Complementation analysis at the ery-M1 locus in Chlamydomonas reinhardi

Summary Previous studies with haploid erythromycin-resistant mutants mapping to the Mendelian locus ery-M1 in Chlamydomonas reinhardi have revealed the presence of an altered chloroplast ribosomal protein (LC6) (Mets and Bogorad, 1971, 1972; Davidson et al., 1974). Vegetative diploids of C. reinhardi heterozygous at the ery-M1 locus have now been constructed. Chloroplast ribosomes from such diploids contain 60–70% wild-type form of protein LC6 and 30–40% altered form of LC6. Growth assays show that these diploids are partially resistant to erythromycin. Whether the diploids are grown in the presence or absence of erythromycin, the same ratio of wild-type: altered form of LC6 in chloroplast ribosomes is observed. Therefore, resistant chloroplast ribosomes must be able to carry out protein synthesis even when many of the sensitive chloroplast ribosomes are blocked by erythromycin.The presence of both the altered and wild-type forms of LC6 in diploids heterozygous at the ery-M1 locus is further evidence that a nuclear gene codes directly for a chloroplast ribosomal protein.An abstract of this work appeared in Genetics 80, S40 (1975)     

Sphingosine-1-Phosphate Induces the Migration of Thyroid Follicular Carcinoma Cells through the MicroRNA-17/PTK6/ERK1/2 Pathway

Sphingosine-1-phosphate (S1P) is a bioactive lipid known to play a role in tumorigenesis and cancer progression. However, the molecular mechanisms of S1P regulated migration of papillary thyroid cancer cells are still unknown. In this study, we showed that S1P induced PTK6 mRNA and protein expression in two thyroid follicular cancer cell lines (ML-1 and FTC-133). Further studies demonstrated that induced PTK6 and its downstream signal component (ERK1/2) are involved in S1P-induced migration. Upon investigating the mechanisms behind this event, we found that miR-17 inhibited the expression of PTK6 through direct binding to its 3’-UTR. Through overexpression and knockdown studies, we found that miR-17 can significantly inhibit S1P-induced migration in thyroid follicular cancer cells. Interestingly, overexpression or knockdown of PTK6 or ERK1/2 effectively removed the inhibition of S1P-induced migration by miR-17. Furthermore, we showed that S1P decreased miR-17 expression levels. Meanwhile, in papillary thyroid cancers, miR-17 is downregulated and negatively associated with clinical staging, whereas PTK6 is upregulated and positively associated with clinical stages. Collectively, our work defines a novel signaling pathway implicated in the control of thyroid cancer migration.     

Cot/tpl2-MKK1/2-Erk1/2 controls mTORC1-mediated mRNA translation in Toll-like receptor-activated macrophages

Cot/tpl2 is the only MAP3K that activates MKK1/2-Erk1/2 in Toll-like receptor–activated macrophages. Here we show that Cot/tpl2 regulates RSK, S6 ribosomal protein, and 4E-BP phosphorylation after stimulation of bone marrow–derived macrophages with lipopolysaccharide (LPS), poly I:C, or zymosan. The dissociation of the 4E-BP–eIF4E complex, a key event in the cap-dependent mRNA translation initiation, is dramatically reduced in LPS-stimulated Cot/tpl2-knockout (KO) macrophages versus LPS-stimulated wild-type (Wt) macrophages. Accordingly, after LPS activation, increased cap-dependent translation is observed in Wt macrophages but not in Cot/tpl2 KO macrophages. In agreement with these data, Cot/tpl2 increases the polysomal recruitment of the 5´ TOP eEF1α and eEF2 mRNAs, as well as of inflammatory mediator gene–encoding mRNAs, such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and KC in LPS-stimulated macrophages. In addition, Cot/tpl2 deficiency also reduces total TNFα, IL-6, and KC mRNA expression in LPS-stimulated macrophages, which is concomitant with a decrease in their mRNA half-lives. Macrophages require rapid fine control of translation to provide an accurate and not self-damaging response to host infection, and our data show that Cot/tpl2 controls inflammatory mediator gene–encoding mRNA translation in Toll-like receptor–activated macrophages.     

MicroRNA-370 carried by M2 macrophage-derived exosomes alleviates asthma progression through inhibiting the FGF1/MAPK/STAT1 axis

Emerging evidence has suggested the functions of exosomes in allergic diseases including asthma. By using a mouse model with asthma induced by ovalbumin (OVA), we explored the roles of M2 macrophage-derived exosomes (M2Φ-Exos) in asthma progression. M2Φ-Exos significantly alleviated OVA-induced fibrosis and inflammatory responses in mouse lung tissues, as well as inhibited abnormal proliferation, invasion, and fibrosis-related protein production in platelet derived growth factor (PDGF-BB) treated primary mouse airway smooth muscle cells (ASMCs). The OVA administration in mice or the PDGF-BB treatment in ASMCs reduced the expression of miR-370, which was detected in M2Φ-Exos by miRNA sequencing. However, treating the mice or ASMCs with M2Φ-Exos reversed the inhibitory effect of OVA or PDGF-BB on miR-370 expression. We identified that the target of miR-370 was fibroblast growth factor 1 (FGF1). Downregulation of miR-370 by Lv-miR-370 inhibitor or overexpression of FGF1 by Lv-FGF1 blocked the protective roles of M2Φ-Exos in asthma-like mouse and cell models. M2Φ-Exos were found to inactivate the MAPK signaling pathway, which was recovered by miR-370 inhibition or FGF1 overexpression. Collectively, we conclude that M2Φ-Exos carry miR-370 to alleviate asthma progression through downregulating FGF1 expression and the MAPK/STAT1 signaling pathway. Our study may offer a novel insight into asthma treatment.     

Shogaol potentiates sevoflurane mediated neuroprotection against ischemia/reperfusion-induced brain injury via regulating apoptotic proteins and PI3K/Akt/mTOR/s6K signalling and HIF-1α/HO-1 expression

The current research was intended to evaluate the impact of 6-shogaol in rodent model of ischemic-reperfusion induced- brain injury and also assessed 6-shogaol enhanced sevoflurane''s neuroprotective effects. Ischemic-Reperfusion (I/R) injury was induced by middle cerebral artery occlusion (MCAO) method in Sprague-Dawley rats. A separate group of animal was exposed to sevoflurane (2.5%) post-conditioning for 1 h immediately after reperfusion. The 6-shogaol (25 mg or 50 mg/kg body weight) was orally administered to treatment group rats for 14 days and then subjected to I/R. The 6-shogaol treatment along with/without sevoflurane post-conditioning reduced the number of apoptotic cell counts, brain edema and cerebral infarct volume. The western blotting analysis revealed a significant stimulation of the PI3K/Akt/mTOR signal pathway. RT-PCR and western blotting studies revealed improved expressions of HIF-1α and HO-1 at both gene level and protein levels. I/R induced neurological deficits were also alleviated on sevoflurane post-conditioning with/without 6-shogaol treatment. The present findings revealed that pre-treatment with 6-shogoal enhanced the neuroprotective properties of sevoflurane post-conditioning, illustrated the efficacy of the compound against I/R injury.     

白细胞介素-6通过AMPK/SIRT1通路下调eNOS Thr495/497

该文探讨了白细胞介素-6(interleukin-6,IL-6)对牛主动脉内皮细胞(bovine aortic endo-thelial cells,BAECs)的内皮型一氧化氮合成酶(endothelial nitric oxide synthase,eNOS)的影响及其可能的发生机制.在原代BAECs细胞培养基础上...     

Fish oil supplementation reduces excretion of 2,3-dinor-6-oxo-PGF1α and the 11-dehydrothromboxane B2/2,3-dinor-6-oxo-PGF1α excretion ratio in adult men

Dietary supplementation with a fish oil concentrate (FOC) reduced the endogenous synthesis of prostacyclin (PGI₂), as measured by the excretion of its major urinary catabolite, 2,3-dinor-6-oxo-PGF_1α (PGI₂-M). Thirty-four healthy men (24–57 years old) were given controlled diets and supplements that provided 40% of the energy from fat and a minimum of 22 mg/d of α-tocopherol for two consecutive experimental periods of 10 weeks each. During the experimental periods, the men received capsules containing 15 g/d of a placebo oil (PO) (period 1) or 15 g/d of the FOC (period 2). In addition to the PO or FOC, capsules contained 1 mg of α-tocopherol per g of fat as an antioxidant. The average daily excretion of PGI₂-M during the last week of FOC supplementation (period 2) was 22% less (P = 0.0001) than at the end of the first period. These results are at variance with those reported in comparable human studies conducted by other investigators during the middle and late 1980s. A 20% reduction (P = 0.003) in the 11-dehydrothromboxane B₂ to 2,3-dinor-6-oxo-PGF_1α excretion ratio at the end of period 2 in this study demonstrates that a shift of the n-6 to n-3 polyunsaturated fatty acid ratio from 12.5 to 2.3 brings about a substantial modulation of the eicosanoid system.     

