be tried to detect the different radical adducts which are otherwise not observable due to the in vivo reduction of radical adducts.     

Stable transfection of calbindin-D28k into the GH3 cell line alters calcium currents and intracellular calcium homeostasis.

Previous work demonstrating the presence and differential distribution of Ca(2+)-binding proteins in the CNS has led to the proposal that cytosolic proteins, such as calbindin-D28k (CB), may play a pivotal role in neurons. We have used a retrovirus containing the full-length cDNA for CB to transfect the pituitary tumor cell line GH3, to generate CB-expressing GH3 cells and to investigate whether ionic channel activities as well as the concentration of intracellular free Ca2+ ([Ca2+]i) homeostasis could be altered by the presence of this Ca(2+)-binding protein. We show that CB-transfected GH3 cells exhibited lower Ca2+ entry through voltage-dependent Ca2+ channels and were better able to reduce [Ca2+]i transients evoked by voltage depolarizations than the wild-type parent cell line. These observations provide a mechanism by which CB may protect tissues against Ca(2+)-mediated excitotoxicity.     

The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function

We describe an organotypic model of human skin comprised of a stratified layer of human epidermal keratinocytes and dermal fibroblasts within a contracted collagen lattice. Feasible and reproducible production of the skin construct has required the use of traditional as well as specialized culture techniques. The configuration of the construct has been engineered to maintain polarity and permit extended culture at the air-liquid interface. Morphological, biochemical and kinetic parameters were assessed and functional assays were performed to determine the degree of similarity to human skin. Light and ultrastructural morphology of the epidermis closely resembled human skin. The immunocytochemical localization of a number of differentiation markers and extracellular matrix proteins was also similar to human skin. Kinetic data showed a transition of the epidermal layer to a morein vivo-like growth rate during the development of the construct at the air-liquid interface. The barrier properties of the construct also increased with time reaching a permeability to water of less than 2%·h after approximately 2 weeks at the air-liquid interface which is still on average 30-fold more water-permeable than normal human skin. The construct is currently used forin vitro research and testing and is also being tested in clinical applications.     

Root Flooding of Muskmelon (Cucumis melo L.) Affects Fruit Sugar Concentration But Not Leaf Carbon Exchange Rate

Flooding the roots of greenhouse-grown muskmelon (Cucumis meloL. cv. Noy Yizreel) plants for 4 days reduced sucrose accumulation36% in the inner mesocarp and 88% in the outer mesocarp of developingfruit. Concentration of the translocated sugars raffinose andstachyose were also lower in fruit on flooded plants than inthose from nonflooded plants. In contrast, fruit hexose concentrationwas similar in both flooded and nonflooded plants. There wasno alteration in activities of enzymes associated with sucrosemetabolism in the fruit which could explain the decreased sucroseconcentration. Four days of root flooding caused no reductionin leaf carbon exchange rate or assimilate export rate, indicatingthat the reduction in fruit sucrose accumulation was not dueto source limitation. Root respiration, measured as CO₂ evolution,was approximately 30% lower in anaerobic roots than in aerobicroots. When viewed as carbohydrate consumed, a doubling of glycolyticactivity occurred in the anaerobic root mass. Increased demandfor carbohydrates by anaerobic roots may lead to a reductionin translocated carbohydrates available for sucrose biosynthesisin the developing fruit. (Received August 29, 1990; Accepted February 21, 1991)     

Polyphosphoinositol lipids inChlamydomonas eugametos gametes

InChlamydomonas eugametos gametes, phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) comprised 0.4 and 0.3% of the whole-cell phospholipids. They were concentrated in the plasma membrane around the cell body and were present in low concentrations in the flagellar membrane. When gametes were fed³²PO ₄ ^- , the label was rapidly incorporated into PtdInsP and PtdInsP₂ and only slowly incorporated into structural lipids such as phosphatidylethanolamine and phosphatidylglycerol. Similarly, when a pulse of³²PO ₄ ^- was chased with PO ₄ ^- , the label was rapidly lost from the polyphosphoinositol lipids but not from the structural lipids. The major fatty acids in the polyphosphoinositides were C-22 carbon polyenoic acids (70%). The significance of these results in relationship to intracellular signalling via inositol phosphates and Ca²⁺ is discussed.Abbreviations InsP₃ inositol 1,4,5-trisphosphate - mt^–/mt⁺ mating-type plus or minus - PtdA phosphatidic acid - PtdEtn phosphatidylethanolamine - PtdGro phosphatidylglycerol - PtdIns phosphatidylinositol - PtdInsP phosphatidylinositol 4-phosphate; - PtdInsP₂ phosphatidylinositol 4,5-bisphosphate - TCA trichloroacetic acid We thank Frank Schuring for Fig. 5A and Susan Kenter, Hans Kruisselbrink, Saskia Bijvank and Nelleke Corbett for their enthousiastic assistance.