Excessive Na+/H+ Exchange in Disruption of Dendritic Na+ and Ca2+ Homeostasis and Mitochondrial Dysfunction following in Vitro Ischemia

Neuronal dendrites are vulnerable to injury under diverse pathological conditions. However, the underlying mechanisms for dendritic Na⁺ overload and the selective dendritic injury remain poorly understood. Our current study demonstrates that activation of NHE-1 (Na⁺/H⁺ exchanger isoform 1) in dendrites presents a major pathway for Na⁺ overload. Neuronal dendrites exhibited higher pH_i regulation rates than soma as a result of a larger surface area/volume ratio. Following a 2-h oxygen glucose deprivation and a 1-h reoxygenation, NHE-1 activity was increased by ∼70–200% in dendrites. This elevation depended on activation of p90 ribosomal S6 kinase. Moreover, stimulation of NHE-1 caused dendritic Na⁺_i accumulation, swelling, and a concurrent loss of Ca²⁺_i homeostasis. The Ca²⁺_i overload in dendrites preceded the changes in soma. Inhibition of NHE-1 or the reverse mode of Na⁺/Ca²⁺ exchange prevented these changes. Mitochondrial membrane potential in dendrites depolarized 40 min earlier than soma following oxygen glucose deprivation/reoxygenation. Blocking NHE-1 activity not only attenuated loss of dendritic mitochondrial membrane potential and mitochondrial Ca²⁺ homeostasis but also preserved dendritic membrane integrity. Taken together, our study demonstrates that NHE-1-mediated Na⁺ entry and subsequent Na⁺/Ca²⁺ exchange activation contribute to the selective dendritic vulnerability to in vitro ischemia.     

Immunospecificity of nuclear antigens in chicken erythroid cells

Summary Specific antisera were produced against chicken reticulocyte dehistonized chromatin. The antisera reacts strongly with chicken reticulocyte chromatin, but only marginally with chicken erythrocyte chromatin. There is no reticulocyte antigen detected in chicken liver. Reticulocyte maturation is accompanied by a gradual decrease in the chromatin immunological activity and template capacity. The reduction of immunological activity is due to the change of chromatin conformation during erythrocyte maturation. Dehistonization and sonication of erythrocyte chromatin raises the erythrocyte chromatin immunological activity to levels similar to those of reticulocyte chromatin. The erythrocyte nuclear antigens are class specific, not being found in frog erythroid cell or murine Friend leukemia cell chromatins.     

Phosphatidyl glycerolphosphate serves as glycerolphosphate donor in polymer synthesis

Phosphatidyl glycerolphosphate was found to serve as the glycerolphosphate donor for polymer synthesis. When CDP-diglyceride and radiolabeled glycerolphosphate were incubated with the membrane enzyme prepared from Streptococcus sanguis, active syntheses of radiolabeled lipids and polymers were observed. The synthesis of polymer was not inhibited by low concentration of unlabeled phosphatidylglycerol. When [3H, 32P]glycerolphosphate was used, the polymer synthesized contained both 3H and 32P. The lipids formed were characterized as phosphatidylglycerol and phosphatidyl glycerolphosphate. The polymers formed from the latter were characterized as lipoteichoic acid like compounds by sodium dodecylsulfate-polyacrylamide gel electrophoresis.     

Mitochondrial DNA phylogeography of Semisulcospira libertina (Gastropoda: Cerithioidea: Pleuroceridae): implications the history of landform changes in Taiwan