Characterization of the Replication Initiator Orc1/Cdc6 from the Archaeon Picrophilus torridus

Eukaryotic DNA replication is preceded by the assembly of prereplication complexes (pre-RCs) at or very near origins in G₁ phase, which licenses origin firing in S phase. The archaeal DNA replication machinery broadly resembles the eukaryal apparatus, though simpler in form. The eukaryotic replication initiator origin recognition complex (ORC), which serially recruits Cdc6 and other pre-RC proteins, comprises six components, Orc1-6. In archaea, a single gene encodes a protein similar to both the eukaryotic Cdc6 and the Orc1 subunit of the eukaryotic ORC, with most archaea possessing one to three Orc1/Cdc6 orthologs. Genome sequence analysis of the extreme acidophile Picrophilus torridus revealed a single Orc1/Cdc6 (PtOrc1/Cdc6). Biochemical analyses show MBP-tagged PtOrc1/Cdc6 to preferentially bind ORB (origin recognition box) sequences. The protein hydrolyzes ATP in a DNA-independent manner, though DNA inhibits MBP-PtOrc1/Cdc6-mediated ATP hydrolysis. PtOrc1/Cdc6 exists in stable complex with PCNA in Picrophilus extracts, and MBP-PtOrc1/Cdc6 interacts directly with PCNA through a PIP box near its C terminus. Furthermore, PCNA stimulates MBP-PtOrc1/Cdc6-mediated ATP hydrolysis in a DNA-dependent manner. This is the first study reporting a direct interaction between Orc1/Cdc6 and PCNA in archaea. The bacterial initiator DnaA is converted from an active to an inactive form by ATP hydrolysis, a process greatly facilitated by the bacterial ortholog of PCNA, the β subunit of Pol III. The stimulation of PtOrc1/Cdc6-mediated ATP hydrolysis by PCNA and the conservation of PCNA-interacting protein motifs in several archaeal PCNAs suggest the possibility of a similar mechanism of regulation existing in archaea. This mechanism may involve other yet to be identified archaeal proteins.     

AtPTR4 and AtPTR6 are differentially expressed,tonoplast-localized members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family

Members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family in plants transport a variety of substrates like nitrate, di- and tripepetides, auxin and carboxylates. We isolated two members of this family from Arabidopsis, AtPTR4 and AtPTR6, which are highly homologous to the characterized di- and tripeptide transporters AtPTR1, AtPTR2 and AtPTR5. All known substrates of members of the PTR/NRT1 family were tested using heterologous expression in Saccharomyces cerevisiae mutants and oocytes of Xenopus laevis, but none could be identified as substrate of AtPTR4 or AtPTR6. AtPTR4 and AtPTR6 show distinct expression patterns, while AtPTR4 is expressed in the vasculature of the plants, AtPTR6 is highly expressed in pollen and during senescence. Phylogenetic analyses revealed that AtPTR2, 4 and 6 belong to one clade of subgoup II, whereas AtPTR1 and 5 are found in a second clade. Like AtPTR2, AtPTR4-GFP and AtPTR6-GFP fusion proteins are localized at the tonoplast. Vacuolar localization was corroborated by co-localization of AtPTR2-YFP with the tonoplast marker protein GFP-AtTIP2;1 and AtTIP1;1-GFP. This indicates that the two clades reflect different intracellular localization at the tonoplast (AtPTR2, 4, 6) and plasma membrane (AtPTR1, 5), respectively.     

Shogaol potentiates sevoflurane mediated neuroprotection against ischemia/reperfusion-induced brain injury via regulating apoptotic proteins and PI3K/Akt/mTOR/s6K signalling and HIF-1α/HO-1 expression

The current research was intended to evaluate the impact of 6-shogaol in rodent model of ischemic-reperfusion induced- brain injury and also assessed 6-shogaol enhanced sevoflurane's neuroprotective effects. Ischemic-Reperfusion (I/R) injury was induced by middle cerebral artery occlusion (MCAO) method in Sprague-Dawley rats. A separate group of animal was exposed to sevoflurane (2.5%) post-conditioning for 1 h immediately after reperfusion. The 6-shogaol (25 mg or 50 mg/kg body weight) was orally administered to treatment group rats for 14 days and then subjected to I/R. The 6-shogaol treatment along with/without sevoflurane post-conditioning reduced the number of apoptotic cell counts, brain edema and cerebral infarct volume. The western blotting analysis revealed a significant stimulation of the PI3K/Akt/mTOR signal pathway. RT-PCR and western blotting studies revealed improved expressions of HIF-1α and HO-1 at both gene level and protein levels. I/R induced neurological deficits were also alleviated on sevoflurane post-conditioning with/without 6-shogaol treatment. The present findings revealed that pre-treatment with 6-shogoal enhanced the neuroprotective properties of sevoflurane post-conditioning, illustrated the efficacy of the compound against I/R injury.     

