Characterization of alphaT3-1 cells stably transfected with luteininzing hormone beta-subunit complementary deoxyribonucleic acid

Luteinizing hormone (LH) consists of alpha- and beta-subunits, and synthesis and secretion of LH are regulated by gonadotropin-releasing hormone (GnRH). In order to examine the molecular mechanisms by which GnRH regulates LH secretion, we transfected alphaT3-1 cells with rat LHbeta-subunit cDNA under the control of a constitutive promoter and established a stable cell line of LH2 cells which secreted LH in response to GnRH. Pulsatile and continuous GnRH pretreatments increased gene expression of the alpha-subunit and synthesis of LH, and enhanced the LH secretion by brief treatments with GnRH and 56 mM KCl. The LH secretions were partially blocked by elimination of extracellular Ca2+. GnRH-induced LH secretion was completely inhibited by calphostin C (a protein kinase C inhibitor) and 1 microM wortmannin. In contrast to the GnRH induction, high K+-induced LH secretion was inhibited by KN93, a Ca2+/calmodulin-dependent protein kinase II inhibitor, as well as by 1 microM wortmannin. We also confirmed that activation of cAMP-pathway induced LH secretion, but activation of mitogen-activated protein (MAP) kinase pathway was not involved in LH secretion. These results suggest that GnRH directly regulates LH secretion as well as LHbeta-subunit synthesis, and that LH2 cells are a useful model for the study of LH secretion induced by several secretagogues.     

Sequential analysis of amino acid substitutions with hepatitis B virus in association with nucleoside/nucleotide analog treatment detected by deep sequencing

Taking nucleoside/nucleotide analogs is a major antiviral therapy for chronic hepatitis B infection. The problem with this treatment is the selection for drug‐resistant mutants. Currently, identification of genotypic drug resistance is conducted by molecular cloning sequenced by the Sanger method. However, this methodology is complicated and time‐consuming. These limitations can be overcome by deep sequencing technology. Therefore, we performed sequential analysis of the frequency of drug resistance in one individual, who was treated with lamivudine on‐and‐off therapy for 2 years, by deep sequencing. The lamivudine‐resistant mutations at rtL180M and rtM204V and the entecavir‐resistant mutation at rtT184L were detected in the first subject. The lamivudine‐ and entecavir‐resistant strain was still detected in the last subject. However, in the deep sequencing analysis, rt180 of the first subject showed a mixture in 76.9% of the methionine and in 23.1% of the leucine, and rt204 also showed a mixture in 69.0% of the valine and 29.8% of the isoleucine. During the treatment, the ratio of resistant mutations increased. At rt184, the resistant variants were detectable in 58.7% of the sequence, with the replacement of leucine by the wild‐type threonine in the first subject. Gradually, entecavir‐resistant variants increased in 82.3% of the leucine in the last subject. In conclusion, we demonstrated the amino acid substitutions of the serial nucleoside/nucleotide analog resistants. We revealed that drug‐resistant mutants appear unchanged at first glance, but actually there are low‐abundant mutations that may develop drug resistance against nucleoside/nucleotide analogs through the selection of dominant mutations.     

Upregulated Expression of AQP 7 in the Skeletal Muscles of Obese ob/ob Mice

Aquaporin (AQP) is suggested to be regulated by leptin through the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway. AQP7 and AQP9 are membrane proteins with water and glycerol channels, the latter of which is essential for triglyceride synthesis. We conjectured that the expression of AQP7 and AQP9 would be altered in the skeletal myofibers in obese leptin deficient ob/ob mice as compared with that of wild mice. RNA and protein levels were studied in the quadriceps femoris muscles of ob/ob and wild mice. Real time quantitative RT-PCR analysis showed that mouse AQP7 mRNA levels in skeletal muscles were significantly higher in ob/ob mice than in wild mice (P<0.01), whereas mouse AQP9 mRNA level was not different between the two groups (P>0.05). Histologically the type 1 myofibers of ob/ob mice contained numerous lipid droplets in oil red O stain samples. Immunohistochemical staining of ob/ob mouse muscles revealed enhanced expression of AQP7 at myofiber surface membranes, while AQP9 expression appeared to be similar to that of wild mice. The findings suggest that the upregulated expression of AQP7 in ob/ob mouse muscles facilitates the secretion of glycerol from myocytes.     

