Effect of FK506 in reducing scar formation by inducing fibroblast apoptosis after sciatic nerve injury in rats

We previously demonstrated that FK506, a generally applied immunosuppressant in organ transplantation, could promote peripheral nerve regeneration through reducing scar formation. However, little is known about how FK506 reduces scar formation. Herein we investigated the influence of FK506 on fibroblast proliferation and its correlation with scar formation after sciatic nerve injury in rats, and further explored the effect of FK506 on fibroblast proliferation and apoptosis in vitro. Masson staining and immunohistochemistry revealed that scar area and fibroblast number in the nerve anastomosis of sciatic nerve-injured rats were significantly reduced after FK506 administration. The scar area had a significant positive correlation with the fibroblast number, as detected by linear correlation analysis. CCK-8 assay and flow cytometry indicated that FK506 also inhibited proliferation and induced apoptosis of fibroblasts in vitro. It was primarily phosphorylation of JNK and ERK that were activated during the apoptosis of fibroblast. Pretreatment of cells with JNK inhibitor, SP600125, or ERK inhibitor, PD98059, could inhibit FK506-induced fibroblast apoptosis, respectively. Moreover, simultaneous application of both inhibitors had additive roles in cell protection from apoptosis. These results suggest that FK506-induced fibroblast apoptosis contributes to the suppression of fibroblast proliferation and then results in the reduction of scar formation in sciatic nerve-injured rat, and that JNK and ERK are involved in FK506-induced fibroblast apoptosis.     

A Hybrid Mechanism for the Synechocystis Arsenate Reductase Revealed by Structural Snapshots during Arsenate Reduction

Evolution of enzymes plays a crucial role in obtaining new biological functions for all life forms. Arsenate reductases (ArsC) are several families of arsenic detoxification enzymes that reduce arsenate to arsenite, which can subsequently be extruded from cells by specific transporters. Among these, the Synechocystis ArsC (SynArsC) is structurally homologous to the well characterized thioredoxin (Trx)-coupled ArsC family but requires the glutaredoxin (Grx) system for its reactivation, therefore classified as a unique Trx/Grx-hybrid family. The detailed catalytic mechanism of SynArsC is unclear and how the “hybrid” mechanism evolved remains enigmatic. Herein, we report the molecular mechanism of SynArsC by biochemical and structural studies. Our work demonstrates that arsenate reduction is carried out via an intramolecular thiol-disulfide cascade similar to the Trx-coupled family, whereas the enzyme reactivation step is diverted to the coupling of the glutathione-Grx pathway due to the local structural difference. The current results support the hypothesis that SynArsC is likely a molecular fossil representing an intermediate stage during the evolution of the Trx-coupled ArsC family from the low molecular weight protein phosphotyrosine phosphatase (LMW-PTPase) family.     

Electrostatic coupling of spectrin dimers to phosphatidylserine containing lipid lamellae

We studied the interaction of spectrin dimers from human erythrocytes with (bilayer and monolayer) model membranes of mixtures of dimyristoylphosphatidylethanolamine, dimyristoylphosphatidylcholine, and dimyristoylphosphatidylserine (DMPS) by densitometric evaluation of phase transitions and phase boundaries, film balance experiments, and microfluorescence. We demonstrate that spectrin readily adsorbs to mixed bilayers and monolayers even in the presence of small DMPS concentrations (30 mol %) whereas no appreciable interaction with lamellae containing zwitterionic lipids alone is observed. The selectivity of the DMPS/spectrin interaction is established by quantitative evaluation of the shifts of the phase boundaries (liquidus and solidus line) caused by the lipid/protein interaction as a function of the composition of the binary lipid mixtures. Quantitative information about the free energy of the lipid/protein interaction is obtained by computer simulation of the phase diagram of the lipid mixture in the absence or in the presence of a very small molar fraction of the protein and comparison of calculated and measured shifts. A binding energy of about 10(-17) J per spectrin molecule is found. The present perturbation method can be generalized to study selective lipid/protein interaction mechanisms in ternary or higher component mixtures. The present results provide evidence that in addition to the binding to band III, spectrin may also couple directly to the lipid moiety of the inner monolayer of erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