低氧经mTOR/4E-BP1和FoxO1/Atrogin-1通路影响骨骼肌蛋白质积累

低氧暴露对骨骼肌蛋白质合成/分解的影响受到广泛关注,但该过程中相关调控通路的研究仍十分有限。本研究拟通过蛋白质相对积累量来研究合成和分解通路的变化。将骨骼肌细胞置于低氧环境中培养,分别在0 h、6 h、12 h和24 h收集细胞,并进行检测。免疫荧光观察肌球蛋白(myosin),翻译表面感应检测蛋白质合成,Western印迹法测试蛋白质合成相关基因(ERK1/2、p-ERK1/2、mTOR、p-mTOR、4E-BP1、p-4E-BP1)、蛋白质分解相关基因(泛素、FoxO1、p-FoxO1、MuRF1和Atrogin-1)表达量。结果发现,随着低氧干预时间延长,肌纤维直径和骨骼肌细胞中蛋白质相对积累量随时间逐渐减小(P<0.01)。与0 h相比,6 h p-4E-BP1/4E-BP1和Atrogin-1的表达显著上调(P<0.05),p-mTOR表达显著高于0 h(P<0.01);6 h和24 h p-mTOR/mTOR的比值显著大于0 h(P<0.05),而p-FoxO1/FoxO1的比值随时间逐渐减小(P<0.01)。上述结果表明,低氧干预能够使骨骼肌细胞直径减少、骨骼肌细胞蛋白质积累减少,并且低氧打破骨骼肌细胞蛋白质合成和分解的平衡,可能是通过调节mTOR/4E-BP1通路活性和FoxO1/Atrogin-1通路的活性实现的。     

Protein kinase C beta relieves autism-like behavior in EN2 knockout mice via upregulation of the FTO/PGC-1α/UCP1 axis

Increasing evidence suggests that disruption of neuron activity contributes to the autistic phenotype. Thus, we aimed in this study to explore the role of protein kinase C beta (PKCβ) in the regulation of neuron activity in an autism model. The expression of PKCβ in the microarray data of autism animal models was obtained from the Gene Expression Omnibus database. Then, mice with autism-like behavior were prepared in EN2 knockout (^−/−) mice. The interaction between PKCβ on fat mass and obesity-associated protein (FTO) as well as between PGC-1α and uncoupling protein 1 (UCP1) were characterized. The effect of FTO on the N⁶-methyladenosine (m6A) modification level of proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) was assayed. Following transfection of overexpressed PKCβ and/or silenced UCP1, effects of PKCβ and UCP1 in autism-like behaviors in EN2^−/− mice were analyzed. Results showed that PKCβ was downregulated in EN2^−/− mouse brain tissues or neurons. PKCβ promoted the expression and stability of FTO, which downregulated the m6A modification level of PGC-1α to promote its expression. Moreover, PGC-1α positively targeted the expression of UCP1. PKCβ knockdown enhanced sociability and spatial exploration ability, and reduced neuron apoptosis in EN2^−/− mouse models of autism, which was reversed by UCP1 overexpression. Collectively, PKCβ overexpression leads to activation of the FTO/m6A/PGC-1α/UCP1 axis, thus inhibiting neuron apoptosis and providing neuroprotection in mice with autism-like behavior.     

Generation of a 'humanized' hCYP1A1_1A2_Cyp1a1/1a2(-/-)_Ahrd mouse line harboring the poor-affinity aryl hydrocarbon receptor

Herein, we describe generation of the hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahr^d mouse line, which carries human functional CYP1A1 and CYP1A2 genes in the absence of mouse Cyp1a1 and Cyp1a2 genes, in a (>99.8%) background of the C57BL/6J genome and harboring the poor-affinity aryl hydrocarbon receptor (AHR) from the DBA/2J mouse. We have characterized this line by comparing it to our previously created hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahr^b1 line—which carries the same but has the high-affinity AHR of the C57BL/6J mouse. By quantifying CYP1A1 and CYP1A2 mRNA in liver, lung and kidney of dioxin-treated mice, we show that dose-response curves in hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahr^d mice are shifted to the right of those in hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahr^b1 mice—similar to, but not as robust as, dose-response curves in DBA/2J versus C57BL/6J mice. This new mouse line is perhaps more relevant than the former to human risk assessment vis-à-vis human CYP1A1 and CYP1A2 substrates, because poor-affinity rather than high-affinity AHR occurs in the vast majority of the human population.     

