Ultrastructural visualization of the internalization of low density lipoprotein by human placental cells

Low density lipoproteins (LDL) were conjugated to colloidal gold to visualize the route for internalization of LDL in the cultured cells of human term placenta. Cells were obtained from placental villi (caesarian section) by a standard trypsin-DNase dispersion method followed in some cases by a Percoll gradient centrifugation step. Employing electron microscopy it was observed that after 3 days of culture, cells obtained by trypsin-DNase dispersion were a mixture of macrophages, mononucleated cells and large multinucleated cells. When the cells were incubated for 3 days after the Percoll purification, essentially multinucleated cells identical to the syncytiotrophoblast were present. The number of LDL receptor was increased by preincubation in medium with lipoprotein depleted serum. In binding experiments cells incubated at 4 degrees C for 2 h with medium containing gold LDL conjugates showed gold LDL attached to the plasma membrane without characteristic localization. After incubation with gold LDL at 37 degrees C for various times, the three cellular types showed ligand internalization. Gold LDL endocytosis involved first coated pits but also uncoated plasmalemmal invaginations. Then gold LDL was further observed in coated and non coated vesicles, smooth walled endosomes, multivesicular bodies and tubular vesicles. Lastly free gold particles were observed in lysosome like dense bodies. These results prove the internalization of gold LDL conjugates by human cultured placental cells, particularly by syncytiotrophoblast like multinucleated cells. This accumulation of LDL (the major cholesterol carrying protein in humans) is recognised to be responsible for the exogenous cholesterol supply indispensable to the progesterone biosynthesis and cellular growth of the placenta.     

Prevention of insulin resistance by environmental manipulation as young rats mature

This study was initiated in an attempt to see if the insulin resistance associated with maturation in young rats could be prevented by environmental manipulation. Consequently, seven week-old rats were either housed in standard laboratory cages and fed a calorie-restricted diet or placed individually in exercise wheel cages and allowed to eat chow ad lib. A control group of rats was housed in standard laboratory cages from seven weeks to five months of age, and also allowed to eat chow ad lib. When studied at five months of age, the chow-fed rats weighed more (624 +/- 8 g) than either the calorie restricted (479 +/- 9 g) or exercise trained (485 +/- 13 g) rats. Insulin action was compared in the three groups by assessing the steady-state serum glucose (SSSG) and insulin (SSSI) concentrations achieved during a continuous intravenous infusion of glucose and exogenous insulin. The results of these studies indicated that SSSG concentration was significantly higher (P less than 0.001) in chow-fed rats than in the two experimental groups. Since SSSI concentrations were the same in all three groups, lower SSSG concentrations in calorie-restricted and exercise trained rats indicates that insulin-stimulated glucose uptake was preserved in these two groups as compared to the chow-fed population. In an attempt to understand why exercise training and calorie restriction prevented the development of insulin resistance, muscle glycogen synthase activity and muscle capillary density were compared in the three groups of five month-old rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of adrenalectomy on in vivo glucose metabolism in insulin resistant Zucker obese rats

Lean (Fa/?) and obese (fa/fa) Zucker rats were adrenalectomized (ADX) in order to assess the contribution of adrenal hormones to insulin resistance of the obese Zucker rat. Glucose utilization was measured using an insulin suppression test. Sham-operated obese rats gained almost twice as much weight as sham-operated lean littermates. However, body weight gain of ADX animals was comparable in both genotypes. It was significantly less than that of the respective sham-operated controls. Body weight differences can be accounted for almost entirely by a marked loss of adipose tissue. Although insulin resistance may be attributable to obesity in part, steroid hormones are thought to be directly antagonistic to insulin for glucose metabolism. Adrenalectomy resulted in a decrease in serum glucose concentrations for both lean and obese Zucker rats compared with their respective sham-operated groups. Serum insulin concentration of lean ADX rats was 23% of sham-operated controls; in obese ADX rats, it was 9% of controls. Elevated levels of steady state serum glucose (SSSG) levels in sham-operated obese rats demonstrate a marked resistance to insulin induced glucose uptake compared with sham-operated lean animals. Adrenalectomy caused a marked improvement in insulin sensitivity of obese rats. The hyperglycemic SSSG levels of the obese rats were reduced 2.5 times by ADX. These results indicate that insulin resistance of Zucker obese rats can be ameliorated by ADX, suggesting adrenal hormones contribute to insulin resistance in these animals.     

Method for generation of human hyperdiversified antibody fragment library

The selection of antibody fragments from libraries using in vitro screening technologies has proven to be a very good alternative to the classical hybridoma technology, and has overcome the laborious process of antibody humanization. However, the complexity of the library is critical in the probability of being able to directly isolate a high affinity antibody specific to a target. We report a method to make hyperdiversified antibody fragment libraries, based on human immunoglobulin variable genes mimicking the somatic hypermutation process. This mutagenesis technology, MutaGen, was used for the first time on the entire variable domain (frameworks and CDRs) of large repertoires of human variable antibody domains. Our MutaGen process uses low-fidelity human polymerases, known as mutases, suggested to be involved in the somatic hypermutation process of immunoglobulin genes. Depending on the mutases used, we generated complementary mutation patterns with randomly distributed mutations. The libraries were generated with an average of 1.8 mutations per 100 amino acids. The hyperdiversified antibody fragment libraries constructed with our process should enable the selection of antibody fragments specific to virtually any target.     

