Structural analysis of the alpha N-terminal region of erythroid and nonerythroid spectrins by small-angle X-ray scattering

We used SpalphaI-1-156 peptide, a well-characterized model peptide of the alphaN-terminal region of erythrocyte spectrin, and SpalphaII-1-149, an alphaII brain spectrin model peptide similar in sequence to SpalphaI-1-156, to study their association affinities with a betaI-spectrin peptide, SpbetaI-1898-2083, by isothermal titration calorimetry. We also determined their conformational flexibilities in solution by small-angle X-ray scattering (SAXS) methods. These two peptides exhibit sequence homology and could be expected to exhibit similar association affinities with beta-spectrin. However, our studies show that the affinity of SpalphaII-1-149 with SpbetaI-1898-2083 is much higher than that of SpalphaI-1-156. Our SAXS findings also indicate a significantly more extended conformation for SpalphaII-1-149 than for SpalphaI-1-156. The radius of gyration values obtained by two different analyses of SAXS data and by molecular modeling all show a value of about 25 A for SpalphaI-1-156 and of about 30 A for SpalphaII-1-149, despite the fact that SpalphaI-1-156 has seven amino acid residues more than SpalphaII-1-149. For SpalphaI-1-156, the SAXS results are consistent with a flexible junction between helix C' and the triple helical bundle that allows multiple orientations between these two structural elements, in good agreement with our published NMR analysis. The SAXS findings for SpalphaII-1-149 support the hypothesis that this junction region is rigid (and probably helical) for alphaII brain spectrin. The nature of the junction region, from one extreme as a random coil (conformationally mobile) segment in alphaI to another extreme as a rigid segment in alphaII, determines the orientation of helix C' relative to the first structural domain. We suggest that this particular junction region in alpha-spectrin plays a major role in modulating its association affinity with beta-spectrins, and thus regulates spectrin tetramer levels. We also note that these are the first conformational studies of brain spectrin.     

Gbeta gamma isoforms selectively rescue plasma membrane localization and palmitoylation of mutant Galphas and Galphaq

Mutation of Galpha(q) or Galpha(s) N-terminal contact sites for Gbetagamma resulted in alpha subunits that failed to localize at the plasma membrane or undergo palmitoylation when expressed in HEK293 cells. We now show that overexpression of specific betagamma subunits can recover plasma membrane localization and palmitoylation of the betagamma-binding-deficient mutants of alpha(s) or alpha(q). Thus, the betagamma-binding-defective alpha is completely dependent on co-expression of exogenous betagamma for proper membrane localization. In this report, we examined the ability of beta(1-5) in combination with gamma(2) or gamma(3) to promote proper localization and palmitoylation of mutant alpha(s) or alpha(q). Immunofluorescence localization, cellular fractionation, and palmitate labeling revealed distinct subtype-specific differences in betagamma interactions with alpha subunits. These studies demonstrate that 1) alpha and betagamma reciprocally promote the plasma membrane targeting of the other subunit; 2) beta(5), when co-expressed with gamma(2) or gamma(3), fails to localize to the plasma membrane or promote plasma membrane localization of mutant alpha(s) or alpha(q); 3) beta(3) is deficient in promoting plasma membrane localization of mutant alpha(s) and alpha(q), whereas beta(4) is deficient in promoting plasma membrane localization of mutant alpha(q); 4) both palmitoylation and interactions with betagamma are required for plasma membrane localization of alpha.     

Neuroprotective effect of curcumin in middle cerebral artery occlusion induced focal cerebral ischemia in rats

Free radical induced neuronal damage is implicated in cerebral ischemia reperfusion (IR) injury and antioxidants are reported to have neuroprotective activity. Several in vitro and in vivo studies have proved the antioxidant potential of curcumin and its metabolites. Hence, in the present study the neuroprotective potential of curcumin was investigated in middle cerebral artery occlusion (MCAO) induced focal cerebral IR injury. 2 h of MCAO and 22 h of reperfusion resulted in the infarct volume of 210.39 +/- 31.25 mm3. Administration of curcumin 100 and 300 mg/kg, i.p. 30 min. after MCAO produced 37.23 +/- 5.10% and 46.39 +/- 10.23% (p < 0.05) reduction in infarct volume, respectively. Ischemia induced cerebral edema was reduced in a dose dependent manner. Curcumin at 300 mg/kg, i.p. produced 50.96 +/- 6.04% reduction in edema (p < 0.05) volume. Increase in lipid peroxidation after MCAO in ipsilateral and contralateral hemisphere of brain was observed, which was reduced by curcumin (300 mg/kg, i.p.)-treatment. Decrease in superoxide dismutase and glutathione peroxidase activity was observed in ipsilateral hemisphere of MCAO animal. Curcumin-treatment (300 mg/kg, i.p.) prevented IR injury mediated fall in glutathione peroxide activity. Peroxynitrite measured using rhodamine123 fluorescence and anti-nitrotyrosine immunofluorescence indicated increased peroxynitrite formation after IR insult. Curcumin-treatment reduced peroxynitrite formation and hence the extent of tyrosine nitration in the cytosolic proteins. These results suggest the neuroprotective potential of curcumin in cerebral ischemia and is mediated through its antioxidant activity.     

