Mass-spectrometric characterization of cisplatin binding sites on native and denatured ubiquitin

Because interactions between cisplatin and plasma proteins contribute to drug efficacy and side effects, it is important to understand both the binding sites of cisplatin on the proteins and the formation of protein–cisplatin adducts. Previous results suggest that cisplatin preferentially binds to residues on the protein surface. The present work employed electrospray ionization mass spectrometry (MS) to identify such sites on both native and denatured ubiquitin (Ub). Fourier transform (FT) MS and tandem MS (MS/MS and MS³) enable analysis of Ub–cisplatin adduct digests to locate specific cisplatin binding sites. Results indicate that there are three such binding sites, i.e., M1, T12 and T14, and D32, on native Ub. The intensity of the relevant peaks in the FT-MS spectrum of the native Ub adduct digest demonstrates that residues T12 and T14 comprise the primary cisplatin binding site under the native conditions rather than residue M1 as reported in previous research studies. It is found in the present work, however, that M1 is the primary binding site on denatured Ub. Comparison of cisplatin binding sites on native and denatured Ub in this research demonstrates that the conformation of a protein significantly influences the preference of cisplatin for specific binding sites.     

Invasion of the African sharp-tooth catfish <Emphasis Type=Italic>Clarias gariepinus</Emphasis> (Burchell, 1822) in South China

We record here the invasion of the African sharp-tooth catfish Clarias gariepinus in the South China biodiversity hotspot, an area rich in endemic and endangered fish fauna. C. gariepinus was introduced into the natural systems as escapees from aquaculture ponds. These catfishes are very large, top predators, and thus have the potential to cause serious threats to the native fish fauna. The impact of C. gariepinus needs more investigation with emphasis on developing techniques for controlling its dispersal.     

A Durable Alternative for Proton‐Exchange Membranes: Sulfonated Poly(Benzoxazole Thioether Sulfone)s

To develop a durable proton‐exchange membrane (PEM) for fuel‐cell applications, a series of sulfonated poly(benzoxazole thioether sulfone)s ( SPTESBOs) are designed and synthesized, with anticipated good dimensional stability (via acid–base cross linking), improved oxidative stability against free radicals (via incorporation of thioether groups), and enhanced inherent stability (via elimination of unstable end groups) of the backbone. The structures and the degree of sulfonation of the copolymers are characterized using Fourier‐transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy (¹H NMR and ¹⁹F NMR). The electrochemical stabilities of the monomers are examined using cyclic voltammetry in a typical three‐electrode cell configuration. The physicochemical properties of the membranes vital to fuel‐cell performance are also carefully evaluated under conditions relevant to fuel‐cell operation, including chemical and thermal stability, proton conductivity, solubility in different solvents, water uptake, and swelling ratio. The new membranes exhibit low dimensional change at 25°C to 90°C and excellent thermal stability up to 250°C. Upon elimination of unstable end groups, the co‐polymers display enhanced chemical resistance and oxidative stability in Fenton's test. Further, the SPTESBO‐HFB‐60 (HFB‐60=hexafluorobenzene, 60 mol% sulfone) membrane displays comparable fuel‐cell performance to that of an NRE 212 membrane at 80°C under fully humidified condition, suggesting that the new membranes have the potential to be more durable but less expensive for fuel‐cell applications.     

Sonic hedgehog is involved in formation of the ventral optic cup by limiting Bmp4 expression to the dorsal domain

Accumulating evidence suggests that Sonic hedgehog (Shh) signaling plays a crucial role in eye vesicle patterning in vertebrates. Shh promotes expression of Pax2 in the optic stalk and represses expression of Pax6 in the optic cup. Shh signaling contributes to establishment of both proximal–distal and dorsal–ventral axes by activating Vax1, Vax2, and Pax2. In the dorsal part of the developing retina, Bmp4 is expressed and antagonizes the ventralizing effects of Shh signaling through the activation of Tbx5 expression in chick and Xenopus. To examine the roles of Shh signaling in optic cup formation and optic stalk development, we utilized the Smoothened (Smo) conditional knockout (CKO) mouse line. Smo is a membrane protein which mediates Shh signaling into inside of cells. Cre expression was driven by Fgf15 enhancer. The ventral evagination of the optic cup deteriorated from E10 in the Smo-CKO, whereas the dorsal optic cup and optic stalk develop normally until E11. We analyzed expression of various genes such as Pax family (Pax2/Pax6), Vax family (Vax1/Vax2) and Bmp4. Bmp4 expression was greatly upregulated in the optic vesicle by the 21-somite stage. Then Vax1/2 expression was decreased at the 20- to 24-somite stages. Pax2/6 expression was affected at the 27- to 32-somite stages. Our data suggest that the effects of the absence of Shh signaling on Vax1/Vax2 are mediated through increased Bmp4 expression throughout the optic cup. Also unchanged patterns of Raldh2 and Raldh3 suggest that retinoic acid is not the downstream to Shh signaling to control the ventral optic cup morphology.     

