Solution structure of the N‐terminal domain of DC‐UbP/UBTD2 and its interaction with ubiquitin

DC‐UbP/UBTD2 is a ubiquitin (Ub) domain‐containing protein first identified from dendritic cells, and is implicated in ubiquitination pathway. The solution structure and backbone dynamics of the C‐terminal Ub‐like (UbL) domain were elucidated in our previous work. To further understand the biological function of DC‐UbP, we then solved the solution structure of the N‐terminal domain of DC‐UbP (DC‐UbP_N) and studied its Ub binding properties by NMR techniques. The results show that DC‐UbP_N holds a novel structural fold and acts as a Ub‐binding domain (UBD) but with low affinity. This implies that the DC‐UbP protein, composing of a combination of both UbL and UBD domains, might play an important role in regulating protein ubiquitination and delivery of ubiquitinated substrates in eukaryotic cells.     

Involvement of aquaporin in thromboxane A2 receptor-mediated, G12/13/RhoA/NHE-sensitive cell swelling in 1321N1 human astrocytoma cells

The physiological role of the thromboxane A₂ (TXA₂) receptor expressed on glial cells remains unclear. We previously reported that 1321N1 human astrocytoma cells pretreated with dibutyryl cyclic AMP (dbcAMP) became swollen in response to U46619, a TXA₂ analogue. In the present study, we examined the detailed mechanisms of TXA₂ receptor-mediated cell swelling in 1321N1 cells. The cell swelling caused by U46619 was suppressed by expression of p115-RGS, an inhibitory peptide of Gα_12/13 pathway and C3 toxin, an inhibitory protein for RhoA. The swelling was also inhibited by treatment with Y27632, a Rho kinase inhibitor and 5-(ethyl-N-isopropyl)amiloride (EIPA), a Na⁺/H⁺-exchanger inhibitor. Furthermore, cell swelling was suppressed by the pretreatment with aquaporin inhibitors mercury chloride or phloretin in a concentration-dependent manner, suggesting that aquaporins are involved in U46619-induced 1321N1 cell swelling. In fact, U46619 caused [³H]H₂O influx into the cells, which was inhibited by p115-RGS, C3 toxin, EIPA, mercury chloride and phloretin. This is the first report that the TXA₂ receptor mediates water influx through aquaporins in astrocytoma cells via TXA₂ receptor-mediated activation of Gα_12/13, Rho A, Rho kinase and Na⁺/H⁺-exchanger.     

Influence of ionic strength and pH on the interaction between high-affinity heparin and antithrombin

Binding constants for the binding of high-affinity heparin to antithrombin at different ionic strengths were determined by fluorescence titrations and were also estimated from dissociation curves of the heparin-antithrombin complex. These curves were monitored by near-ultraviolet circular dichroism or fluorescence. The dependence of the binding constant on the activity of NaCl suggested that maximally 5–6 charged groups are directly involved in the interaction between the two macromolecules. Major pH-dependent changes of the interaction, as evident by changes of the spectroscopic properties of the complex between the molecules, were found to occur below pH 5.5 and above pH 8.5. The acid change, which was irreversible, was most likely caused by an irreversible conformational change of antithrombin. At alkaline pH, however, the gross conformation of antithrombin was stable up to pH 12, while the affinity of high-affinity heparin for antithrombin began to decrease markedly at pH 8.5. The dissociation curve, which was reversible, had a midpoint around pH 9.5. This is compatible with the loss of affinity being caused by either a local conformational change, by ionization of tyrosine or by titration of one or more amino groups.     

Flux analysis of recombinant Saccharomyces cerevisiae YPB-G utilizing starch for optimal ethanol production

Genetically modified Saccharomyces cerevisiae strain (YPB-G) which secretes a bifunctional fusion protein that contains both Bacillus subtilis -amylase and Aspergillus awamori glucoamylase activities was used for the direct conversion of starch into ethanol. Starch was either supplied initially to different nutrient media or added instantaneously to the reactor at various discrete time instants (pulse feeding). Stoichiometric modeling was used to investigate the effects of initial substrate concentration and growth rate of the recombinant yeast culture on ethanol production. Reaction stoichiometries describing both the anabolism and catabolism of the microorganism were used as an input to flux balance analysis (FBA), the preferred metabolic modeling approach since the constructed stoichiometric network was underdetermined. Experiments for batch and fed-batch systems at different substrate concentrations were analyzed theoretically in terms of flux distributions using ethanol production rate as the maximization criteria. Calculated ethanol rates were in agreement with experimental measurements, suggesting that this recombinant microorganism is sufficiently evolved to optimize its ethanol production. The function of the main pathways of yeast metabolism (PPP, EMP, TCA) are discussed together with the node analyses of glucose-6-P and pyruvate branch points. Theoretical node analysis revealed that if the split ratio in G6P branch point is changed by genetic manipulations, the ethanol yield would be affected considerably.     