Valproic acid,a histone deacetylase inhibitor,regulates cell proliferation in the adult zebrafish optic tectum

Background: Valproic acid (VPA) has been used to treat epilepsy and bipolar disorder. Several reports have demonstrated that VPA functions as a histone deacetylase (HDAC) inhibitor. While VPA is known to cause teratogenic changes in the embryonic zebrafish brain, its effects on neural stem cells (NSCs) in both the embryonic and adult zebrafish are not well understood. Results: In this study, we observed a proliferative effect of VPA on NSCs in the embryonic hindbrain. In contrast, VPA reduced cell proliferation in the adult zebrafish optic tectum. Treatment with HDAC inhibitors showed a similar inhibitory effect on cell proliferation in the adult zebrafish optic tectum, suggesting that VPA reduces cell proliferation through HDAC inhibition. Cell cycle progression was also suppressed in the optic tectum of the adult zebrafish brain because of HDAC inhibition. Recent studies have demonstrated that HDAC inhibits the Notch signaling pathway; hence, adult zebrafish were treated with a Notch inhibitor. This increased the number of proliferating cells in the adult zebrafish optic tectum with down‐regulated expression of her4, a target of Notch signaling. Conclusions: These results suggest that VPA inhibits HDAC activity and upregulates Notch signaling to reduce cell proliferation in the optic tectum of adult zebrafish. Developmental Dynamics 243:1401–1415, 2014. © 2014 Wiley Periodicals, Inc.     

Protein aggregation can inhibit clathrin-mediated endocytosis by chaperone competition

Protein conformational diseases exhibit complex pathologies linked to numerous molecular defects. Aggregation of a disease-associated protein causes the misfolding and aggregation of other proteins, but how this interferes with diverse cellular pathways is unclear. Here, we show that aggregation of neurodegenerative disease-related proteins (polyglutamine, huntingtin, ataxin-1, and superoxide dismutase-1) inhibits clathrin-mediated endocytosis (CME) in mammalian cells by aggregate-driven sequestration of the major molecular chaperone heat shock cognate protein 70 (HSC70), which is required to drive multiple steps of CME. CME suppression was also phenocopied by HSC70 RNAi depletion and could be restored by conditionally increasing HSC70 abundance. Aggregation caused dysregulated AMPA receptor internalization and also inhibited CME in primary neurons expressing mutant huntingtin, showing direct relevance of our findings to the pathology in neurodegenerative diseases. We propose that aggregate-associated chaperone competition leads to both gain-of-function and loss-of-function phenotypes as chaperones become functionally depleted from multiple clients, leading to the decline of multiple cellular processes. The inherent properties of chaperones place them at risk, contributing to the complex pathologies of protein conformational diseases.Many neurodegenerative diseases are characterized by protein misfolding and aggregation (1–5). Although the underlying disease origins may be genetically inherited or manifest sporadically, as exemplified by Huntington disease and ALS, respectively, the pathologies of these maladies all share the common molecular occurrence of protein aggregation (6). A network of protein folding and clearance mechanisms (the proteostasis network) is proposed to maintain a healthy proteome for normal cellular function (7, 8). Central to the proteostasis network are molecular chaperones and cochaperones, a diverse group of proteins that modulate the synthesis, folding, transport, and degradation of proteins (7). The conformations of aggregation-prone proteins are subject to multiple layers of regulation by the proteostasis network; however, as evidenced by the widespread pathologies of protein conformational diseases, the aggregation propensity of proteins associated with these diseases ultimately overwhelms the proteostasis machineries, thus initiating a cascade of cellular dysfunction (9–11).It is increasingly common for diseases of protein aggregation to be described as the result of gain-of-function toxicity. This toxicity is largely attributed to the dominant appearance of diverse aggregate species and the subsequent aberrant association of various proteostasis network components and other metastable proteins with these aggregates. This position is supported by experiments using immunohistochemical, biochemical, and MS methods on diseased patient tissues, as well as on numerous cellular and animal model systems (12–16). Some of these molecular interactions, such as those between aggregates and proteasomal subunits, appear irreversible, suggesting a permanent sequestration of these proteins. The association of molecular chaperones with aggregates, on the other hand, appears transient (17, 18), indicating that chaperones may be functionally recognizing aggregates as substrates for potential disaggregation and refolding (19).Beyond refolding of toxic misfolded proteins, chaperones are also essential for the folding of endogenous metastable client proteins, as well as in the assembly and disassembly of functional protein complexes. Thus, chaperones regulate a wide range of essential cellular processes, including gene expression, vesicular trafficking, and signal transduction (20–25). This dual role of chaperones suggests that a “competition” may arise between aggregates and endogenous protein clients for finite chaperone resources in situations where aggregates have accumulated. It has been proposed that such an imbalance may trigger the onset of many neurodegenerative diseases (10, 26), and recent studies report that polyglutamine (polyQ)-based aggregates can sequester and inhibit the function of a low-abundance cochaperone, Sis1p/DNAJB1, in protein degradation (27).Here, we show that diverse disease-associated aggregates sequester the highly abundant major chaperone heat shock cognate protein 70 (HSC70) to the point of functional collapse of an essential cellular process, clathrin-mediated endocytosis (CME). Importantly, aggregate-driven CME inhibition is reversible and can be rescued by nominally increasing HSC70 levels. Aggregate-driven chaperone depletion may help explain the phenotypic complexities displayed in protein conformational diseases.