A unique AtSar1D-AtRabD2a nexus modulates autophagosome biogenesis in Arabidopsis thaliana

In eukaryotes, secretory proteins traffic from the endoplasmic reticulum (ER) to the Golgi apparatus via coat protein complex II (COPII) vesicles. Intriguingly, during nutrient starvation, the COPII machinery acts constructively as a membrane source for autophagosomes during autophagy to maintain cellular homeostasis by recycling intermediate metabolites. In higher plants, essential roles of autophagy have been implicated in plant development and stress responses. Nonetheless, the membrane sources of autophagosomes, especially the participation of the COPII machinery in the autophagic pathway and autophagosome biogenesis, remains elusive in plants. Here, we provided evidence in support of a novel role of a specific Sar1 homolog AtSar1d in plant autophagy in concert with a unique Rab1/Ypt1 homolog AtRabD2a. First, proteomic analysis of the plant ATG (autophagy-related gene) interactome uncovered the mechanistic connections between ATG machinery and specific COPII components including AtSar1d and Sec23s, while a dominant negative mutant of AtSar1d exhibited distinct inhibition on YFP-ATG8 vacuolar degradation upon autophagic induction. Second, a transfer DNA insertion mutant of AtSar1d displayed starvation-related phenotypes. Third, AtSar1d regulated autophagosome progression through specific recognition of ATG8e by a noncanonical motif. