Stuttgart, Univ., Diss., 2002.

In the case of sudden death or injury stallion reproductive system or necessary castration preventing the collection of semen can to preserve the genetic potential of exceptional individuals take advantage of epididymal sperm. Virtually the only way, how to preserve fertilizing sperm POTENTIAL for long time IS cryopreservation. However, this process induces irreversible changes that are the cause of the reduced number of sperm capable of fertilization after thawing. Epididymal sperm cells have properties different from the ejaculated, harder to keep. Therefore, they are constantly looking for ways to streamline their cryopreservation. GENERAL most commonly used cryoprotectants include glycerol and Egg yolk. Despite the positive effects is glycerol toxic to cells and Egg yolk also contains ingredients that act on sperm metabolism negatively. Increasing the efficiency of cryopreservation protocols themselves likely to be achieved by replacing toxic components while leveraging industry-produced active yolk fractions. Submitted dissertation topic itself will deal with the use of cholesterol-loaded cyclodextrins Along with low density lipoproteins, the cryopreservation of epididymal sperm. Main method: HPLC, Western blot, electrophoresis, fluorescent labels, computer-assisted sperm analysis (CASA).

Plasma triacylglycerols (TG) are elevated in diabetes, metabolic syndrome, obesity, and dyslipidemia. Very-low density lipoprotein (VLDL) is the main plasma carrier of TG and the direct metabolic precursor of low-density lipoprotein (LDL), the main carrier of plasma cholesterol and the major causative risk factor for atherosclerosis. Binding of LDL to heparan sulfate on the arterial wall initiates retention and modifications of LDL in the arterial intima, triggering atherosclerosis. Studies presented in this dissertation show that variations in TG levels and lipoprotein binding to heparin, a model for heparan sulfate, alter the structural and biochemical stability of VLDL and LDL, and increase their atherogenic potential. The molecular consequences of variations in the lipoprotein TG content and LDL-heparin binding were determined by combining heparin affinity chromatography with biochemical, spectroscopic and electron microscopic techniques. Remodeling of human VLDL and LDL by thermal denaturation was used to mimic key aspects of lipoprotein remodeling in vivo. Our studies revealed that increasing the TG content in VLDL promotes changes in the lipoprotein size and release of the exchangeable apolipoproteins. Similarly, increased TG content in LDL promotes lipoprotein remodeling and fusion. Additionally, an increase in TG content increases lipoprotein susceptibility to oxidation and lipolysis, thereby promoting the generation of free fatty acids that augment fusion. Consequently, TG-induced destabilization may be a general property of plasma lipoproteins. Our studies showed that binding to heparin initiates irreversible pro-atherogenic remodeling of human LDL. As a result of heparin binding, LDL showed decreased structural stability and increased susceptibility to hydrolysis and fusion. Further, phospholipid hydrolysis and/or glycation of LDL (as occurs in diabetes) increased the proteolytic susceptibility of apolipoprotein (apo)B (the major apolipoprotein of VLDL and LDL) and its heparin binding affinity. LDL derived from hyperglycemic patients with type-2 diabetes, became particularly destabilized following heparin binding causing apoB fragmentation and LDL fusion. In summary, binding to heparin alters apoB conformation and triggers pro-atherogenic LDL modifications including proteolysis, lipolysis and structural destabilization. Furthermore, phospholipid lipolysis and glycation of LDL in vitro strengthen its binding to heparin. Together, these findings help establish a mechanistic link between diabetes and atherosclerosis.

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No / Acid phosphatases hydrolyse phosphomonoesters at acidic pH in a variety of physiological contexts. The recently defined class C family of acid phosphatases includes the 32 kDa LppC lipoprotein of Streptococcus equisimilis. To define further the distribution of acid phosphatases in the genus Streptococcus we have examined the equine pathogens Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus. Whole cell assays indicated that these organisms possess two acid phosphatases with activity optima at pH 5.0 and pH 6.0-6.5 and that only the former of these was, like LppC, resistant to EDTA. Western blotting with a polyclonal anti-LppC antiserum revealed the presence of a cross-reactive 32 kDa protein in both organisms. The cross-reactive protein in S. equi was shown to be a surface accessible lipoprotein as its processing was inhibited by the antibiotic globomycin and it was released from whole cells by treatment with trypsin. The presence of DNA sequences homologous to the S. equisimilis lppC gene were confirmed by PCR. These data strongly suggest that Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus produce a lipoprotein acid phosphatase homologous to LppC of S. equisimilis.

Elevated plasma concentrations of lipoprotein(a) [Lp(a)] have been identified as an independent risk factor for vascular diseases including coronary heart disease and stroke. In the current study, we have examined the binding and degradation of recombinant forms of apolipoprotein(a) [r-apo(a)], the unique kringle-containing moiety of Lp(a), using a cultured cell model. We found that the incubation of human hepatoma (HepG2) cells with an iodinated 17 kringle-containing (17K) recombinant form of apo(a) resulted in a two-component binding system characterized by a high affinity (Kd = 12 nM), low capacity binding site, and a low affinity (Kd = 249 nM), high capacity binding site. We subsequently determined that the high affinity binding site on HepG2 cells corresponds to the LDL receptor. In the HepG2 cell model, association of apo(a) with the LDL receptor was shown to be dependent on the formation of Lp(a) particles from endogenous LDL. Using an apo(a) mutant incapable of binding to the high affinity site through its inability to form Lp(a) particles (17KΔLBS7,8), we further demonstrated that the LDL receptor does not participate in Lp(a) catabolism. The low affinity binding component observed on HepG2 cells, familial hypercholesterolemia (FH) fibroblasts and human embryonic kidney (HEK) 293 cells may correspond to a member(s) of the plasminogen receptor family, as binding to this site(s) was decreased by the addition of the lysine analogue epsilon-aminocaproic acid. The lysine-dependent nature of the low affinity binding site was further confirmed in HepG2 binding studies utilizing r-apo(a) species with impaired lysine binding ability. We observed a reduction maximum binding capacity for 17K r-apo(a) variants lacking the strong lysine binding site (LBS) in kringle IV type 10 (17KΔAsp) and the very weak LBS in kringle V (17KΔV). Degradation of Lp(a)/apo(a) was found to be mediated exclusively by the low affinity component on both HepG2 cells and FH fibroblasts. Fluorescence confocal microscopy, using the 17K r-apo(a) variant fused to green fluorescent protein, further confirmed that degradation by the low affinity component on HepG2 cells does not proceed by the activity of cellular lysosomes. Taken together, these data suggest a potentially significant route for Lp(a)/apo(a) clearance in vivo. / Thesis (Master, Biochemistry) -- Queen's University, 2009-09-26 02:15:50.754

The Drosophila Lipoprotein particles bear lipid-linked morphogens on their surface and are required for long-range signaling activity of Wingless and Hedgehog. They also bind a wide variety of gpi-linked proteins. Whether any of these proteins affect morphogen signaling is unknown. Here, I show that the gpi-linked heparan sulfate proteoglycan Dally is released from cell membranes and binds to lipoprotein particles both with and without its lipid anchor. Hedgehog signaling efficiency is reduced in Dally mutant discs, but can be rescued non-autonomously by expression of non-gpi-modified Dally. This Dally isoform colocalizes with Hedgehog, Patched and Lipophorin in endosomes and increases Hedgehog signaling efficiency without affecting Hedgehog distribution. These data show that Hedgehog signaling activity can be influenced by other Lipophorin-associated proteins, and suggest Lipoproteins provide a platform for regulation of morphogen signaling.

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No description available.

[Truncated abstract] Dyslipidaemia in obesity and the metabolic syndrome is typically characterized by elevated plasma concentrations of apolipoprotein (apo) B and chylomicron remnants, and low apoA-I levels. This may account for the increased risk of cardiovascularrelated diseases. Although the precise mechanisms whereby visceral obesity confers the onset of dyslipidaemia have not been fully established, it may relate chiefly to insulin resistance. Insulin resistance leads to increased hepatic secretion of very low density lipoprotein (VLDL) apoB, as well as impaired catabolism of VLDL, intermediate density lipoprotein (IDL), low-density lipoprotein (LDL) and chylomicron remnants, and high density lipoprotein (HDL) apoA-I. This thesis tests the unifying hypothesis that lipoprotein metabolism, in particular apoB, chylomicron remnants and apoA-I, is abnormal in the metabolic syndrome, and that medical intervention can correct for these abnormalities. The primary objectives were to examine firstly, the kinetics of apoB and apoA-I by stable isotope technology and secondly, chylomicron remnant kinetics by using an indirect assessment of a new breath test. Six observational statements and related hypotheses were constructed and derived from the unifying hypothesis that examine the kinetics of lipoprotein metabolism, adipose tissue mass compartments and liver fat accumulation, as well as the impact of plasma adipocytokines in subjects with visceral adiposity and features of the metabolic syndrome. The first four observational statements related to cross-sectional studies of lipoprotein kinetics, adipose tissue mass distribution and liver fat accumulation as well as plasma adipocytokines in both obese and non-obese men. The latter two observational statements related to the effect of statin therapy and dietary weight loss on the improvement of lipoprotein kinetics in obesity. The findings from these studies collectively support the unifying hypothesis. The kinetics of apoB in VLDL, IDL and LDL, and apoA-I in HDL were assessed by gas-chromatography mass spectrometry following either a primed-constant infusion of 13C-leucine or an intravenous bolus injection of d3-leucine. ... This is the first study to examine the effects of dietary weight loss on LDL and HDL metabolism and the relationships with adipocytokines in men with the metabolic syndrome. The data support the unifying hypothesis that medical intervention with dietary weight loss could correct the kinetic abnormalities in VLDL, LDL and HDL. The aforementioned studies showed that plasma lipid and lipoprotein abnormalities in visceral obesity are chiefly regulated by the combination of hepatic over-secretion of VLDL particles, and catabolic defects in apoB and chylomicron remnants as well as apoA-I-containing lipoprotein particles. These kinetic defects may also relate to low and high plasma adiponectin and RBP-4 levels, respectively. The data arising from the thesis are consistent with the unifying hypothesis and support the role of dietary intervention and pharmacotherapy as a recommended treatment in correcting the abnormalities in lipoprotein metabolism within the metabolic syndrome.