Studies on the enzymatic and physicochemical behaviour of the trichloroacetic acid-treated and untreated bovine pancreatic ribonuclease

Exposure of ribonuclease A to 5% trichloroacetic acid inactivates the enzyme partially. One of the possible reasons for such inactivation might be the exposure of one of the buried tyrosyl groups to the outside surface of the molecule (Sagar and Pandit (1983) Biochim. Biophys. Acta 743, 303-309). The trichloroacetic acid-treated enzyme hydrolysed 2':3'-cCMP with an efficiency of about 60%; while with rRNA as substrate, it is about 45%. Results indicate that apart from the reduction in the activity on trichloroacetic acid treatment, the enzyme possesses a reduced ability to break down the secondary structures of substrates such as rRNA in the first phase of the reaction. Thermal unfolding of ribonuclease A was followed by various physicochemical techniques such as UV absorbance, CD-spectroscopy and differential scanning microcalorimetry. The results indicate that the enzyme, after trichloroacetic acid-treatment, has a less ordered structure when compared to that of untreated enzyme. Thermal unfolding profiles reveal that trichloroacetic acid-treated ribonuclease A, like the untreated enzyme, follows a one-step transition with relatively lower transition temperature (Tm). NMR-spectral data suggests perturbations in the histidyl environment at the active site.     

Incorporation of Host Complement Regulatory Proteins into Newcastle Disease Virus Enhances Complement Evasion

Newcastle disease virus (NDV), an avian paramyxovirus, is inherently tumor selective and is currently being considered as a clinical oncolytic virus and vaccine vector. In this study, we analyzed the effect of complement on the neutralization of NDV purified from embryonated chicken eggs, a common source for virus production. Fresh normal human serum (NHS) neutralized NDV by multiple pathways of complement activation, independent of neutralizing antibodies. Neutralization was associated with C3 deposition and the activation of C2, C3, C4, and C5 components. Interestingly, NDV grown in mammalian cell lines was resistant to complement neutralization by NHS. To confirm whether the incorporation of regulators of complement activity (RCA) into the viral envelope afforded complement resistance, we grew NDV in CHO cells stably transfected with CD46 or HeLa cells, which strongly express CD46 and CD55. NDV grown in RCA-expressing cells was resistant to complement by incorporating CD46 and CD55 on virions. Mammalian CD46 and CD55 molecules on virions exhibited homologous restriction, since chicken sera devoid of neutralizing antibodies to NDV were able to effectively neutralize these virions. The incorporation of chicken RCA into NDV produced in embryonated eggs similarly provided species specificity toward chicken sera.     

Human breast milk stimulates low density lipoprotein (LDL) binding to human skin fibroblasts in culture

Human breast milk incorporated at 1% concentration into the culture medium significantly (p less than 0.05) increased the binding of 125I-LDL to receptors of human skin fibroblasts in culture. Homogenized cows milk and infant formula (Similac) also possessed this stimulating property. The stimulating activity of milk persisted after dialysis and extraction with cold acetone. These preliminary studies suggest that milk might contain potent factor(s) influencing cholesterol metabolic process in early life.     

Incorporation of 14C-mevalonate into biliary cholesterol and bile acids by perfused rat liver: Effect of plasma and individual lipoproteins

Effect of plasma and individual lipoproteins on the incorporation of ¹⁴C-mevalonate into biliary bile acids and cholesterol by perfused rat liver was investigated. Use of plasma free perfysate (instead of whole blood) gave similar rates of incorporation of ¹⁴ C-mevalonate into biliary bile acids, but showed a decrease in percent distribution in cholic acid and a increase in β-muricholic acid. Addition of VLDL (isolated from Zucker rats) into the plasma free perfusate caused a significant increase in the incorporation of the label into bile acids, but LDL and HDL had no effect. HDL caused an increase in biliary excretion of ¹⁴C-cholesterol.     

Metabolic utilization of human osteoblast cell line hFOB 1.19 under normoxic and hypoxic conditions: A phenotypic microarray analysis

Osteoblasts play an important role in bone regeneration and repair. The hypoxia condition in bone occurs when bone undergoes fracture, and this will trigger a series of biochemical and mechanical changes to enable bone repair. Hence, it is interesting to observe the metabolites and metabolism changes when osteoblasts are exposed to hypoxic condition. This study has looked into the response of human osteoblast hFOB 1.19 under normoxic and hypoxic conditions by observing the cell growth and utilization of metabolites via Phenotype MicroArrays™ under these two different oxygen concentrations. The cell growth of hFOB 1.19 under hypoxic condition showed better growth compared to hFOB 1.19 under normal condition. In this study, osteoblast used glycolysis as the main pathway to produce energy as hFOB 1.19 in both hypoxic and normoxic conditions showed cell growth in well containing dextrin, glycogen, maltotriose, D-maltose, D-glucose-6-phospate, D-glucose, D-mannose, D-Turanose, D-fructose-6-phosphate, D-galactose, uridine, adenosine, inosine and α-keto-glutaric acid. In hypoxia, the cells have utilized additional metabolites such as α-D-glucose-1-phosphate and D-fructose, indicating possible activation of glycogen synthesis and glycogenolysis to metabolize α-D-glucose-1-phosphate. Meanwhile, during normoxia, D-L-α-glycerol phosphate was used, and this implies that the osteoblast may use glycerol-3-phosphate shuttle and oxidative phosphorylation to metabolize glycerol-3-phosphate.     

Bile acid and cholesterol excretion in the pregnant guinea pig: Studies on the hypercholesterolemia of pregnancy

The relationship of bile acid and cholesterol excretion to changes in plasma cholesterol during pregnancy were studied in guinea pigs. Plasma cholesterol level increased in the first trimester of pregnancy, reached to a peak during the second trimester and decreased in the third trimester reaching the lowest level at one week prior to parturition. Cholesterol level returned to the control level after parturition. Plasma triglyceride level followed a similar trend attaining peak values at second trimester and gradually returned to the control level at the third trimester of pregnancy. Bile acid and total sterol excretion were significantly higher in guinea pigs during the last phase of pregnancy while they remained unchanged during early stage of pregnancy.