Purification of galactose-1-phosphate uridylyltransferase from human placenta

Galactose-1-phosphate uridylyltransferase (uridine diphosphoglucose: α-d-galactose-1-phosphate uridylyltransferase, EC 2.7.7.12) has been purified 4000-fold from human placenta in four chromatographic steps using DEAE-cellulose, hydrocylapatite, ethyliminohexylagarose, and Sephacryl S-200. The specific activity of the homogeneous enzyme was 56 units/mg protein. The placental enzyme consists of two similar subunits, each of molecular weight about 48,000. The placental enzyme was similar to published results for the red cell enzyme (V. P. Williams, Arch. Biochem. Biophys., 1978, 191, 182–191) with respect to subunit molecular weight, electrophoretic migration, and immunological properties. The more purified fractions of the placental enzyme invariably contained a glycoprotein which was removed in the gel filtration step. After this glycoprotein was removed, the enzyme was very labile and only about 20% of the catalytic activity was recovered.     

BRE over-expression promotes growth of hepatocellular carcinoma

BRE, also known as TNFRSF1A modulator and BRCC45, is an evolutionarily highly conserved protein. It is a death receptor-associated protein in cytoplasm and a component of BRCA1/2-containing DNA repair complex in nucleus. BRE was found to have anti-apoptotic activity. Over-expression of BRE by transfection promoted survival of cell lines against apoptotic induction; whereas depletion of the protein by siRNA resulted in the opposite. In vivo anti-apoptotic activity of BRE was demonstrated by significant attenuation of Fas-induced acute fulminant hepatitis in transgenic mice expressing the human protein specifically in the liver. BRE was also implicated in tumor promotion by the accelerated tumor growth of Lewis Lung carcinoma transfected with human BRE; and by high expression of BRE specifically in the tumoral regions of human hepatocellular carcinoma (HCC). The present study was to test directly if transgenic expression of BRE in livers could promote HCC development in neonatal diethylnitrosamine model. By 8 months after tumor induction, the maximal sizes of tumor nodules of transgenic mice were significantly larger than those of the non-transgenic controls, although the numbers of tumor nodules between the two groups did not significantly differ. Importantly, as in human HCC, the mouse endogenous BRE level was up-regulated in mouse HCC nodules. These results show that BRE over-expression can indeed promote growth, though not initiation, of liver tumors. Furthermore, the common occurrence of BRE over-expression in human and mouse HCC suggests that up-regulation of BRE is functionally important in liver tumor development.     

CaV1.2 I-II linker structure and Timothy syndrome

Ca_V channels are multi-subunit protein complexes that enable inward cellular Ca²⁺ currents in response to membrane depolarization. We recently described structure-function studies of the intracellular α1 subunit domain I-II linker, directly downstream of domain IS6. The results show the extent of the linker’s helical structure to be subfamily dependent, as dictated by highly conserved primary sequence differences. Moreover, the difference in structure confers different biophysical properties, particularly the extent and kinetics of voltage and calcium-dependent inactivation. Timothy syndrome is a human genetic disorder due to mutations in the Ca_V1.2 gene. Here, we explored whether perturbation of the I-II linker helical structure might provide a mechanistic explanation for a Timothy syndrome mutant’s (human Ca_V1.2 G406R equivalent) biophysical effects on inactivation and activation. The results are equivocal, suggesting that a full mechanistic explanation for this Timothy syndrome mutation requires further investigation.     

Survival Expectations of the Obese: Is Excess Mortality Reflected in Perceptions?