Two oligosaccharides from Marsdenia roylei

Phytochemical analysis of dried twigs of Marsdenia roylei (family Asclepiadaceae) has resulted in the isolation of a trisaccharide, maryal, and a diglycoside, rolinose. Their structures were determined as O-beta-D-oleandropyranosyl-(1-->4)-O-beta-D-digitoxopyranosyl++ +-(1-->4)-D- cymaral and ethyl O-beta-D-oleandropyranosyl-(1-->4)-O-3-O-methyl-6-deoxy-beta-D- allopyranoside, respectively, by chemical degradation and spectroscopic methods.     

Isolation of lactoperoxidase, lactoferrin, α-lactalbumin, β-lactoglobulin B and β-lactoglobulin A from bovine rennet whey using ion exchange chromatography

A mild and rapid method is described for isolating various milk proteins from bovine rennet whey. β-Lactoglobulin from bovine rennet whey was easily adsorbed on and desorbed from a weak anion exchanger, diethylaminoethyl-Toyopearl. However, α-lactalbumin could not be adsorbed onto the resin. α-Lactalbumin and β-lactoglobulin from rennet whey could also be adsorbed and separated using a strong anion exchanger, quaternary aminoethyl-Toyopearl. The rennet whey was passed through a strong cation exchanger, sulphopropyl-Toyopearl, to separate lactoperoxidase and lactoferrin. α-Lactalbumin and β-lactoglobulin were adsorbed onto quaternary aminoethyl-Toyopearl. α-Lactalbumin was eluted using a linear (0–0.15 M) concentration gradient of NaCl in 0.05 M Tris–HCl buffer (pH 8.5). Subsequently, β-lactoglobulin B and β-lactoglobulin A were eluted from the column with 0.05 M Tris–HCl (pH 6.8), using a linear (0.1–0.25 M) concentration gradient of NaCl. The yields were 1260 mg α-lactalbumin, 1290 mg β-lactoglobulin B and 2280 mg β-lactoglobulin A from 1 l rennet whey.     

Arginase,an s-phase enzyme in a human cell line

Suspension cultures of ‘Chang liver’ cells were synchronized by preincubation in a glutamine-deficient medium or by thymidine blockade. Specific arginase activity varied in the synchronized cultures, being high when the number of S-phase cells was maximal. A relationship between high arginase activity and a high percentage of (S+G₂) cells was also found when unsynchronized cells were separated by velocity sedimentation. The increase in arginase activity near the G₁/S border was totally inhibited in the presence of cycloheximide. The rate of decrease in activity after addition of the drug indicated that the variations in the rate of synthesis of the enzyme, while the rate of degradation was more or less constant, corresponding to 4–6% per h. The role of arginase in cells lacking a urea cycle and the regulation of arginase activity in ‘Chang liver’ cells is discussed.     

A Comparison of Bone Mineral Density and Its Predictors in HIV-Infected and HIV-Uninfected Older Men

ObjectiveBone health in older individuals with HIV infection has not been well studied. This study aimed to compare bone mineral density (BMD), trabecular bone score (TBS), and bone markers between HIV-infected men and age- and body mass index (BMI)-matched HIV-uninfected men aged ≥60 years. We investigated the associations of risk factors related to fracture with BMD, TBS, and bone markers in HIV-infected men.MethodsThis cross-sectional study included 45 HIV-infected men receiving antiretroviral therapy and 42 HIV-uninfected men. Medical history, BMD and TBS measurements, and laboratory tests related to bone health were assessed in all the participants. HIV-related factors known to be associated with bone loss were assessed in the HIV-infected men.ResultsThe mean BMD, TBS, and osteopenia or osteoporosis prevalence were similar among the cases and controls. The HIV-infected men had significantly higher mean N-terminal propeptide of type 1 procollagen and C-terminal cross-linking telopeptide of type I collagen levels. Stepwise multiple linear regression analysis demonstrated that low BMI (lumbar spine, P = .015; femoral neck, P = .018; and total hip, P = .005), high C-terminal cross-linking telopeptide of type I collagen concentration (total hip, P = .042; and TBS, P = .010), and low vitamin D supplementation (TBS, P = .035) were independently associated with low BMD and TBS.ConclusionIn older HIV-infected men with a low fracture risk, the mean BMD and TBS were similar to those of the age- and BMI-matched controls. The mean bone marker levels were higher in the HIV group. Traditional risk factors for fracture, including low BMI, high C-terminal cross-linking telopeptide of type I collagen level, and low vitamin D supplementation, were significant predictors of low BMD and TBS.     

Mechano- and pH-sensing convergence on Ca2+-mobilising proteins – A recipe for cancer?

Alterations in the (bio)chemical and physical microenvironment of cells accompany and often promote disease formation and progression. This is particularly well established for solid cancers, which are typically stiffer than the healthy tissue in which they arise, and often display profound acidification of their interstitial fluid. Cell surface receptors can sense changes in the mechanical and (bio)chemical properties of the surrounding extracellular matrix and fluid, and signalling through these receptors is thought to play a key role in disease development and advancement. This review will look at ion channels and G protein coupled receptors that are activated by mechanical cues and extracellular acidosis, and stimulation of which results in increases in intracellular Ca²⁺ concentrations. Cellular Ca²⁺ levels are dysregulated in cancer as well as cancer-associated cells, and mechano- and proton-sensing proteins likely contribute to these aberrant intracellular Ca²⁺ signals, making them attractive targets for therapeutic intervention.