Differential levels of glutamate dehydrogenase 1 (GLUD1) in Balb/c and C57BL/6 mice and the effects of overexpression of the Glud1 gene on glutamate release in striatum

We have previously shown that overexpression of the Glud1 (glutamate dehydrogenase 1) gene in neurons of C57BL/6 mice results in increased depolarization-induced glutamate release that eventually leads to selective neuronal injury and cell loss by 12 months of age. However, it is known that isogenic lines of Tg (transgenic) mice produced through back-crossing with one strain may differ in their phenotypic characteristics from those produced using another inbred mouse strain. Therefore, we decided to introduce the Glud1 transgene into the Balb/c strain that has endogenously lower levels of GLUD1 (glutamate dehydrogenase 1) enzyme activity in the brain as compared with C57BL/6. Using an enzyme-based MEA (microelectrode array) that is selective for measuring glutamate in vivo, we measured depolarization-induced glutamate release. Within a discrete layer of the striatum, glutamate release was significantly increased in Balb/c Tg mice compared with wt (wild-type) littermates. Furthermore, Balb/c mice released approx. 50–60% of the amount of glutamate compared with C57BL/6 mice. This is similar to the lower levels of endogenous GLUD1 protein in Balb/c compared with C57BL/6 mice. The development of these Glud1-overexpressing mice may allow for the exploration of key molecular events produced by chronic exposure of neurons to moderate, transient increases in glutamate release, a process hypothesized to occur in neurodegenerative disorders.     

Synthesis of the four isomers of 5-hydroxy-PGI1

We wish to report here the syntheses of (5$\text{S}$, 6$\text{R}$)-5-hydroxy-, (5$\text{R}$, 6$\text{R}$)-5-hydroxy-, (5$\text{R}$, 6$\text{S}$)-5-hydroxy-, and (5$\text{S}$, 6$\text{S}$)-5-hydroxy-PGI₁ and their methyl ester derivatives. Treatment of (5$\text{R}$, 6$\text{S}$)-5-epoxy- and (5$\text{S}$, 6$\text{R}$)-5-epoxy-PFG_1α methyl esters with acid washed silica gel afforded (5$\text{R}$, 6$\text{R}$)-5-hydroxy- and (5$\text{S}$, 6$\text{S}$)-5-hydroxy-PGI₁ methyl esters; correspondingly, silica promoted cyclization of (5$\text{S}$, 6$\text{S}$)-epoxy- and (5$\text{S}$, 6$\text{R}$)-5-epoxy- PGF₁ methyl esters yielded (5$\text{S}$, 6$\text{R}$)-5-hydroxy- and (5$\text{R}$, 6$\text{S}$)-5-hydroxy- PGI₁ methyl esters. Alternatively, the 5-hydroxyl group was introduced into the PGI₁ skeleton $\text{via}$ reaction of the 5-mercuric halides with sodium borohydride in the presence of oxygen. Stereochemical assignments were based on their mode of synthesis and ¹H nmr shift differences.     

Chronological expression of Wnt target genes Ccnd1, Myc, Cdkn1a, Tfrc, Plf1 and Ramp3

The canonical Wnt pathway regulates several biological processes including development, cell growth and proliferation via consecutive gene regulation. A high number of target genes of the Wnt pathway has been identified, but the chronological order of target gene expression is still elusive. This order is supposed to be crucial for the controlled course of events downstream of the activated Wnt pathway. Here we present the expression chronologies of the target genes Ccnd1 (encoding for cyclin D1), Myc (c-Myc), Cdkn1a (p21^CIP1/WAF1), Tfrc (Transferrin receptor 1), Plf1 (Proliferin-1) and Ramp3 (Receptor activity-modifying protein 3) in C57MG cells after stimulation with Wnt-3a. We discriminated between immediate (below 1 h), early (between 1 and 6 h), intermediate (between 6 and 12 h) and late (after 12 h) targets. According to this classification Myc and Tfrc belong to the immediate target genes; Ccnd1, Plf1 and Ramp3 are early target genes and Cdkn1a is an intermediate target gene.