Objective: This study compared self‐reported subjective life expectancy (i.e., probability of living to age 75) for normal‐weight, overweight, and obese weight groups to examine whether individuals are internalizing information about the health risks due to excessive weight. Research Methods and Procedures: Using data from the Health and Retirement Study, a total of 9035 individuals 51 to 61 years old were analyzed by BMI category. The primary outcome measure was individuals’ reports about their own expectations of survival to age 75. Absolute and relative risks of survival were compared with published estimates of survival to age 75. Results: Consistently, higher levels of BMI were associated with lower self‐estimated survival probabilities. Differences relative to normal weight ranged from 4.9% (p < 0.01) for male nonsmokers to 8.8% (p < 0.001) for female nonsmokers. However, these differences were substantially less than those obtained from published survival curve estimates, suggesting that obese individuals tended to underestimate mortality risks. Discussion: Individuals appeared to underestimate the mortality risks of excessive weight; thus, knowledge campaigns about the risks of obesity should remain a top priority.     

Competitive and facilitative relationships among three shrub species,and the role of browsing intensity and rooting depth in the Succulent Karoo,South Africa

Abstract. The nearest‐neighbour technique is used to infer competition and facilitation between the three most abundant species in a semi‐arid region of western South Africa. Relationships among the shrubs Leipoldtia schultzei and Ruschia robusta, which are leaf‐succulent members of the Mesembryanthemaceae (‘mesembs’) and Hirpicium alienatum a non‐succulent Asteraceae, were compared on two adjacent sites with different histories of browsing intensity. Competition was more prevalent and more important than facilitation. The only evidence for facilitation was found at the heavily‐browsed site where the palatable Hirpicium was larger under the unpalatable Leipoldtia. Generally the prevalence and importance of competition was reduced at the heavily‐browsed site. Strong evidence was obtained for intraspecific competition in each of the three species; also, competition was evident between the two mesembs, where Leipoldtia was competitively dominant over Ruschia, although neither species inhibited Hirpicium. Minimal competition between the mesembs and the asteraceous shrub was interpreted in terms of differentiation in rooting depth, and competition within the mesembs, in terms of overlap in rooting depth. The mesembs had the bulk of their roots in the top 5 cm of soil, while the asteraceous shrub had the bulk of its roots, and all its fine roots, at greater depths. The shallow‐rooted morphology of the mesembs is well adapted to utilize small rainfall events, which occur frequently in the Succulent Karoo, and do not penetrate the soil deeply. Modifications of existing methods are applied for analysing nearest‐neighbour interactions.     

Coupling of polyphosphoinositide breakdown with calcium efflux in formyl-methionyl-leucyl-phenylalanine-stimulated rabbit neutrophils

Exposure of rabbit neutrophils to formyl-methionyl-leucyl-phenylalanine (FMLP) induced the efflux of 45Ca2+ from pre-labeled cells which was almost complete within 30 s. On the other hand, FMLP-induced 45Ca2+ influx did not become apparent until 60 s after stimulation. When [3H]arachidonic acid-labeled neutrophils were stimulated with FMLP, the radioactivities in phosphatidylinositol 4,5-biphosphate (TPI) and phosphatidylinositol 4-phosphate (DPI) significantly decreased in parallel with the induction of 45Ca2+ efflux. In contrast, degradation of polyphosphoinositides in [3H]glycerol-labeled neutrophils was not significant until 60 s. Taken together, these results indicate that the early degradation of polyphosphoinositides, especially of those rich in arachidonic acid is closely associated with the initial efflux of calcium in FMLP-stimulated rabbit neutrophils. The study of resynthesis of polyphosphoinositides by measuring 32Pi incorporation into these lipids is also presented.     

High affinity specific antiestrogen binding sites are concentrated in rough microsomal membranes of rat liver

Saturation and competitive binding analyses demonstrated the presence of a high affinity (K_D = 0.92 nM), specific antiestrogen binding site (AEBS) in rat liver microsomes and at least 75% of total liver AEBS was recovered in this fraction. When microsomes were further separated into smooth and rough fractions, AEBS was concentrated in the latter. Subsequent dissociation of ribosomes from the rough membranes revealed that AEBS was associated with the membrane and not the ribosomal fraction. Antiestrogen binding activity could not be extracted from membranes with 1 M KCl or 0.5 M acetic acid but could be solubilized with sodium cholate. These data indicate that AEBS is an integral membrane component of the rough microsomal fraction of rat liver.