Non-random, individual-specific methylation profiles are present at the sixth CTCF binding site in the human H19/IGF2 imprinting control region

Expression of imprinted genes is classically associated with differential methylation of specific CpG-rich DNA regions (DMRs). The H19/IGF2 locus is considered a paradigm for epigenetic regulation. In mice, as in humans, the essential H19 DMR--target of the CTCF insulator--is located between the two genes. Here, we performed a pyrosequencing-based quantitative analysis of its CpG methylation in normal human tissues. The quantitative analysis of the methylation level in the H19 DMR revealed three unexpected discrete, individual-specific methylation states. This epigenetic polymorphism was confined to the sixth CTCF binding site while a unique median-methylated profile was found at the third CTCF binding site as well as in the H19 promoter. Monoallelic expression of H19 and IGF2 was maintained independently of the methylation status at the sixth CTCF binding site and the IGF2 DMR2 displayed a median-methylated profile in all individuals and tissues analyzed. Interestingly, the methylation profile was genetically transmitted. Transgenerational inheritance of the H19 methylation profile was compatible with a simple model involving one gene with three alleles. The existence of three individual-specific epigenotypes in the H19 DMR in a non-pathological situation means it is important to reconsider the diagnostic value and functional importance of the sixth CTCF binding site.     

Towards a stable noeuromycin analog with a D-manno configuration: synthesis and glycosidase inhibition of D-manno-like tri- and tetrahydroxylated azepanes

Noeuromycin is a highly potent albeit unstable glycosidase inhibitor due to its hemiaminal function. While stable D-gluco-like analogs have been reported, no data are available for D-manno-like structures. A series of tri- and tetrahydroxylated seven-membered iminosugars displaying either a D-manno-or a L-gulo-like configuration, were synthesized from methyl α-D-mannopyranoside using a reductive amination-mediated ring expansion as the key step. Screening towards a range of commercial glycosidases demonstrated their potency as competitive glycosidase inhibitors while cellular assay showed selective albeit weak glycoprotein processing mannosidase inactivation.     

Combinatorial engineering to enhance thermostability of amylosucrase

Amylosucrase is a transglucosidase that catalyzes amylose-like polymer synthesis from sucrose substrate. About 60,000 amylosucrase variants from two libraries generated by the MutaGen random mutagenesis method were submitted to an in vivo selection procedure leading to the isolation of more than 7000 active variants. These clones were then screened for increased thermostability using an automated screening process. This experiment yielded three improved variants (two double mutants and one single mutant) showing 3.5- to 10-fold increased half-lives at 50 degrees C compared to the wild-type enzyme. Structural analysis revealed that the main differences between wild-type amylosucrase and the most improved variant (R20C/A451T) might reside in the reorganization of salt bridges involving the surface residue R20 and the introduction of a hydrogen-bonding interaction between T451 of the B' domain and D488 of flexible loop 8. This double mutant is the most thermostable amylosucrase known to date and the only one usable at 50 degrees C. At this temperature, amylose synthesis by this variant using high sucrose concentration (600 mM) led to the production of amylose chains twice as long as those obtained by the wild-type enzyme at 30 degrees C.     

Detection of distinct receptors for native and acetylated low-density lipoproteins in human placental microvilli by ligand-immunoblotting

Ligand-immunoblotting was used to detect distinct receptors for native low-density lipoprotein and for acetylated low-density lipoprotein on microvillous membranes from human term placentas. Antisera directed against native and modified low-density lipoproteins were prepared in rabbits and their specificities were assessed by immunodiffusion and immunoelectrophoresis. The receptor for low-density lipoprotein was detected as a 160 kDa protein and that for acetylated low-density lipoprotein as a 200 kDa protein. These receptors were compared with their counterparts in cultured human skin fibroblasts, bovine adrenal cortex and J774 macrophage-like cells. This is the first investigation that visualizes the presence of receptors for both native and modified low-density lipoproteins in a steroidogenic tissue.     

A cytoplasmic protein-protein interaction detection method based on reporter translation

One approach to drug discovery involves the targeting of abnormal protein-protein interactions that lead to pathology. We present a new technology allowing the detection of such interactions within the cytoplasm in a yeast-based system. The interaction detection is based on the sequestration of a translation termination factor involved in stop codon recognition. This sequestration inhibits the activity of the factor, thereby permitting the translation of a reporter gene harboring a premature stop codon. This novel cytoplasmic protein-protein interaction (CPPI) detection system should prove to be useful in the characterization of proteins as well as in partner identification, interaction mapping, and drug discovery applications.