Antifibrotic effect of the Chinese herbs, Astragalus mongholicus and Angelica sinensis, in a rat model of chronic puromycin aminonucleoside nephrosis

Nephrotic syndrome has long been treated in China with two herbs, Astragalus mongholicus and Angelica sinensis, which may have antifibrotic effects. METHODS: Rats with chronic puromycin-induced nephrosis were treated with Astragalus and Angelica 3 mL/d (n = 7) or enalapril 10 mg/kg/d (n = 7). Normal control rats (n = 7) received saline rather than puromycin, and an untreated control group (n = 7) received puromycin but no treatment. After 12 weeks, stained sections of the glomerulus and tubulointerstitium were evaluated for injury. Immunohistochemistry staining measured extracellular matrix components, transforming growth factor-beta1 (TGFbeta1), osteopontin, ED-1-positive cells, and alpha-actin. TGFbeta1 mRNA was assessed by in situ hybridization. Renin, ACE activity, angiotensin, and aldosterone were measured by radioimmunoassay or colorimetry. In the untreated rats, chronic renal injury progressed to marked fibrosis at 12 weeks. Astragalus and Angelica significantly reduced deterioration of renal function and histologic damage. Expressions of type III and IV collagen, fibronectin, and laminin also decreased significantly. This anti-fibrotic effect was similar to that of enalapril. The herbs had no effect on the renin-angiotensin system but did reduce the number of ED-1-positive, and alpha-actin positive cells and expression of osteopontin compared to untreated controls. The combination of Astragalus and Angelica retarded the progression of renal fibrosis and deterioration of renal function with comparable effects of enalapril. These effects were not caused by blocking the intrarenal renin-angiotensin system, but associated with suppression of the overexpression of TGFbeta1 and osteopontin, reduction of infiltrating macrophages, and less activation of renal intrinsic cells [corrected].     

20(S)-Protopanaxadiol Inhibition of Progression and Growth of Castration-Resistant Prostate Cancer

Castration-resistant progression of prostate cancer after androgen deprivation therapies remains the most critical challenge in the clinical management of prostate cancer. Resurgent androgen receptor (AR) activity is an established driver of castration-resistant progression, and upregulation of the full-length AR (AR-FL) and constitutively-active AR splice variants (AR-Vs) has been implicated to contribute to the resurgent AR activity. We reported previously that ginsenoside 20(S)-protopanaxadiol-aglycone (PPD) can reduce the abundance of both AR-FL and AR-Vs. In the present study, we further showed that the effect of PPD on AR expression and target genes was independent of androgen. PPD treatment resulted in a suppression of ligand-independent AR transactivation. Moreover, PPD delayed castration-resistant regrowth of LNCaP xenograft tumors after androgen deprivation and inhibited the growth of castration-resistant 22Rv1 xenograft tumors with endogenous expression of AR-FL and AR-Vs. This was accompanied by a decline in serum prostate-specific antigen levels as well as a decrease in AR levels and mitoses in the tumors. Notably, the 22Rv1 xenograft tumors were resistant to growth inhibition by the next-generation anti-androgen enzalutamide. The present study represents the first to show the preclinical efficacy of PPD in inhibiting castration-resistant progression and growth of prostate cancer. The findings provide a rationale for further developing PPD or its analogues for prostate cancer therapy.     

Molecular cloning, characterization and expression of a C-type lectin cDNA in Chinese mitten crab, Eriocheir sinensis

C-type lectins are pattern-recognition proteins which are functionally important for pathogen recognition and immune regulation in vertebrates and invertebrates. In this study, a lectin cDNA named as Es-Lectin was cloned and characterized from the Chinese mitten crab, Eriocheir sinensis. The full-length sequence of this Es-Lectin cDNA was 651 bp, including an open reading frame of 483 bp encoding 160 amino acids. The predicted molecular weight of the Es-Lectin was 11.8 kDa. A typical signal peptide of 21 amino acids was deduced at the N-terminus of the predicted protein. This Es-Lectin belongs to a C-type lectin and contains six cysteines, a conserved EPN motif (Glu-Pro-Asn) and an imperfect WND (Trp-Asn-Asp) motif (FND, Phe-Asn-Asp). This Es-Lectin had 55% and 32% identity with other two C-type lectins in E. sinensis, and 29-36% homology with decapods. Although the Es-Lectin was also expressed in gill, hepatopancreas, intestine, muscle and stomach, its expression in haemocytes was the greatest. The expression of Es-Lectins in haemocytes increased at 1.5 h after the Aeromonas hydrophila challenge. After a slight decrease, the Es-Lectin expression in haemocytes significantly increased at 48 h post-challenge. The diverse distribution of Es-Lectin and its enhancement by bacterial challenge indicate that C-type lectins are important in the innate immune response to bacterial infection, and can be activated for innate immune response in crab at the initial stage after pathogen infection.     

Structural and functional analysis of the single-strand origin of replication from the lactococcal plasmid pWV01

The single-strand origin (SSO) of the rolling-circle (RC), broad-host-range lactococcal plasmid pWVO1 was functionally characterized. The activity of this SSO in the conversion of single-stranded DNA to double-stranded DNA was tested both in vivo and in vitro. In addition, the effect of this SSO on plasmid maintenance was determined. The functional pWVO1 SSO comprises a 250 by region, containing two inverted repeats (IRs). The activity of each IR was tested, separately and in combination, in a plasmid derivative that was otherwise completely devoid of structures that might function as SSO. One of the IRs (IR 1) showed some homology with other previously described SSOs of the SSO_A type, as well as with the conversion signal of the Escherichia coli phage X174. This IR was shown to have a partial, RNA polymerise-independent activity in complementary strand synthesis, both in vivo and in vitro. The second IR, which had no activity of its own, was required for full SSO activity, both in vivo and in vitro. The conversion of single-stranded DNA to the double-stranded form by the complete SSO was only partly sensitive to inhibition by rifampicin, indicating the existence of an RNA polymerase-independent pathway for this event. The results suggest that the pWVO1 SSO can be activated by two different routes: an RNA polymerise-dependent one (requiring the entire SSO), and an RNA polymerase-independent one (requiring only IR I).