Cation-distribution in developing follicles of the fruit fly Drosophila melanogaster

Abstract. Cations were precipitated with potassium antimonate in ovarian follicles of Drosophila and the distribution of the formed precipitates was studied. The precipitates were analyzed with a laser microprobe mass analyzer (LAMMA) and found to contain a high concentration of calcium; potassium and sodium were also detected. On counting the antimon precipitates in stage 10B follicles with the electron microscope, few precipitates per unit area were found in anterior nurse cells, but more in posterior nurse cells; the highest precipitate density occurred consistently in the oocyte. When follicles of different stages were compared, the precipitate density was found to increase in the ooplasm and in the posterior nurse cells during vitellogenesis, whereas it remained nearly constant in the anterior nurse cells. Thus, the ratio of precipitates between the posterior and anterior end of the follicle increases during vitellogenesis. It begins to decrease at the time when the nurse cells collapse. These results suggest that the electrical polarity observed in polytrophic ovarioles may be based on differences in the cation distribution along the antero-posterior axis of the follicle.     

Genetic studies of human apolipoproteins. V. A novel rapid procedure to screen apolipoprotein E polymorphism

A simple and new method has been developed to detect apolipoprotein E polymorphism directly from plasma or serum without prior ultracentrifugation and delipidation. The method combines the use of dialyzed plasma or serum samples with or without neuraminidase treatment followed by monodimensional isoelectric focusing in simple or 3 M urea gels at a constant low power and progressively increasing voltage over a 3-hr period, and finally protein blotting to a nitrocellulose membrane. Apolipoprotein E phenotypes are identified immunologically using a double antibody reaction, the primary antibody being a monospecific, polyclonal goat anti-apolipoprotein E, and the secondary antibody being a rabbit anti-goat IgG conjugated with alkaline phosphatase. The method was employed to screen apolipoprotein E polymorphism in two white populations in the United States. The frequency values are comparable to those reported previously by other investigators using conventional detection methods. The procedure is simple, accurate, suitable for large scale epidemiologic, clinical, and genetic studies.     

A novel in vitro bovine cartilage punch model for assessing the regeneration of focal cartilage defects with biocompatible bacterial nanocellulose

Introduction

Current therapies for articular cartilage defects fail to achieve qualitatively sufficient tissue regeneration, possibly because of a mismatch between the speed of cartilage rebuilding and the resorption of degradable implant polymers. The present study focused on the self-healing capacity of resident cartilage cells in conjunction with cell-free and biocompatible (but non-resorbable) bacterial nanocellulose (BNC). This was tested in a novel in vitro bovine cartilage punch model.

Methods

Standardized bovine cartilage discs with a central defect filled with BNC were cultured for up to eight weeks with/without stimulation with transforming growth factor-β1 (TGF-β1. Cartilage formation and integrity were analyzed by histology, immunohistochemistry and electron microscopy. Content, release and neosynthesis of the matrix molecules proteoglycan/aggrecan, collagen II and collagen I were also quantified. Finally, gene expression of these molecules was profiled in resident chondrocytes and chondrocytes migrated onto the cartilage surface or the implant material.

Results

Non-stimulated and especially TGF-β1-stimulated cartilage discs displayed a preserved structural and functional integrity of the chondrocytes and surrounding matrix, remained vital in long-term culture (eight weeks) without signs of degeneration and showed substantial synthesis of cartilage-specific molecules at the protein and mRNA level. Whereas mobilization of chondrocytes from the matrix onto the surface of cartilage and implant was pivotal for successful seeding of cell-free BNC, chondrocytes did not immigrate into the central BNC area, possibly due to the relatively small diameter of its pores (2 to 5 μm). Chondrocytes on the BNC surface showed signs of successful redifferentiation over time, including increase of aggrecan/collagen type II mRNA, decrease of collagen type I mRNA and initial deposition of proteoglycan and collagen type II in long-term high-density pellet cultures. Although TGF-β1 stimulation showed protective effects on matrix integrity, effects on other parameters were limited.

Conclusions

The present bovine cartilage punch model represents a robust, reproducible and highly suitable tool for the long-term culture of cartilage, maintaining matrix integrity and homoeostasis. As an alternative to animal studies, this model may closely reflect early stages of cartilage regeneration, allowing the evaluation of promising biomaterials with/without chondrogenic factors.     

The matrilins--adaptor proteins in the extracellular matrix

The matrilins form a four-member family of modular, multisubunit matrix proteins, which are expressed in cartilage but also in many other forms of extracellular matrix. They participate in the formation of fibrillar or filamentous structures and are often associated with collagens. It appears that they mediate interactions between collagen-containing fibrils and other matrix constituents, such as aggrecan. This adaptor function may be modulated by physiological proteolysis that causes the loss of single subunits and thereby a decrease in binding avidity. Attempts to study matrilin function by gene inactivation in mouse have been frustrating and so far not yielded pronounced phenotypes, presumably because of the extensive redundancy within the family allowing compensation by one family member for another. However, mutations in matrilin-3 in humans cause different forms of chondrodysplasias and perhaps also hand osteoarthritis. As loss of matrilin-3 is not critical in mouse, these phenotypes are likely to be caused by dominant negative effects.     

