A Novel miRNA-hlo-miR-2-Serves as a Regulatory Factor That Controls Molting Events by Targeting CPR1 in Haemaphysalis longicornis Nymphs.

Successful completion of the molting process requires new epidermal growth and ecdysis of the old cuticle in Haemaphysalis longicornis (H. longicornis). MicroRNAs (miRNAs) participate in the development of organisms by inhibiting the expression of their target mRNAs. In this study, a novel tick-specific miRNA was identified and denoted hlo-miR-2 that serves as a novel regulator of molting events in H. longicornis nymphs by targeting a cuticular protein. The full length of this cuticular protein was first obtained and named it CPR1. A qRT-PCR analysis showed that hlo-miR-2 and CPR1 exhibit significant tissue and temporal specificity and that their transcription levels are negatively correlated during the molting process. CPR1, as a direct target of hlo-miR-2, was identified by a luciferase reporter assay in vitro. Agomir treatment indicated that the overexpression of hlo-miR-2 significantly reduced the protein expression level of CPR1, decreased the molting rate and delayed the molting time point in H. longicornis nymphs. RNA interference (RNAi) experiments demonstrated that CPR1 was significantly associated with the molting process in H. longicornis nymphs. Phenotypic rescue experiments convincingly showed that hlo-miR-2 participated in molting events by targeting CPR1 in H. longicornis nymphs. In summary, we present evidence demonstrating that miRNAs constitute a novel important regulator of molting events in addition to hormones. The described functional evidence implicating CPR1 in molting events contributes to an improved understanding of the distinct functions of the CPR family in ticks and will aid the development of a promising application of cuticular protein RNAi in tick control.

7,8-dihydroxyflavone protects PC12 cells against 6-hydroxydopamine-induced cell death through modulating PI3K/Akt and JNK pathways.

We have recently shown that 7,8-dihydroxyflavone (7,8-DHF) protects PC12 cells against 6-OHDA-induced cytotoxicity through its antioxidant activity. In the present study, we investigated the molecular mechanisms underlying the neuronal protective activity of 7,8-DHF. Western blot analysis showed that 6-OHDA (100µM, 24h) enhanced the phosphorylation of JNK and ERK1/2, but it markedly suppressed the expression of p-Akt, implying that 6-OHDA induces PC12 cell death through activating the pro-apoptotic MAPKs pathway but suppressing the survival PI3K/Akt pathway. More importantly, addition of 7,8-DHF fully prevented the activation of JNK and suppression of Akt induced by 6-OHDA. Interestingly, pretreatment with the PI3K-specific inhibitor LY294002 largely blocked 7,8-DHF function in protecting PC12 cells from 6-OHDA-induced cell death. In contrast, the MEK inhibitor PD98059 showed little effect on the protective activity of 7,8-DHF. These results suggest that 7,8-DHF might protect PC12 cells from 6-OHDA-induced cell death through activating PI3K/Akt pathway and inhibiting JNK pathway.

Activation of bestrophin Cl- channels is regulated by C-terminal domains.

Bestrophins (VMD2, VMD2L1, VMD2L2, and VMD2L3) are a new family of anion channels. The mechanisms of their regulation are not yet well understood. Recently, we found that a domain (amino acids 356-364) in the C terminus of mouse VMD2L3 (mBest3) inhibited channel activity when it was expressed in HEK293 cells (Qu, Z., Cui, Y., and Hartzell, H. C. (2006) FEBS Lett. 580, 2141-2214). Here we show that this auto-inhibitory (AI) domain in mBest3 and human (h)Best3 is composed of seven critical residues, (356)IPSFLGS(362). Replacement of any residue (except Pro(357)) in the domain with alanine activated Cl(-) currents. Substitution of Pro(357) with other amino acids, especially phenylalanine, did activate currents. Membrane biotinylation demonstrated that nonfunctional mBest3 protein was trafficked to the plasma membrane, implying that the AI domain inhibited channel gating but not trafficking. mBest3-F359A and hBest3-G361A mutations induced outwardly rectifying currents, suggesting that the AI domain is associated with the channel pore or gating mechanism. Supporting this suggestion, the mBest3 AI domain was demonstrated to be located within a membrane-associated region. When the wild-type mBest3 C terminus (amino acids 292-669) was expressed in HEK293 cells, the protein was located mainly in the particulate fraction, but it became soluble when a sequence containing the AI domain was deleted (Delta353-404). There is an AI domain ((357)QPSFQGS(363)) in mouse VMD2L1 (mBest2) as well, but its inhibitory effect is competed by a downstream facilitatory sequence (amino acids 405-454). These results suggest that an auto-inhibitory mechanism in C termini may be universal among bestrophins investigated in the study.