Effective RNA interference in cultured silkworm cells mediated by overexpression of Caenorhabditis elegans SID-1

RNA interference (RNAi) is a conserved mechanism that catalyzes sequence-specific gene silencing and has been used for loss-of-function genetic screens in many organisms. Here, we demonstrated that the expression of Caenorhabditis elegans SID-1 (CeSID-1) could trigger effective gene silencing in the cultured silkworm cell line, BmN4 (BmN4-SID1). Soaking the BmN4-SID1 in dsRNA corresponding to endogenous target genes induced a significant decrease of the amount of mRNA or protein. A small amount of dsRNA was enough to silence the target gene in a few days. Overexpression of CeSID-1 did not affect the cell viability. Our results suggest that BmN4-SID1 can be used in many applications in silkworm cells and will become a valuable resource for gene analysis.     

Mutations in the gene PRRT2 cause paroxysmal kinesigenic dyskinesia with infantile convulsions

Paroxysmal Kinesigenic Dyskinesia with Infantile Convulsions (PKD/IC) is an episodic movement disorder with autosomal dominant inheritance and high penetrance, but the causative gene is unknown. We have now identified four truncating mutations involving the PRRT2 gene in the vast majority (24/25) of well characterized families with PKD/IC. PRRT2 truncating mutations were also detected in 28 of 78 additional families. The PRRT2 gene encodes a proline-rich transmembrane protein of unknown function that has been reported to interact with the t-SNARE, SNAP25. PRRT2 localizes to axons but not to dendritic processes in primary neuronal culture and mutants associated with PKD/IC lead to dramatically reduced PRRT2 protein levels leading ultimately to neuronal hyperexcitability that manifests in vivo as PKD/IC.     

TNF-α and Tumor Lysate Promote the Maturation of Dendritic Cells for Immunotherapy for Advanced Malignant Bone and Soft Tissue Tumors

Background

Dendritic cells (DCs) play a pivotal role in the immune system. There are many reports concerning DC-based immunotherapy. The differentiation and maturation of DCs is a critical part of DC-based immunotherapy. We investigated the differentiation and maturation of DCs in response to various stimuli.

Methods

Thirty-one patients with malignant bone and soft tissue tumors were enrolled in this study. All the patients had metastatic tumors and/or recurrent tumors. Peripheral blood mononuclear cells (PBMCs) were suspended in media containing interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF). These cells were then treated with or without 1) tumor lysate (TL), 2) TL + TNF-α, 3) OK-432. The generated DCs were mixed and injected in the inguinal or axillary region. Treatment courses were performed every week and repeated 6 times. A portion of the cells were analyzed by flow cytometry to determine the degree of differentiation and maturation of the DCs. Serum IFN-γ and serum IL-12 were measured in order to determine the immune response following the DC-based immunotherapy.

Results

Approximately 50% of PBMCs differentiated into DCs. Maturation of the lysate-pulsed DCs was slightly increased. Maturation of the TL/TNF-α-pulsed DCs was increased, commensurate with OK-432-pulsed DCs. Serum IFN-γ and serum IL-12 showed significant elevation at one and three months after DC-based immunotherapy.

Conclusions

Although TL-pulsed DCs exhibit tumor specific immunity, TL-pulsed cells showed low levels of maturation. Conversely, the TL/TNF-α-pulsed DCs showed remarkable maturation. The combination of IL-4/GM-CSF/TL/TNF-α resulted in the greatest differentiation and maturation for DC-based immunotherapy for patients with bone and soft tissue tumors.     