CXCL3 overexpression promotes the tumorigenic potential of uterine cervical cancer cells via the MAPK/ERK pathway

CXCL3 belongs to the CXC-type chemokine family and is known to play a multifaceted role in various human malignancies. While its clinical significance and mechanisms of action in uterine cervical cancer (UCC) remain unclear. This investigation demonstrated that the UCC cell line HeLa expressed CXCL3, and strong expression of CXCL3 was detected in UCC tissues relative to nontumor tissues. In addition, CXCL3 expression was strongly correlated with CXCL5 expression in UCC tissues. In vitro, HeLa cells overexpressing CXCL3, HeLa cells treated with exogenous CXCL3 or treated with conditioned medium from WPMY cells overexpressing CXCL3, exhibited enhanced proliferation and migration activities. In agreement with these findings, CXCL3 overexpression was also associated with the generation of HeLa cell tumor xenografts in athymic nude mice. Subsequent mechanistic studies demonstrated that CXCL3 overexpressing influenced the expression of extracellular signal-regulated kinase (ERK) signaling pathway associated genes, including ERK1/2, Bcl-2, and Bax, whereas the CXCL3-induced proliferation and migration effects were attenuated by exogenous administration of the ERK1/2 blocker PD98059. The data of the current investigation support that CXCL3 appears to hold promise as a potential tumor marker and interference target for UCC.     

Synthesis and SAR study of novel pseudo-steroids as potent and selective progesterone receptor antagonists

Synthesis of novel 7-pseudo-steroids 1c has been achieved from trenbolone 3 via an efficient 14 step sequence with overall yields of 10–15%. Various substitutions were incorporated at both the aromatic side chain as well as the D ring. The orientation of aromatic side chain at C10 plays a crucial role for progesterone receptor (PR) activity. Compound 2a (T47D = 1 nM) with –NMe₂ para to the aromatic group along with spirofurane groups in the D ring was the optimal substitution. All compounds were also evaluated for glucocorticoid receptor (GR) antagonist activities in vivo in a rat and found efficacious in uterine complement C3 assay via the oral route of administrations.     

Localization of the steroid-binding site of the human sex steroid-binding protein of plasma (SBP or SHBG) by site-directed mutagenesis.

The amino-terminal region of the human sex steroid-binding protein of plasma (SBP or SHBG) containing K134 and M139 was found to represent part of the steroid-binding site. This was accomplished by constructing and expressing site-directed mutants having the following replacements: M139L, M139K, M139S, K134A, H235S, and Y57F. The results indicated that M139L and H235S were fully-active, K134A and Y57F were 50 and 67% active, M139K was 7% active, and M139S was inactive. These results support affinity-labeling data indicating that both K134 and M139 are located in or near the site, and suggest that Y57 may play a role in steroid binding. The fully active H235S mutant reveals that H235 is not involved in the steroid-binding process.     

Structural basis of Zika virus helicase in recognizing its substrates

The recent explosive outbreak of Zika virus (ZIKV) infection has been reported in South and Central America and the Caribbean. Neonatal microcephaly associated with ZIKV infection has already caused a public health emergency of international concern. No specific vaccines or drugs are currently available to treat ZIKV infection. The ZIKV helicase, which plays a pivotal role in viral RNA replication, is an attractive target for therapy. We determined the crystal structures of ZIKV helicase-ATP-Mn2+ and ZIKV helicase-RNA. This is the first structure of any flavivirus helicase bound to ATP. Comparisons with related flavivirus helicases have shown that although the critical P-loop in the active site has variable conformations among different species, it adopts an identical mode to recognize ATP/Mn2+. The structure of ZIKV helicase-RNA has revealed that upon RNA binding, rotations of the motor domains can cause significant conformational changes. Strikingly, although ZIKV and dengue virus (DENV) apo-helicases share conserved residues for RNA binding, their different manners of motor domain rotations result in distinct individual modes for RNA recognition. It suggests that flavivirus helicases could have evolved a conserved engine to convert chemical energy from nucleoside triphosphate to mechanical energy for RNA unwinding, but different motor domain rotations result in variable RNA recognition modes to adapt to individual viral replication.