The mitochondrial DNA cytochrome c oxidase subunit I sequences from 95 specimens of Semisulcospira libertina in Taiwan were identified as two major phylogroups, exhibiting a southern and northern distribution, north of Formosa Bank and south of Miaoli Plateau. The genetic distance between these two phylogroups was 12.20 %, and the distances within-phylogroups were 4.97 and 5.56 %. According to a molecular clock of 1.56 % per lineage per million years, the divergence time between these two major phylogroups was estimated at 4.94 million years ago (mya), with the two phylogroups forming at 3.64 and 3.75 mya, respectively. Moreover, the geological events have suggested that Taiwan Island emerged above sea level at 4–5 mya, and became its present shape at 2 mya. These results suggested that these two phylogroups might originate from two independent ancestral populations or divergent before colonizing Taiwan. Within South phylogroup, the initial colonization was hypothesized to be in Kaoping River (WT), followed by its northward. The high divergence between south- and north of WT River was influenced by the formation of the Kaoping foreland basins. Within North phylogroup, the colonization was from central sub-region through paleo-Miaoli Plateau to northern and northeastern sub-regions. This study showed that the landform changes might have shaped the genetic structure of S. libertina in concert. Apparently, two cryptic species or five different genetic stocks of S. libertina could be identified; these results are useful for the evaluation and conservation of S. libertina in Taiwan.     

Preclinical pharmacokinetics of MHAA4549A,a human monoclonal antibody to influenza A virus,and the prediction of its efficacious clinical dose for the treatment of patients hospitalized with influenza A

MHAA4549A is a human immunoglobulin G1 (IgG1) monoclonal antibody that binds to a highly conserved epitope on the stalk of influenza A hemagglutinin and blocks the hemagglutinin-mediated membrane fusion in the endosome, neutralizing all known human influenza A strains. Pharmacokinetics (PK) of MHAA4549A and its related antibodies were determined in DBA/2J and Balb-c mice at 5 mg/kg and in cynomolgus monkeys at 5 and 100 mg/kg as a single intravenous dose. Serum samples were analyzed for antibody concentrations using an ELISA and the PK was evaluated using WinNonlin software. Human PK profiles were projected based on the PK in monkeys using species-invariant time method. The human efficacious dose projection was based on in vivo nonclinical pharmacological active doses, exposure in mouse infection models and expected human PK. The PK profiles of MHAA4549A and its related antibody showed a linear bi-exponential disposition in mice and cynomolgus monkeys. In mice, clearance and half-life ranged from 5.77 to 9.98 mL/day/kg and 10.2 to 5.76 days, respectively. In cynomolgus monkeys, clearance and half-life ranged from 4.33 to 4.34 mL/day/kg and 11.3 to 11.9 days, respectively. The predicted clearance in humans was ～2.60 mL/day/kg. A single intravenous dose ranging from 15 to 45 mg/kg was predicted to achieve efficacious exposure in humans. In conclusion, the PK of MHAA4549A was as expected for a human IgG1 monoclonal antibody that lacks known endogenous host targets. The predicted clearance and projected efficacious doses in humans for MHAA4549A have been verified in a Phase 1 study and Phase 2a study, respectively.     

Increase of reactive oxygen species (ROS) in endothelial cells by shear flow and involvement of ROS in shear-induced c-fos expression

Intracellular reactive oxygen species (ROS) may participate in cellular responses to various stimuli including hemodynamic forces and act as signal transduction messengers. Human umbilical vein endothelial cells (ECs) were subjected to laminar shear flow with shear stress of 15, 25, or 40 dynes/cm² in a parallel plate flow chamber to demonstrate the potential role of ROS in shear-induced cellular response. The use of 2′,7′-dichlorofluorescin diacetate (DCFH-DA) to measure ROS levels in ECs indicated that shear flow for 15 minutes resulted in a 0.5- to 1.5-fold increase in intracellular ROS. The levels remained elevated under shear flow conditions for 2 hours when compared to unsheared controls. The shear-induced elevation of ROS was blocked by either antioxidant N-acetyl-cysteine (NAC) or catalase. An iron chelator, deferoxamine mesylate, also significantly reduced the ROS elevation. A similar inhibitory effect was seen with a hydroxyl radical (^·OH) scavenger, 1,3-dimethyl-2-thiourea (DMTU), suggesting that hydrogen peroxide (H₂O₂), ^·OH, and possibly other ROS molecules in ECs were modulated by shear flow. Concomitantly, a 1.3-fold increase of decomposition of exogenously added H₂O₂ was observed in extracts from ECs sheared for 60 minutes. This antioxidant activity, abolished by a catalase inhibitor (3-amino-1,2,4-triazole), was primarily due to the catalase. The effect of ROS on intracellular events was examined in c-fos gene expression which was previously shown to be shear inducible. Decreasing ROS levels by antioxidant (NAC or catalase) significantly reduced the induction of c-fos expression in sheared ECs. We demonstrate for the first time that shear force can modulate intracellular ROS levels and antioxidant activity in ECs. Furthermore, the ROS generation is involved in mediating shear-induced c-fos expression. Our study illustrates the importance of ROS in the response and adaptation of ECs to shear flow. J. Cell. Physiol. 175:156–162, 1998. © 1998 Wiley-Liss, Inc.     

