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Why regeneration does not occur in mammals remains elusive. In lower vertebrates, epimorphic regeneration of the limb is directed by the wound epidermis, which controls blastema formation to promote regrowth of the appendage. Herein, we report that knockout (KO) or inhibition of Apoptosis Signal-regulated Kinase-1 (ASK1), also known as mitogen-activated protein kinase kinase kinase 5 (MAP3K5), after full thickness ear punch in mice prolongs keratinocyte activation within the wound epidermis and promotes regeneration of auricular cartilage. Histological analysis showed the ASK1 KO ears displayed enhanced protein markers associated with blastema formation, hole closure and regeneration of auricular cartilage. At seven days after punch, the wound epidermis morphology was markedly different in the KO, showing a thickened stratum corneum with rounded cell morphology and a reduction of both the granular cell layer and decreased expression of filament aggregating protein. In addition, cytokeratin 6 was expressed in the stratum spinosum and granulosum. Topical application of inhibitors of ASK1 (NQDI-1), the upstream ASK1 activator, calcium activated mitogen kinase 2 (KN93), or the downstream target, c-Jun N-terminal kinase (SP600125) also resulted in enhanced regeneration; whereas inhibition of the other downstream target, the p38 α/β isoforms, (SB203580) had no effect. The results of this investigation indicate ASK1 inhibition prolongs keratinocyte and blastemal cell activation leading to ear regeneration. [ABSTRACT FROM AUTHOR]

Objectives: To identify the negative effect on treatment results of reserving damaged intervertebral discs when treating type B and type C spinal fracture-dislocations through a one-stage posterior approach. Methods: This is a retrospective review of 53 consecutive patients who were treated in our spine surgery center from January 2005 to May 2012 due to severe thoracolumbar spinal fracture-dislocation. The patients in Group A (24 patients) underwent long-segment instrumentation laminectomy with pedicle screw-rod fixators for neural decompression. In Group B (29 patients), the patients underwent long-segment instrumentation laminectomy with pedicle screw-rod fixators for neural decompression evacuating of the ruptured disc and inserting of a bone graft into the evacuated disc space for interbody fusion. The mean time between injury and operation was 4.1 days (range 2–15 days). The clinical, radiologic and complication outcomes were analyzed retrospectively. Results: Periodic follow-ups were carried out until an affirmative union or treatment failure took place. A progressive kyphosis angle larger than 10°, loss of disc height, pseudoarthrosis, recurrence of dislocation or subluxation, or instrument failure before fusion were considered treatment failures. Treatment failures were detected in 13 cases in Group A (failure rate was 54.2%). In Group B, there were 28 cases in which definitive bone fusion was demonstrated on CT scans, and CT scans of the other cases demonstrated undefined pseudoarthrosis without hardware failure. There were statistically significant differences between the two groups (p<0.001 chi-square test). The neurologic recoveries, assessed by the ASIA scoring system, were not satisfactory for the neural deficit patients in either group, indicating there was no significant difference with regard to neurologic recovery between the two groups (p>0.05 Fisher's exact test). Conclusion: Intervertebral disc damage is a common characteristic in type B and C spinal fracture-dislocation injuries. The damaged intervertebral disc should be removed and substituted with a bone graft because reserving the damaged disc in situ increases the risk of treatment failure. [ABSTRACT FROM AUTHOR]

Glycyrrhetic acid (GA) exerts synergistic anti-asthmatic effects via a β2-adrenergic receptor (β2AR)-mediated pathway. Cholesterol is an important component of the structure and function of lipid rafts, which play critical roles in the β2AR-Gs-adenylate cyclase (AC)-mediated signaling pathway. Owing to the structural similarities between GA and cholesterol, we investigated the possibility that GA enhances β2AR signaling by altering cholesterol distribution. Azide-terminal GA (ATGA) was synthesized and applied to human embryonic kidney 293 (HEK293) cells expressing fusion β2AR, and the electron spin resonance (ESR) technique was utilized. GA was determined to be localized predominantly on membrane and decreased their cholesterol contents. Thus, the fluidity of the hydrophobic region increased but not the polar surface of the cell membrane. The conformations of membrane proteins were also changed. GA further changed the localization of Gαs from lipid rafts to non-raft regions, resulting the binding of β2AR and Gαs, as well as in reduced β2AR internalization. Co-localization of β2AR, Gαs, and AC increased isoproterenol-induced cAMP production and cholesterol reloading attenuated this effect. A speculation wherein GA enhances beta-adrenergic activity by increasing the functional linkage between the subcomponents of the membrane β2AR-protein kinase A (PKA) signaling pathway was proposed. The enhanced efficacy of β2AR agonists by this novel mechanism could prevent tachyphylaxis. [ABSTRACT FROM AUTHOR]