The cellular structure of the Hartig net: coenocytic and transfer cell-like organization

The organization of the Hartig net in the mature state was studied in mycorrhizas of Amanita muscaria (Pers. ex. Fries) Hook. Picea abies (L.) Karst. grown in vitro. The tips of the fan-like branched hyphae contain dense cytoplasm with a large number of mitochondria and rER frequently stretched in the direction of the hyphal growth, indicating that active transfer of solutes between host and fungus is localized here. Lack of septation of the hyphae and their intimate juxtaposition, leaving no space in between, result in a coenocytic, transfer cell-like structure of the Hartig net. The multinucleate status of the cells is shown. The advantage of this organization for nutrient exchange between host and fungus and for nutrient transport within the hyphal system is discussed.     

Metabolism of apolipoprotein E-containing human plasma lipoproteins by rat and human cells in culture.

Cultured preadipocytes from rat epididymal fat pads were able to bind, internalize, and degrade human plasma very-low-density lipoproteins (VLDL) more efficiently than low-density lipoproteins (LDL). VLDL, but not LDL, activated acyl-CoA: cholesterol acyltransferase (ACAT) and increased cholesterol accumulation in these cells. However, trypsin-treated VLDL (T-VLDL) lost the capacity to bind, activate ACAT, and increase cholesterol accumulation. After the treatment of VLDL with trypsin, SDS/polyacrylamide-gel electrophoresis and immunoblotting showed that apolipoprotein E (apo E) was completely degraded, whereas apolipoprotein CII (apo C-II) was preserved. ApoE complexed with dimyristoyl phosphatidylcholine (DMPC) was able to complete with VLDL for binding to the cells. Although T-VLDL did not bind to the preadipocytes, these cells accumulate triacylglycerols from T-VLDL, presumably after lipolysis, as efficiently as from native VLDL. Rat smooth muscle cells and skin fibroblasts also bind and metabolize human VLDL better than LDL. However, human skin fibroblasts and omental preadipocytes metabolized LDL better than VLDL. These studies indicate that rat tissues can recognize and metabolize apoE-containing human plasma VLDL although they cannot recognize human LDL.     

Bipolar anastomosis technique with removable instruments: an easy, fast, and reliable technique for vascular anastomosis

The interrupted suture technique is most commonly used for microsurgical vascular anastomosis. For several reasons (e.g., exposure of suture material to blood, time needed), many attempts have been made to find other solutions. This article describes a new means of performing a microsurgical vascular anastomosis. The aim of this study was to show the feasibility and possible advantages of this new technique. The basic components at work here are a modified cuff and electrically generated heat used to unite the vessel walls. In this way, both endothelial layers are adapted without manipulating the inside of the vessel or leaving behind foreign matter. Various energy/coagulation time settings were used to perform arterial anastomoses (n = 42) in an isogeneic abdominal aorta interposition model in the rat. The quality of anastomosis was evaluated at days 1, 10, 21, and 120. Immediately after the welding process all anastomoses (n = 42) were patent. No stenosis was found at any observation time. Anastomosis time ranged from 3 to 18 minutes (average, 11 minutes). This new technique permits a vascular anastomosis to be performed easily and reliably with a high patency rate. With this technique, the authors are convinced that a skilled surgeon can create a high-quality anastomosis in a fraction of the time needed to sew an anastomosis.     

Conformational gating of dimannose binding to the antiviral protein cyanovirin revealed from the crystal structure at 1.35 A resolution

Cyanovirin (CV-N) is a small lectin with potent HIV neutralization activity, which could be exploited for a mucosal defense against HIV infection. The wild-type (wt) protein binds with high affinity to mannose-rich oligosaccharides on the surface of gp120 through two quasi-symmetric sites, located in domains A and B. We recently reported on a mutant of CV-N that contained a single functional mannose-binding site, domain B, showing that multivalent binding to oligomannosides is necessary for antiviral activity. The structure of the complex with dimannose determined at 1.8 A resolution revealed a different conformation of the binding site than previously observed in the NMR structure of wt CV-N. Here, we present the 1.35 A resolution structure of the complex, which traps three different binding conformations of the site and provides experimental support for a locking and gating mechanism in the nanoscale time regime observed by molecular dynamics simulations.     

Altered integration of matrilin-3 into cartilage extracellular matrix in the absence of collagen IX

The matrilins are a family of four noncollagenous oligomeric extracellular matrix proteins with a modular structure. Matrilins can act as adapters which bridge different macromolecular networks. We therefore investigated the effect of collagen IX deficiency on matrilin-3 integration into cartilage tissues. Mice harboring a deleted Col9a1 gene lack synthesis of a functional protein and produce cartilage fibrils completely devoid of collagen IX. Newborn collagen IX knockout mice exhibited significantly decreased matrilin-3 and cartilage oligomeric matrix protein (COMP) signals, particularly in the cartilage primordium of vertebral bodies and ribs. In the absence of collagen IX, a substantial amount of matrilin-3 is released into the medium of cultured chondrocytes instead of being integrated into the cell layer as in wild-type and COMP-deficient cells. Gene expression of matrilin-3 is not affected in the absence of collagen IX, but protein extraction from cartilage is greatly facilitated. Matrilin-3 interacts with collagen IX-containing cartilage fibrils, while fibrils from collagen IX knockout mice lack matrilin-3, and COMP-deficient fibrils exhibit an intermediate integration. In summary, the integration of matrilin-3 into cartilage fibrils occurs both by a direct interaction with collagen IX and indirectly with COMP serving as an adapter. Matrilin-3 can be considered as an interface component, capable of interconnecting macromolecular networks and mediating interactions between cartilage fibrils and the extrafibrillar matrix.