A single nucleotide polymorphism within the novel sex-linked testis-specific retrotransposed PGAM4 gene influences human male fertility

BACKGROUND: The development of novel fertilization treatments, including in vitro fertilization and intracytoplasmic injection, has made pregnancy possible regardless of the level of activity of the spermatozoa; however, the etiology of male-factor infertility is poorly understood. Multiple studies, primarily through the use of transgenic animals, have contributed to a list of candidate genes that may affect male infertility in humans. We examined single nucleotide polymorphisms (SNPs) as a cause of male infertility in an analysis of spermatogenesis-specific genes. METHODS AND FINDING: We carried out the prevalence of SNPs in the coding region of phosphoglycerate mutase 4 (PGAM4) on the X chromosome by the direct sequencing of PCR-amplified DNA from male patients. Using RT-PCR and western blot analyses, we identified that PGAM4 is a functional retrogene that is expressed predominantly in the testes and is associated with male infertility. PGAM4 is expressed in post-meiotic stages, including spermatids and spermatozoa in the testes, and the principal piece of the flagellum and acrosome in ejaculated spermatozoa. A case-control study revealed that 4.5% of infertile patients carry the G75C polymorphism, which causes an amino acid substitution in the encoded protein. Furthermore, an assay for enzymatic activity demonstrated that this polymorphism decreases the enzyme's activity both in vitro and in vivo. CONCLUSION: These results suggest that PGAM4, an X-linked retrogene, is a fundamental gene in human male reproduction and may escape meiotic sex chromosome inactivation. These findings provide fresh insight into elucidating the mechanisms of male infertility.     

Combination treatment of rosuvastatin or atorvastatin, with regular exercise improves arterial wall stiffness in patients with coronary artery disease

Objective

Statin- and exercise-therapy are both clinically beneficial by preventing cardiovascular events in patients with coronary artery disease (CAD). However, there is no information on the vascular effects of the combination of statins and exercise on arterial wall stiffness in CAD patients.

Methods

The present study is a sub-analysis of PRESET study that determined the effects of 20-week treatment with statins (rosuvastatin, n = 14, atorvastatin, n = 14) combined with regular exercise on arterial wall stiffness assessed by measurement of brachial and ankle pulse wave velocity (baPWV) in CAD patients.

Results

The combination of statins and regular exercise significantly improved exercise capacity, lipid profile, including low- and high-density lipoprotein cholesterol, and high-sensitivity C-reactive protein (hs-CRP), baPWV (baseline: 1747±355, at 20 weeks of treatment: 1627±271 cm/s, p = 0.008), and basophil count (baseline: 42±32, 20 weeks: 26±15 cells/µL, p = 0.007), but had no effect on blood pressure (baseline: 125±22, 20 weeks: 121±16 mmHg). Changes in baPWV correlated significantly with changes in basophil count (r = 0.488, p = 0.008), but not with age, lipids profile, exercise capacity, or hs-CRP.

Conclusion

In CAD patients, the combination treatment with statins and exercise resulted in significant amelioration of arterial wall stiffness, at least in part, through reduction of circulating basophils.     

Sex hormones induce a gender-related difference in renal expression of a novel prostaglandin transporter, OAT-PG, influencing basal PGE2 concentration

Based on the nucleotide sequence of a mouse prostaglandin-specific transporter (mOAT-PG), we identified a rat homolog (rOAT-PG) which shares 80% identity with mOAT-PG in a deduced amino acid sequence. rOAT-PG transports PGE(2) and colocalizes with 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a metabolic enzyme for PGs, in proximal tubules, suggesting that rOAT-PG is involved in PGE(2) clearance to regulate its physiological function in the renal cortex. We found that the expression level of rOAT-PG in the renal cortex was much higher in male rats than in female rats whereas there was no gender difference in the expression level of cyclooxygenase-2, a key enzyme producing PGE(2), and 15-PGDH in the renal cortex. Tissue PGE(2) concentration in the renal cortex was lower in male rats than in female rats, suggesting that renocortical PGE(2) concentration is primarily determined by the expression level of OAT-PG, which is regulated differently between male and female rats. Castration of male rat led to a remarkable reduction in OAT-PG expression and a significant increase in renocortical PGE(2) concentration. These alterations were recovered by testosterone supplementation. These results suggest that OAT-PG is involved in local PGE(2) clearance in the renal cortex. Although the physiological importance of the gender difference in local PGE(2) clearance is still unclear, these findings might be a key to clarifying the physiological roles of PGE(2) in the kidney.     