β2 adrenergic receptor (β2AR) is a prototypical G- protein coupled receptor that stimulates the classic cAMP-protein kinase A (PKA) signaling pathway. Recent studies indicate that the cAMP-PKA activities are spatiotemporally regulated in part due to dynamic association of β2AR with phosphodiesterase 4D (PDE4D), a group of cAMP degradation enzymes. Here, we demonstrate that in cardiomyocytes, palmitoylation of β2AR, the covalent acylation of cysteine residue 341, plays a critical role in shaping subcellular cAMP-PKA activities in cardiomyocytes via regulating β2AR association with arrestin/PDE4D. Replacing cysteine 341 on β2AR with alanine (C341A) leads to an impaired binding to β arrestin 2. Surprisingly, the C341A mutant is able to internalize via an arrestin-independent pathway at saturated concentration of agonist stimulation; the internalization becomes caveolae-dependent and requires dynamin GTPase. However, the impaired binding to β arrestin 2 also leads to an impaired recruitment of PDE4D to the C341A mutant. Thus, the mutant C341A β2AR is transported alone from the plasma membrane to the endosome without recruiting PDE4D. This alteration leads to an enhanced cytoplasmic cAMP signal for PKA activation under β2AR stimulation. Functionally, Mutation of the C341 residue or inhibition of palmitoylation modification of β2AR enhances the receptor- induced PKA activities in the cytoplasm and increases in myocyte contraction rate. Our data reveal a novel function of palmitoylation in shaping subcellular cAMP-PKA signaling in cardiomyocytes via modulating the recruitment of β arrestin 2-PDE4D complexes to the agonist-stimulated β2AR. [ABSTRACT FROM AUTHOR]

Tylophorine analogs exhibit a broad range of pharmacological activities, including anti-cancer, anti-inflammatory, antiautoimmune, and anti-virus effects. Structure-activity relationship study of different structure tylophorine analogs can provide further understanding of their biological activity. Modifications on the E ring of the quinolizidine moiety of cryptopleurine analogs changed the potency and the selective inhibitory effect on NF-κB, AP-1, and CRE signaling pathways. Functional cryptopleurine analogs showed potent inhibition of NF-κB signaling pathway in both HepG2 and HEK- 293 cell lines. The E ring structure analogs also differed in suppression of protein translation, and expression of cyclin D1. Our results showed that DCB-3503 or Rac-cryptopleurine could be a scaffold for modification to yield compounds with different mechanisms of action. [ABSTRACT FROM AUTHOR]

During meiotic recombination, induced double-strand breaks (DSBs) are processed into crossovers (COs) and non-COs (NCO); the former are required for proper chromosome segregation and fertility. DNA synthesis is essential in current models of meiotic recombination pathways and includes only leading strand DNA synthesis, but few genes crucial for DNA synthesis have been tested genetically for their functions in meiosis. Furthermore, lagging strand synthesis has been assumed to be unnecessary. Here we show that the Arabidopsis thaliana DNA REPLICATION FACTOR C1 (RFC1) important for lagging strand synthesis is necessary for fertility, meiotic bivalent formation, and homolog segregation. Loss of meiotic RFC1 function caused abnormal meiotic chromosome association and other cytological defects; genetic analyses with other meiotic mutations indicate that RFC1 acts in the MSH4-dependent interference-sensitive pathway for CO formation. In a rfc1 mutant, residual pollen viability is MUS81-dependent and COs exhibit essentially no interference, indicating that these COs form via the MUS81-dependent interference-insensitive pathway. We hypothesize that lagging strand DNA synthesis is important for the formation of double Holliday junctions, but not alternative recombination intermediates. That RFC1 is found in divergent eukaryotes suggests a previously unrecognized and highly conserved role for DNA synthesis in discriminating between recombination pathways. [ABSTRACT FROM AUTHOR]

Background: Natural killer (NK) cells activation has been reported to contribute to inflammation and liver injury during hepatitis B virus (HBV) infection both in transgenic mice and in patients. However, the role of NK cells in the process of HBV-associated hepatocellular carcinoma (HCC) development has not been addressed. Killer cell immunoglobulin-like receptors (KIRs) are involved in regulating NK cell activation through recognition of specific human leukocyte antigen (HLA) class I allotypes. Methodology/Principal Findings: To investigate whether KIR and HLA genes could influence the risk of HBV-associated HCC development, 144 HBV-infected patients with HCC and 189 well-matched HBV infectors with chronic hepatitis or cirrhosis as non-HCC controls were enrolled in this study. The presence of 12 loci of KIR was detected individually. HLA-A, -B, -C loci were genotyped with high-resolution. HLA-C group 1 homozygote (OR = 2.02; p=0.005), HLA-Bw4-80I (OR = 2.67; p=2.0E-04) and combination of full-length form and 22 bp-deleted form of KIR2DS4 (KIR2DS4/1D) (OR = 1.89; p=0.017) were found associated with HCC incidence. When the combined effects of these three genetic factors were evaluated, more risk factors were observed correlating with higher odds ratios for HCC incidence (P trend = 7.4E-05). Because all the risk factors we found have been reported to result in high NK cell functional potential by previous studies, our observations suggest that NK cell activation may contribute to HBV-associated HCC development. Conclusions/Significance: In conclusion, this study has identified significant associations that suggest an important role for NK cells in HCC incidence in HBV-infected patients. Our study is useful for HCC surveillance and has implications for novel personalized therapy strategy development aiming at HCC prevention in HBV-infected patients. [ABSTRACT FROM AUTHOR]