First crystal structures of human carbonic anhydrase II in complex with dual aromatase-steroid sulfatase inhibitors

Carbonic anhydrase (CA) catalyzes the reversible hydration of carbon dioxide to hydrogen carbonate. The role of CA in maintaining pH balance has made it an attractive drug target for the treatment of cancer, and it has recently been implicated in the delivery of sulfamate-containing drugs. With the acceptance of steroid sulfatase as a target for hormone-dependent cancer, novel dual aromatase-steroid sulfatase inhibitors (DASIs) containing a sulfamate group are now being developed. In this study, we show that CA II is potently inhibited by several members of this class of inhibitor. The structures of CA II complexed with 4-[(4-O-sulfamoylbenzyl)(4-cyanophenyl)amino]-4H-[1,2,4]triazole (K(D) = 84 +/- 5 nM) and 4-[(3-bromo-4-O-sulfamoylbenzyl)(4-cyanophenyl)amino]-4H-[1,2,4]triazole (K(D) = 454 +/- 29 nM) are reported to 2.02 and 1.76 A, respectively. Both inhibitors ligate to the active site zinc(II) atom via their sulfamate nitrogen, while the rest of the molecule is contained within the hydrophobic binding pocket. Key protein residues include Val-121, Phe-131, Val-135, Val-143, Leu-141, Leu-198, Pro-202, and Leu-204. Despite being structurally similar, the two ligands experience different types of binding particularly in the sulfamate-containing aromatic ring and the opposite geometric arrangement of the triazole and cyanophenyl groups around the configurationally invertible central nitrogen atom. Small changes in inhibitor structure can cause large changes in binding to CA II, and this underlines the importance of structure-based drug design with this enzyme and other isoforms relevant to potential anticancer therapy. Moreover, these results underpin the idea that binding to erythrocyte CA II may be a general method of stabilizing and delivering sulfamate-based drugs in vivo.     

Mechanisms of contractile ring tension production and constriction

The contractile ring is a remarkable tension-generating cellular machine that constricts and divides cells into two during cytokinesis, the final stage of the cell cycle. Since the ring’s discovery, the parallels with muscle have been emphasized. Both are contractile actomyosin machineries, and long ago, a muscle-like sliding filament mechanism was proposed for the ring. This review focuses on the mechanisms that generate ring tension and constrict contractile rings. The emphasis is on fission yeast, whose contractile ring is sufficiently well characterized that realistic mathematical models are feasible, and possible lessons from fission yeast that may apply to animal cells are discussed. Recent discoveries relevant to the organization in fission yeast rings suggest a stochastic steady-state version of the classic sliding filament mechanism for tension. The importance of different modes of anchoring for tension production and for organizational stability of constricting rings is discussed. Possible mechanisms are discussed that set the constriction rate and enable the contractile ring to meet the technical challenge of maintaining structural integrity and tension-generating capacity while continuously disassembling throughout constriction.     

CD of gels and suspensions: apparent CD in the soret region of sickle hemoglobin gels

CD spectra in the soret region of sickle-cell deoxyhemoglobin (deoxy-HbS) fiber gels are radically different from the CD of deoxy-HbS in solution. An explanation is found using the Stokes–Mueller representation of the interaction of a polarized beam with the instrument optical train and sample to derive expressions for the apparent CD of gels and suspensions of optically active molecules that consist of randomly oriented domains or particles that are linearly dichroic and linearly birefringent. These theoretical considerations show that the apparent CD spectra from such systems have contributions from the LD and birefringence of each domain even if no net linear birefringence and dichroism is apparent in the sample. Thus, the interpretation of the CD from gels and suspensions is problematic, unless it can be demonstrated that each domain or particle has extremely small absorbance or that the LD and birefringence of each is a very small fraction of the total absorbance. As a result, we conclude that the spectra of HbS gels are not due to the CD of the heme per se; rather, they also reflect the randomly oriented domain structure of the gels and the LD and linear birefringence associated with each domain.     

