[Two mutations of the KRT6A gene in Chinese patients with pachyonychia congenita type I].

OBJECTIVE: To investigate the gene mutation in a Chinese pedigree and one sporadic case with pachyonychia congenita type I(PC-1), as well as to explore the relationship between the genotype and phenotype. METHODS: The whole coding region of the KRT16 and KRT6A genes were amplified by long-range polymerase chain reaction (PCR). Six patients with PC-1 were studied, five of them were from a pedigree and the other one was sporadic. One unaffected member in the pedigree and 100 unrelated healthy individuals were also studied in order to exclude polymorphism. PCR products were directly sequenced to detect the mutation. RESULTS: No mutations in the KRT16 gene were observed. All patients harbored a mutation in the KRT6A gene. All five patients in the pedigree had a mutation at codon 465 (TAC to CAC) which substitutes tyrosine (Y) by histidine (H). In the sporadic patient, codon 171 (AAC) was mutated to GAC, which changes the asparagines (N) to aspartic acid (D). No such mutations were found in the unaffected member of the pedigree and the 100 unrelated controls. The mutation of Y465H is located at the end of 2B and N171D at the beginning of 1A domain of KRT6A, both are hotspots for pathogenic keratin mutations. CONCLUSION: The mutations Y465H and N171D of the KRT16A gene were detected in the pedigree and the sporadic case respectively. The Y465H mutation was a novel mutation, and the N171D mutation was reported recently.

Silibinin Induced Human Glioblastoma Cell Apoptosis Concomitant with Autophagy through Simultaneous Inhibition of mTOR and YAP.

Silibinin, also known as silybin, is the major flavonolignan isolated from Silybum marianum. Although previous reports demonstrated that silibinin exhibits significant tumor suppressor activities in various cancers by promoting cell apoptosis, it was also shown to trigger autophagy to counteract apoptosis induced by exogenous stresses in several types of cells. However, there is no report to address the role of silibinin induced autophagy in human A172 and SR glioblastoma cells. Our study showed that silibinin treatment not only inhibited the metabolic activities of glioblastoma cells but also promoted their apoptosis through the regulation of caspase 3 and PARP-1 in concentration- and time-dependent manners. Meanwhile, silibinin induced autophagy through upregulation of microtubule-associated protein a light chain 3- (LC3-) II. And autophagy inhibition with chloroquine, a lysosomotropic agent, significantly enhanced silibinin induced glioblastoma cell apoptosis. Moreover, silibinin dose-dependently downregulated the phosphorylation levels of mTOR at Ser-2448, p70S6K at Thr-389, and 4E-BP1 at Thr-37/46. Furthermore, the expression of YAP, the downstream effector of Hippo signal pathway, was also suppressed by silibinin. These results suggested that silibinin induced glioblastoma cell apoptosis concomitant with autophagy which might be due to simultaneous inhibition of mTOR and YAP and silibinin induced autophagy exerted a protective role against cell apoptosis in both A172 and SR cells.

[Gender-dependent difference of NF-kappaB expression in the hippocampus of prenatally stressed offspring rats].

In this study, immunohistochemistry and Western blot were used to determine whether the expression of NF-kappaB in the hippocampus of prenatally stressed offspring rats is gender-dependent. The results were as follows: In the female offspring rats, the expressions of p65 in the hippocampal dentate gyrus in mid-term stress (MS) and late-term stress (LS) groups were significantly less than that in the control group (P<0.01). There was a significant difference between MS and LS groups (P<0.01). The expressions of p50 in all regions of hippocampus in MS and LS groups were significantly more than that in the control group (P<0.01). A significant difference was also present between MS and LS groups (P<0.01). In the male offspring rats, the expressions of p65 in the hippocampal dentate gyrus in MS and LS groups were evidently more than that in the control group (P<0.01). There was a significant difference between MS and LS groups (P<0.01). The expressions of p50 in all regions of hippocampus in MS and LS groups were significantly less than that in the control group (P<0.05, P<0.01). There was also a significant difference in p65 expression between MS and LS groups (P<0.01). In addition, in the control group the expressions of p65 in the hippocampal dentate gyrus of female offspring rats were significantly more than that of male ones (P<0.01). However, in LS group the expressions of p65 in the hippocampal dentate gyrus of female offspring rats were significantly less than that of male ones (P<0.01). Moreover, there was no significant difference in p65 expression between female and male offspring rats in MS group. In the control group the gender difference in the expression of p50 was only observed in hippocampal CA1 (P<0.01). The expressions of p50 in all regions of hippocampus of female offspring rats were significantly more than that of male ones in LS group (P<0.01). There was no significant difference in p50 expression between female and male offspring rats in MS group. The results of Western blot were similar to those of immunohistochemical study. These results indicate that prenatal stress in different gestational periods significantly affects the expressions of p65 and p50 in hippocampus, and this effect is gender-dependent. This may be one of the mechanisms underlying the gender difference in the ability of learning and memory of the prenatally stressed offspring rats.