miR-146a Enhances the Oncogenicity of Oral Carcinoma by Concomitant Targeting of the IRAK1, TRAF6 and NUMB Genes

MicroRNAs are short non-coding RNAs that regulate gene expression and are crucial to tumorigenesis. Oral squamous cell carcinoma (OSCC) is a prevalent malignancy worldwide. Up-regulation of miR-146 has been identified in OSCC tissues. However, the roles of miR-146 in carcinogenesis are controversial as it is suppressive in many other malignancies. The present study investigated the pathogenic implications of miR-146a in oral carcinogenesis. Microdissected OSCC exhibits higher levels of miR-146a expression than matched adjacent mucosal cells. The plasma miR-146a levels of patients are significantly higher than those of control subjects; these levels decrease drastically after tumor resection. miR-146a levels in tumors and in patients’ plasma can be used to classify OSCC and non-disease status (sensitivity: >0.72). Exogenous miR-146a expression is significantly increased in vitro oncogenic phenotypes as well as during xenograft tumorigenesis and OSCC metastasis. The plasma miR-146a levels of these mice parallel the xenograft tumor burdens of the mice. A miR-146a blocker abrogates the growth of xenograft tumors. miR-146a oncogenic activity is associated with down-regulation of IRAK1, TRAF6 and NUMB expression. Furthermore, miR-146a directly targets the 3′UTR of NUMB and a region within the NUMB coding sequence when suppressing NUMB expression. Exogenous NUMB expression attenuates OSCC oncogenicity. Double knockdown of IRAK1 and TRAF6, and of TRAF6 and NUMB, enhance the oncogenic phenotypes of OSCC cells. Oncogenic enhancement modulated by miR-146a expression is attenuated by exogenous IRAK1 or NUMB expression. This study shows that miR-146a expression contributes to oral carcinogenesis by targeting the IRAK1, TRAF6 and NUMB genes.     

Association of polymorphisms in the Chr18q11.2 locus with tuberculosis in Chinese population

A GWAS study has reported that two single nucleotide polymorphisms (SNPs) were associated with predisposition to tuberculosis (TB) in African populations. These two loci represented the long-waited GWAS hits for TB susceptibility. To determine whether these two SNPs are associated with TB in Chinese population, we attempted an replication in a cohort of over one thousand Chinese TB patients and 1,280 healthy controls using melting temperature shift allele-specific genotyping analysis. We found that only SNP rs4331426 was significantly associated with TB in Chinese population (p = 0.011). However, the effect was opposite. The G allele of the SNP in Chinese population is a protective allele (OR = 0.62, 95 % CI 0.44–0.87), while it was the risk allele for African population (OR = 1.19, 95 % CI 1.12–1.26). No significance was found for SNP rs2335704. The results provided an independent support for a role in susceptibility to TB for SNP rs4331426. However, it also indicated that direct predisposition element to TB and the association effects may vary across ethnic groups.