A role for Rho kinase in vascular contraction evoked by sodium fluoride

Agonist and depolarization-induced vascular smooth muscle contractions involve the activation of Rho-kinase pathway. However, there are no reports addressing the question whether this pathway is involved in NaF-induced vascular contractions. We hypothesized that Rho-kinase plays a role in vascular contraction evoked by sodium fluoride in rat aortae. In both physiological salt solution and calcium-free solution with 2 mM EGTA, cumulative addition of NaF increased vascular tension in concentration-dependent manners. Effects of Rho-kinase inhibitor (Y27632) on phosphorylation of myosin light chain (MLC20) and myosin targeting subunit (MYPT1(Thr696)) of myosin light chain phosphatase as well as NaF-induced contractions were determined using isolated tissue and the Western blot experiments. Y27632 inhibited NaF-induced contractions in a concentration-dependent manner. NaF increased phosphorylation of MLC20 and MYPT1(Thr696), which were also inhibited by Y27632. However, MLCK inhibitor (ML-7) or PKC inhibitor (Ro31-8220) did not inhibit the NaF-induced contraction. These results indicate that activation of Rho-kinase and the subsequent phosphorylation of MYPT1(Thr696) play important roles in NaF-induced contraction of rat aortae.     

Two Rubisco activase isoforms may play different roles in photosynthetic heat acclimation in the rice plant

Studies on some plant species have shown that increasing the growth temperature gradually or pretreating with high temperature can lead to obvious photosynthetic acclimation to high temperature. To test whether this acclimation arises from heat adaptation of ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) activation mediated by Rubisco activase (RCA), gene expression of RCA large isoform (RCA_L) and RCA small isoform (RCA_S) in rice was determined using a 4‐day heat stress treatment [40/30°C (day/night)] followed by a 3‐day recovery under control conditions [30/22°C (day/night)]. The heat stress significantly induced the expression of RCA_L as determined by both mRNA and protein levels. Correlative analysis indicated that RCA_S protein content was extremely significantly related to Rubisco initial activity and net photosynthetic rate (Pn) under both heat stress and normal conditions. Immunoblot analysis of the Rubisco–RCA complex revealed that the ratio of RCA_L to Rubisco increased markedly in heat‐acclimated rice leaves. Furthermore, transgenic rice plants expressing enhanced amounts of RCA_L exhibited higher thermotolerance in Pn and Rubisco initial activity and grew better at high temperature than wild‐type (WT) plants and transgenic rice plants expressing enhanced amounts of RCA_S. Under normal conditions, the transgenic rice plants expressing enhanced amounts of RCA_S showed higher Pn and produced more biomass than transgenic rice plants expressing enhanced amounts of RCA_L and wild‐type plants. Together, these suggest that the heat‐induced RCA_L may play an important role in photosynthetic acclimation to moderate heat stress in vivo, while RCA_S plays a major role in maintaining Rubisco initial activity under normal conditions.     

Analysis of pSC138, the multidrug resistance plasmid of Salmonella enterica serotype Choleraesuis SC-B67

Salmonella enterica serotype Choleraesuis (S. Choleraesuis) usually causes systemic infections in man and needs antimicrobial treatment. Multidrug resistance (MDR) in S. Choleraesuis is thus a great concern in the treatment of systemic non-typhoid salmonellosis. A large plasmid, pSC138, was identified in 2002 from a S. Choleraesuis strain SC-B67 that was resistant to all antimicrobial agents commonly used to treat salmonellosis, including ciprofloxacin and ceftriaxone. Complete DNA sequence of the plasmid had been determined previously (Chiu et al., 2005). In the present study, the sequence of pSC138 was reannotated in detail and compared with several newly sequenced plasmids. Some transposable elements and drug resistance genes were further delineated. Plasmid pSC138 was 138,742 bp in length and consisted of 177 open reading frames (ORFs). While 134 of the ORFs displayed significant identity levels to other plasmid and prokaryotic sequences, the remaining 43 ORFs have not been previously reported. Mobile elements, including two integrons, seven insertion sequences and eight transposons, and a truncated prophage together encompass at least 66,781 bp (48.1%) of the plasmid genome. The sequence of pSC138 consists of three major regions: a large composite transposable region Tn6088 with a Tn21-like backbone inserted by a variety of integrons or transposable elements; a transfer/maintenance region that contains a conserved ISEcp1-mediated transposon-like element Tn6092, carrying an AmpC gene, bla(CMY-2), that confers the ceftriaxone resistance; and a Rep_3 type of replication region. Another seven bacteremic strains of S. Choleraesuis that expressed the same MDR phenotype were identified during 2003-2008. The same Rep_3 type replicase and the bla(CMY-2)-containing, ISEcp1-mediated transposon-like element were found in the MDR isolates, suggesting a successful preservation and dissemination of the MDR plasmid. Comparison of pSC138 with other recently published plasmids revealed a high identity level between partial sequences of pSC138 and plasmids of the same or different incompatibility groups. The large MDR region found in pSC138 may provide a niche for the future evolution of the plasmid by acquisition of relevant resistance genes through the panoply of mobile elements and illegitimate recombination events.     

Release of erythrocytes from the spleen during exercise and splenic constriction by adrenaline infusion in the rainbow trout

Erythrocyte-supplying function of the spleen was examined in the rainbow troutSalmo gairdneri under exercise. The spleen showed remarkable reduction, about 70% in weight and about 85% in hemoglobin content, after forced exercise of 15 min. The amount of erythrocytes released from the spleen was 2.33 ml/kg body, and this amount corresponds to about 20% of the total volume of circulating erythrocytes in resting condition. No damage was observed at the spleen, splenic artery and splenic vein after the exercise. Examination of the vascular system by a corrosion casting method showed that no place other than the venous circulation exists for the erythrocytes released from the contracted spleen. The spleen was strongly constricted by infusion of adrenaline into the organ. These facts imply that the fish spleen supplies stored hemoglobin into the circulating blood in response to an increased demand of oxygen during exercise, under the control of the sympathetic nervous system.     

A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.     

Modification of alternative splicing in bovine somatic cell nuclear transfer embryos using engineered CRISPR-Cas13d

Animal cloning can be achieved by somatic cell nuclear transfer(SCNT), but the resulting live birth rate is relatively low. We previously improved the efficiency of bovine SCNT by exogenous melatonin treatment or by overexpression of lysine-specific demethylase 4D(KDM4D) and 4E(KDM4E). In this study, we revealed abundant alternative splicing(AS) transitions during fertilization and embryonic genome activation, and demonstrated abnormal AS in bovine SCNT embryos compared with in vitro fertilized ...