86Rb+ fluxes in Chinese hamster ovary cells as a function of membrane cholesterol content

Steady-state fluxes of ⁸⁶Rb⁺ (as a tracer for K⁺) were measured in Chinese hamster ovary cells (CHO-K1) and a mutant (CR1) defective in the regulation of cholesterol biosynthesis; the membrane cholesterol content of this mutant was varied by growing it on a range of cholesterol supplements to lipid-free medium (Sinensky, M. (1978) Proc. Natl. Acad. Sci. U.S. 75, 1247–1249).Analogous to previous findings in ascites tumor cells, ⁸⁶Rb⁺ influx in the parent strain was differentiated into a ouabain-inhibitable ‘pump’ flux, furosemide-sensitive, chloride-dependent exchange diffusion, and a residual ‘leak’ flux.On the basis of this flux characterization, ⁸⁶Rb⁺ pump and leak fluxes were measured in the mutant as a function of membrane cholesterol content. Pump and leak fluxes, when expressed per ml cell water, were independent of the cholesterol content of the mutant. Moreover, ⁸⁶Rb⁺ fluxes in the mutant were equal to those in the parent strain. Our data imply that the flux behavior of K⁺ in the steady state is independent of the ordering of membrane lipid acyl chains.     

Production of hyaluronan-binding glycoprotein by human monocytes. Its use as marker in myeloid leukemia]

A hyaluronan-binding protein fraction was isolated by affinity chromatography of peripheral human blood mononuclear cell culture medium through immobilized hyaluronan. The presence of a hyaluronan-binding protein similar to human brain hyaluronectin was demonstrated by (i) the ELISA method on hyaluronan-coated plastic plates using anti-hyaluronectin antibodies, (ii) the lowering of the elution volume of the protein on liquid gel chromatography in the presence of hyaluronan, (iii) the extinction of the reaction to human brain hyaluronectin when antibodies were absorbed out with monocyte hyaluronectin, (iv) western blotting with polyclonal and monoclonal anti-hyaluronectin antibodies. The hyaluronectin-producing cells were adherent (10 min., 37 degrees C) to plastic, esterase (+) and CD 14 (+) cells and had the morphology of monocytes. The protein expression was investigated in leukemic cells by means of the immunocytochemical method. Hyaluronectin expression was restricted to 4/12 of M4 and M5 types of acute myeloid leukemias. Other myeloid leukemia and acute lymphoblastic leukemia cells were negative. The results indicate that hyaluronectin can be produced under free form in the absence of hyaluronan, by human peripheral blood monocytes. It supports the hypothesis that the expression of hyaluronectin in tumour stroma could be due, at least in part, to inflammatory cells of the tumour. The expression of the protein by M4 and M5 acute myeloid leukemia cells suggests that hyaluronectin could be synthesized by immature cells of the monocytic lineage as well as by mature monocytes.     

Programmed cell death in the ovary: insights and future prospects using genetic technologies

Programmed cell death (PCD) plays a prominent role in development of the fetal ovaries and in the postnatal ovarian cycle. As is the case with other major organ systems, an evolutionarily conserved framework of genes and signaling pathways has been implicated in determining whether or not ovarian germ cells and somatic cells will die in response to either developmental cues or pathological insults. However, the identification of increasing numbers of potential ovarian cell death regulatory factors over the past several years has underscored the need for studies to now separate correlation (e.g. endogenous gene expression) from function (e.g. requirement of the gene product for the execution of PCD). In this regard, genetic technologies have recently been used to examine the functional significance of specific proteins and signaling molecules to the regulation of PCD in the female gonad in vivo. In addition to the more classic approaches, such as the use of genetic null and transgenic mice, methods that achieve cell lineage-selective and/or developmentally timed gene targeting are on the horizon for use by reproductive biologists to more accurately dissect the mechanisms by which PCD is controlled in the ovary. This minireview will highlight some of the advances that have already been made using gene knockout and transgenic mice, as well as provide an overview of the current and future status of cell lineage-selective gene disruption, in the context of PCD and ovarian function.     

Microscale autoradiographic method for the qualitative and quantitative analysis of apoptotic DNA fragmentation

A method combining the advantages of electrophoretic DNA fractionation and autoradiography is described for the qualitative and quantitative analysis of internucleosomal DNA fragmentation that occurs during apoptosis, or “programmed cell death”. This procedure utilizes terminal transferase enzyme to uniformly add one molecule of [α ³²P]-to the 3′-of DNA fragments. Following gel electrophoresis and autoradiographic analysis, the total amount of radiolabel incorporated into the low molecular weight DNA fraction can be quantitated and used to estimate the degree of apoptotic DNA fragmentation in any given sample. This method requires as little as 15 ng of total cellular DNA and increases the sensitivity of apoptotic DNA detection by at least 100-fold over the widely used ethidium bromide staining method. The procedure should prove valuable for the analysis of apoptosis in minute quantities of tissues and cultured cells. © 1993 Wiley-Liss, Inc.     

Mass spectrometry based analysis of human plasma‐derived factor X revealed novel post‐translational modifications

Human coagulation factor X is a central component of the blood coagulation cascade that converts, under its activated form, prothrombin into thrombin. Generation of thrombin is the final step of the clotting cascade that leads to the clot by polymerization of fibrinogen molecules into a fibrin network. Today, research of new by‐passing agents of the coagulation may contribute to an increased interest for human factor X, which may, in consequence, lead to the need of a more exhaustive picture of its structural features. Several post‐translational modifications of human factor X such as γ‐carboxylation/β‐hydroxylation of the N‐terminal light chain and N‐/O‐glycosylation of the activation peptide have been described. But, so far as we know, no comprehensive studies of its post‐translational modifications have been reported. In this article we report an exhaustive structural analysis of human factor X by mass spectrometry using successive protein and peptide mapping. Surprisingly, human factor X was found to be mostly O‐glucosylated on its light chain at Ser₁₀₆ position, Ser₉ of its activation peptide is phosphorylated at about 30% and its C‐terminal heavy chain is fully O‐glycosylated at Thr₂₄₉ by a mucin‐type O‐glycan (HexNAc‐Hex‐NeuAc). The knowledge of these post‐translational modifications is mandatory for the development of recombinant molecules.     

Emerging role of autophagy in kidney function,diseases and aging

Autophagy is a highly conserved process that degrades cellular long-lived proteins and organelles. Accumulating evidence indicates that autophagy plays a critical role in kidney maintenance, diseases and aging. Ischemic, toxic, immunological, and oxidative insults can cause an induction of autophagy in renal epithelial cells modifying the course of various kidney diseases. This review summarizes recent insights on the role of autophagy in kidney physiology and diseases alluding to possible novel intervention strategies for treating specific kidney disorders by modifying autophagy.