Role of angiotensin II-mediated AMPK inactivation on obesity-related salt-sensitive hypertension

Salt-sensitive hypertension is a characteristic of the metabolic syndrome. Given the links to cardiovascular events, the mechanisms underlying sodium metabolism may represent an important therapeutic target for this disorder. Angiotensin II (AII) is a key peptide underlying sodium retention. However, 5'AMP-activated protein kinase (AMPK) has also been reported to participate in the regulation of ion transport. In this study we examined the relationship between AII and AMPK on the development of hypertension in two salt-sensitive mouse models. In the first model, the mice were maintained on a high-fat diet (HFD) for 12 weeks, in order to develop features similar to the metabolic syndrome, including salt-sensitive hypertension. HFD-induced obese mice showed elevated systolic blood pressure and lower sodium excretion in response to salt loading, along with an increase in AII contents and inactivation of AMPK in the kidney, which were significantly improved by the treatment of an angiotensin II antagonist, losartan, for 2 weeks. To clarify the effects of AII, a second group of mice was infused with AII via an osmotic pump, which led to higher systolic blood pressure, and decreases in urinary sodium excretion and the expression of AMPK, in a manner similar to those observed in the HFD mice. However, treatment with an AMPK activator, metformin, improved the changes induced by the AII, suggesting that AII induced sodium retention works by acting on AMPK activity. Finally, we evaluated the changes in salt-sensitivity by performing 2-week salt loading experiments with or without metformin. AII infusion elevated blood pressure by salt loading but metformin prevented it. These findings indicate that AII suppresses AMPK activity in the kidney, leading to sodium retention and enhanced salt-sensitivity, and that AMPK activation may represent a new therapeutic target for obesity-related salt-sensitive hypertension.     

Quantitative analysis of extension-torsion coupling of actin filaments

Actin filaments have a double-helix structure consisting of globular actin molecules. In many mechanical cellular activities, such as cell movement, division, and shape control, modulation of the extensional and torsional dynamics of the filament has been linked to regulatory actin-binding protein functions. Therefore, it is important to quantitatively evaluate extension-torsion coupling of filament to better understand the actin filament dynamics. In the present study, the extension-torsion coupling was investigated using molecular dynamics simulations. We constructed a model for the actin filament consisting of 14 actin subunits in an ionic solvent as a minimal functional unit, and analyzed longitudinal and twisting Brownian motions of the filament. We then derived the expected value of energy associated with extension and torsion at equilibrium, and evaluated the extension-torsion stiffness of the filament from the thermal fluctuations obtained from the MD simulations. The results demonstrated that as the analyzed sampling-window duration was increased, the extension-torsion coupling stiffness evaluated on a nanosecond scale tended to converge to a value of 7.6×10(-11) N. The results obtained from this study will contribute to the understanding of biomechanical events, under mechanical tension and torque, involving extension-torsion coupling of filaments.     

Autophagy regulates inflammation in adipocytes

Autophagy is an essential process for both the maintenance and the survival of cells, with homeostatic low levels of autophagy being critical for intracellular organelles and proteins. In insulin resistant adipocytes, various dysfunctional/damaged molecules, organelles, proteins, and end-products accumulate. However, the role of autophagy (in particular, whether autophagy is activated or not) is poorly understood. In this study we found that in adipose tissue of insulin resistant mice and hypertrophic 3T3-L1 adipocytes autophagy was suppressed. Also in hypertrophic adipocytes, autophagy-related gene expression, such as LAMP1, LAMP2, and Atg5 was reduced, whereas gene expression in the inflammatory-related genes, such as MCP-1, IL-6, and IL-1β was increased. To find out whether suppressed autophagy was linked to inflammation we used the autophagy inhibitor, 3-methyladenine, to inhibit autophagy. Our results suggest that such inhibition leads to an increase in inflammatory gene expression and causes endoplasmic reticulum (ER) stress (which can be attenuated by treatment with the ER stress inhibitor, Tauroursodeoxycholic Acid). Conversely, the levels of inflammatory gene expression were reduced by the activation of autophagy or by the inhibition of ER stress. The results indicate that the suppression of autophagy increases inflammatory responses via ER stress, and also defines a novel role of autophagy as an important regulator of adipocyte inflammation in systemic insulin resistance.