[Effects of siRNA on expression of livin and dose- and time-response in human malignant melanoma LiBr cells].

AIM: To investigate the effects of siRNA on expression of livin and dose- and time-response in human malignant melanoma LiBr cells. METHODS: Three chemically synthesized specific siRNAs targeting to livin were transfected to LiBr cells, then the expression of livin was detected both at mRNA level by real-time RT-PCR and at protein level by Western blot, and the effective one was selected for use on observation of dose- and time-response to livin silencing. RESULTS: One of three designed siRNAs could effectively knock down the livin expression both at mRNA and protein levels in dose- and time- dependent manners, 100 nmol/L of which achieved the highest knockdown effect on mRNA at 48 h and on protein at 72 h after transfection. CONCLUSION: Expression of livin in LiBr cells could be efficiently knocked down by siRNA with target site 790-808 of livin mRNA.

Silencing livin gene by siRNA leads to apoptosis induction, cell cycle arrest, and proliferation inhibition in malignant melanoma LiBr cells.

AIM: The aim of the present study was to investigate the effects of silencing the livin gene by small interfering RNA (siRNA) on the expression of livin and the effects on apoptosis, cell cycle, and proliferation in human malignant melanoma LiBr cells. METHODS: Three chemically-synthetic siRNA duplexes targeting livin were transiently transfected into the LiBr cells, and the effects on livin expression were detected both at the mRNA level by real-time RT-PCR and at the protein level by Western blotting. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, flow cytometric analysis, and the expression of procaspase-3 and activated caspase-3 analysis by Western blotting. Cell cycle was analyzed by flow cytometry. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. RESULTS: One of the 3 designed siRNA could effectively knock down the livin expression both at the mRNA and protein levels in dose- and time-dependent manners; 100 nmol/L with maximum downregulation on mRNA at 48 h, and on the protein at 72 h after transfection. Silencing livin could significantly induce apoptosis, arrest cell cycle at the G0/G1 phase, and inhibit proliferation in LiBr cells. Meanwhile, caspase-3 was activated. CONCLUSION: The livin gene could serve as a potential molecular target for gene therapy by siRNA for malignant melanoma.

[Effects of prenatal stress on kinetic properties of high-voltage-activated Ca2+ channel in freshly isolated offspring rat hippocampal CA3 pyramidal neurons].

OBJECTIVE: To investigate the effects of prenatal stress (PNS) on kinetic properties of high-voltage-activated (HVA) Ca(2+) channel in freshly isolated offspring rat hippocampal CA3 pyramidal neurons. METHODS: The pregnant rats were exposed to restraint stress, and the hippocampal CA3 pyramidal neurons were freshly isolated from the offspring rats to record voltage-gate Ca(2+) channel currents in these neurons using whole-cell configuration of patch-clamp technique. RESULTS: PNS significantly increased HVA calcium current density and integral current but did not alter the steady-state activation and steady-state inactivation properties of HVA Ca(2+) channel in the offspring rat CA3 neurons. The maximal HVA calcium current density was -40.89-/+0.31 pA/pF in the control group (n=10) and -49.44-/+0.37 pA/pF in PNS group (n=8, P<0.01). The maximal integral current of the HVA Ca(2+) channel was 106.81-/+4.20 nA*ms in the control group (n=10) and 133.49-/+2.59 nA*ms in the PNS group (n=8, P<0.01). CONCLUSION: Maternal exposure to stress during the critical phase of pregnancy may result in long-lasting effects on the ion channels of the hippocampal neurons in the offspring rats.

[The effects of prenatal stress on Ca2+ channel and K+ channel of hippocampal CA3 pyramidal neurons in rat offspring].

To investigate the effects of prenatal stress (PNS) on high-voltage-activated (HVA) Ca2+ channel and delayed rectifier potassium currents (I(KD)) of freshly isolated rat offspring hippocampal CA3 pyramidal neurons, the pregnant rats of PNS group were exposed to restraint stress and patch clamp technique of whole-cell mode was employed to record HVA Ca2+ and K+ channel currents in hippocampal CA3 pyramidal neurons. It was observed that PNS increased HVA calcium peak current amplitude. The maximal HVA calcium peak current amplitudes were -576.52 +/- 7.03 pA in the control group and -702.05 +/- 6.82 pA in the PNS group respectively (P < 0.01). The conductance-voltage relationship of HVA Ca2+ channel was not changed. Current-voltage relationship and conductance-voltage relationship of I(KD) in offspring CA3 neurons were also not affected by PNS. The data suggested that exposure of pregnant animals to a period of stress could impose lasting effects on the offspring hippocampus CA3 neurons Ca2+ channel during a critical phase of fetal development. The mechanism may include two ways. One was that high CORT led to enhance level of expression for the HVA Ca2+ channel subunit mRNAs and increased HVA calcium currents. Another was that PNS caused an increase in the production of ROS in hippocampal CA3 region and ROS caused an increase in the phosphorylation of HVA Ca2+ channel of offspring hippocampal CA3 neurons.