Identification of a GM1-Binding Protein on the Surface of Murine Neuroblastoma Cells

S20Y murine neuroblastoma cells appear to express a protein component(s) able to adhere specifically to the oligosaccharide portion of GM1 (oligo-GM1). To identify proteins with which the oligo-GM1 becomes closely associated, a radiolabeled (125I), photoactivatable derivative of oligo-GM1 was prepared. This was accomplished by reductive amination of the glucosyl moiety of oligo-GM1 to 1-deoxy-1-aminoglucitol, followed by reaction of the amine with sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD). Crosslinking studies using the photoactivatable probe indicated that it came in close proximity to a protein with an apparent molecular mass of approximately 71 kDa. In competition experiments, as little as a 10-fold molar excess of oligo-GM1 resulted in a selective reduction in labeling of this protein; preincubation with a 200-fold molar excess of siayllactose was necessary to observe the same change in the labeling pattern, lending additional support to the hypothesis that the approximately 71-kDa protein specifically associates with oligo-GM1. Cell surface location of the oligo-GM1 binding protein was confirmed using subcellular fractionation and morphological analyses.     

Structural Insight into Chromatin Recognition by Multiple Domains of the Tumor Suppressor RBBP1

Retinoblastoma-binding protein 1 (RBBP1) is involved in gene regulation, epigenetic regulation, and disease processes. RBBP1 contains five domains with DNA-binding or histone-binding activities, but how RBBP1 specifically recognizes chromatin is still unknown. An AT-rich interaction domain (ARID) in RBBP1 was proposed to be the key region for DNA-binding and gene suppression. Here, we first determined the solution structure of a tandem PWWP-ARID domain mutant of RBBP1 after deletion of a long flexible acidic loop L12 in the ARID domain. NMR titration results indicated that the ARID domain interacts with DNA with no GC- or AT-rich preference. Surprisingly, we found that the loop L12 binds to the DNA-binding region of the ARID domain as a DNA mimic and inhibits DNA binding. The loop L12 can also bind weakly to the Tudor and chromobarrel domains of RBBP1, but binds more strongly to the DNA-binding region of the histone H2A-H2B heterodimer. Furthermore, both the loop L12 and DNA can enhance the binding of the chromobarrel domain to H3K4me3 and H4K20me3. Based on these results, we propose a model of chromatin recognition by RBBP1, which highlights the unexpected multiple key roles of the disordered acidic loop L12 in the specific binding of RBBP1 to chromatin.     

MiR-204 regulates type 1 IP3R to control vascular smooth muscle cell contractility and blood pressure

MiR-204 is expressed in vascular smooth muscle cells (VSMC). However, its role in VSMC contraction is not known. We determined if miR-204 controls VSMC contractility and blood pressure through regulation of sarcoplasmic reticulum (SR) calcium (Ca²⁺) release. Systolic blood pressure (SBP) and vasoreactivity to VSMC contractile agonists (phenylephrine (PE), thromboxane analogue (U46619), endothelin-1 (ET-1), angiotensin-II (Ang II) and norepinephrine (NE) were compared in aortas and mesenteric resistance arteries (MRA) from miR-204^−/− mice and wildtype mice (WT). There was no difference in basal systolic blood pressure (SBP) between the two genotypes; however, hypertensive response to Ang II was significantly greater in miR-204^−/− mice compared to WT mice. Aortas and MRA of miR-204^−/− mice had heightened contractility to all VSMC agonists. In silico algorithms predicted the type 1 Inositol 1, 4, 5-trisphosphate receptor (IP₃R1) as a target of miR-204. Aortas and MRA of miR-204^−/− mice had higher expression of IP₃R1 compared to WT mice. Difference in agonist-induced vasoconstriction between miR-204^−/− and WT mice was abolished with pharmacologic inhibition of IP₃R1. Furthermore, Ang II-induced aortic IP₃R1 was greater in miR-204^−/− mice compared to WT mice. In addition, difference in aortic vasoconstriction to VSMC agonists between miR-204^−/− and WT mice persisted after Ang II infusion. Inhibition of miR-204 in VSMC in vitro increased IP₃R1, and boosted SR Ca²⁺ release in response to PE, while overexpression of miR-204 downregulated IP₃R1. Finally, a sequence-specific nucleotide blocker that targets the miR-204-IP₃R1 interaction rescued miR-204-induced downregulation of IP₃R1. We conclude that miR-204 controls VSMC contractility and blood pressure through IP₃R1-dependent regulation of SR calcium release.     

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.     

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.     

Pharmacological and Physicochemical Properties of Pre-Versus Postsynaptic 5-Hydroxytryptamine1A Receptor Binding Sites in the Rat Brain: A Quantitative Autoradiographic Study

Numerous data suggested that the pharmacological and biochemical properties of 5-hydroxytryptamine1A (5-HT1A) receptors exhibit some regional differences in the CNS, notably within the raphe nuclei compared with various forebrain areas (such as the hippocampus). This possibility has been further investigated in the dorsal raphe nucleus and two areas within the hippocampus, the dentate gyrus and the CA1 area, using the quantitative autoradiographic technique. The potencies of 5'-guanylylimidodiphosphate to inhibit the specific binding of 125I-Bolton-Hunter-8-methoxy-2-(N-propyl-N-propylamino)tetralin (125I-BH-8-MeO-N-PAT) to 5-HT1A sites and of N-ethylmaleimide to block these sites irreversibly were identical in the dorsal raphe nucleus and the hippocampal areas in rat brain sections. In contrast, slight but significant differences were noted in the pH dependence and pharmacological properties of 5-HT1A sites labeled by 125I-BH-8-MeO-N-PAT in these three regions. Similarly, heat denaturation experiments and tissue exposure to either phospholipase A2 or the alkylating agent 8-methoxy-2-(N-2'-chloropropyl,N-propyl)aminotetraline revealed regional differences in the properties of 5-HT1A sites. However, in most cases, the observed variations were of greater amplitude between the CA1 area and the dentate gyrus, where 5-HT1A sites are located postsynaptically, than between any one of these areas and the dorsal raphe nucleus where they act as (presynaptic) somatodendritic autoreceptors. These data further support that subtypes of 5-HT1A receptors probably exist in the rat brain, but this heterogeneity seems unrelated to the pre- or post-synaptic location of these receptors.     

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.     

TRAIL Receptors Serve as Stress-Associated Molecular Patterns to Promote ER-Stress-Induced Inflammation

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