Disease Variants of FGFR3 Reveal Molecular Basis for the Recognition and Additional Roles for Cdc37 in Hsp90 Chaperone System

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Tissue Selective Androgen Receptor Modulators (SARMs) Increase Pelvic Floor Muscle Mass in Ovariectomized Mice

Stress urinary incontinence (SUI), a prevalent condition, is represented by an involuntary leakage of urine that results, at least in part, from weakened or damaged pelvic floor muscles and is triggered by physical stress. Current treatment options are limited with no oral therapies available. The pelvic floor is rich in androgen receptor and molecules with anabolic activity including selective androgen receptor modulators (SARMs) may serve as therapeutic options for individuals with SUI. In this study, two SARMs (GTx‐024 and GTx‐027) were evaluated in a post‐menopausal animal model in order to determine their effect on pelvic floor muscles. Female C57BL/6 mice were ovariectomized and their pelvic muscles allowed to regress. The animals were then treated with vehicle or doses of GTx‐024 or GTx‐027. Animal total body weight, lean body mass, and pelvic floor muscle weights were measured along with the expression of genes associated with muscle catabolism. Treatment with the SARMs resulted in a restoration of the pelvic muscles to the sham‐operated weight. Coordinately, the induction of genes associated with muscle catabolism was inhibited. Although a trend was observed towards an increase in total lean body mass in the SARM‐treated groups, no significant differences were detected. Treatment of an ovariectomized mouse model with SARMs resulted in an increase in pelvic floor muscles, which may translate to an improvement of symptoms associated with SUI and serves as the basis for evaluating their clinical use. J. Cell. Biochem. 118: 640–646, 2017. © 2016 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.     

Proteomic analysis of larvae during development, attachment, and metamorphosis in the fouling barnacle, Balanus amphitrite

The barnacle, Balanus amphitrite, is one of the primary model organisms for rocky-shore ecology studies and biofouling research. This barnacle species has a complex life cycle during which the swimming nauplius molts six times and transforms into a cyprid stage. Cyprids must attach to a surface to metamorphose into a juvenile barnacle. To clarify the overall profile of protein expression during larval development and metamorphosis, 2-DE was used to compare the proteome of the nauplius, the swimming cyprid, the attached cyprid, and the metamorphosed cyprid. The proteome of the swimming cyprid was distinctly different from that of other life stages and had about 400 spots. The proteomes of the attached and metamorphosed cyprids were similar with respect to major proteins but had significantly lower numbers of spots compared to that of swimming larval stages. Obviously, synthesis of most proteins from swimming cyprids was switched off after attachment and metamorphosis. Our advanced MS analysis (MALDI-TOF/TOF MS/MS) allowed us to identify the proteins that were differentially and abundantly expressed in the swimming cyprid. These proteins included signal transduction proteins (adenylate cyclase and calmodulin) and juvenile hormone binding proteins. In summary, for the first time, we have analyzed the global protein expression pattern of fouling marine invertebrate larvae during metamorphosis. Our study provides new insights into the mechanisms of barnacle larval metamorphosis and also provides a foundation for exploring novel targets for antifouling treatments.     

Effect of temperature, salinity and delayed attachment on development of the solitary ascidian Styela plicata (Lesueur)

The solitary ascidian Styela plicata (Lesueur) is a common member of epibenthic marine communities in Hong Kong, where seawater experiences extensive seasonal changes in temperature (18-30 °C) and salinity (22-34‰). In this investigation, the relative sensitivity of different developmental stages (i.e., duration of embryonic development, larval metamorphosis and post-larval growth) to various temperature (18, 22, 26 and 30 °C) and salinity (22‰, 26‰, 30‰ and 34‰) combinations is reported. Fertilized eggs did not develop at lower salinities (22‰ and 26‰). At higher salinities (30‰ and 34‰), the duration of embryonic development increased with decreasing temperature (18 °C: 11.5±0.3 h; 30 °C: 8.5±0.3 h). More than 50% of larvae spontaneously attached and metamorphosed at all the levels of temperature and salinity tested. At higher temperatures (22, 26 and 30 °C) and salinities (30‰ and 34‰), functional siphon developed in about 72 h after hatching, whereas at low temperature (18 °C), siphon developed only in <30% of individuals in about 90 h. However, none of the metamorphosed larvae developed subsequently at low salinity (22‰). When forced to swim (or delayed attachment), larvae lost about 0.27 mJ after 48 h (about 22% of the stored energy). Such a drop in energy reserves, however, was not strong enough to cause a significant impact on post-larval growth. This study suggests that temperature and salinity reductions due to seasonal monsoon may have significant effect on the embryo and post-larval growth of S. plicata in Hong Kong.