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.     

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.     

Plant phosphoglucose isomerase genes lack introns and are expressed in Escherichia coli

Nuclear genes that appear to encode both cytosolic and plastid isozymes of phosphoglucose isomerase (PGI), an essential glycolytic enzyme, have been isolated from three diploid species of the annual wild flower genus Clarkia (Onagraceae). The genes do not contain introns and are expressed to varying degrees in Escherichia coli when cloned in either Charon 35 phage or pUC plasmid vectors. The PGI proteins synthesized in E. coli form dimers, are catalytically active, and their electrophoretic mobilities are similar to those of appropriate Clarkia PGIs. The nucleotide sequence of a gene encoding a plastid isozyme of C. unguiculata is described.     

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.     

Parameters that specify the timing of cytokinesis.

One model for the timing of cytokinesis is based on findings that p34(cdc2) can phosphorylate myosin regulatory light chain (LC20) on inhibitory sites (serines 1 and 2) in vitro (Satterwhite, L.L., M.H. Lohka, K.L. Wilson, T.Y. Scherson, L.J. Cisek, J.L. Corden, and T.D. Pollard. 1992. J. Cell Biol. 118:595-605), and this inhibition is proposed to delay cytokinesis until p34(cdc2) activity falls at anaphase. We have characterized previously several kinase activities associated with the isolated cortical cytoskeleton of dividing sea urchin embryos (Walker, G.R., C.B. Shuster, and D.R. Burgess. 1997. J. Cell Sci. 110:1373-1386). Among these kinases and substrates is p34(cdc2) and LC20. In comparison with whole cell activity, cortical H1 kinase activity is delayed, with maximum levels in cortices prepared from late anaphase/telophase embryos. To determine whether cortical-associated p34(cdc2) influences cortical myosin II activity during cytokinesis, we labeled eggs in vivo with [(32)P]orthophosphate, prepared cortices, and mapped LC20 phosphorylation through the first cell division. We found no evidence of serine 1,2 phosphorylation at any time during mitosis on LC20 from cortically associated myosin. Instead, we observed a sharp rise in serine 19 phosphorylation during anaphase and telophase, consistent with an activating phosphorylation by myosin light chain kinase. However, serine 1,2 phosphorylation was detected on light chains from detergent-soluble myosin II. Furthermore, cells arrested in mitosis by microinjection of nondegradable cyclin B could be induced to form cleavage furrows if the spindle poles were physically placed in close proximity to the cortex. These results suggest that factors independent of myosin II inactivation, such as the delivery of the cleavage stimulus to the cortex, determine the timing of cytokinesis.     

Factors determining the reconstructive denaturation of proteins in sodium dodecyl sulfate solutions. Further circular dichroism studies on structural reorganization of seven proteins

The factors determining the onset and extent of reconstructive denaturation of proteins were considered by comparing circular dichroism (CD) data of seven proteins and previously published findings. The effects of sodium dodecyl sulfate (SDS) on the conformation of the following proteins were tested: lysozyme, the mitogens fromPhytolacca americana (fractions Pa2 and Pa4), lectin fromWistaria floribunda, ovine lutropin, a Bence Jones protein, and histone H2B. While the helix content of lysozyme was raised by SDS slightly, in the Bence Jones protein andW. floribunda lectin it increased from near zero to about 25–30%. In histone H2B the helix content was raised by SDS even to about 48%. However, no clear indication of helix formation could be observed in the mitogens and lutropin, even at low pH or 2.0–2.5. The tertiary structure of the proteins was perturbed by SDS. It was concluded that the reorganization of secondary structure of the proteins was favored by the following factors: (1) presence of helicogenic amino acid sequences in the protein, (2) availability of positively charged sites of the basic amino acids for interactions with the dodecyl ion, (3) absence of a large surplus of negatively charged sites on the surface of protein, and (4) absence of extensive disulfide cross-linking within the macromolecule. Both hydrophobic and electrostatic interactions occur in reconstructive denaturation, and the newly formed helices are stabilized by hydrophobic shielding by the alkyl chains of the alkyl sulfate.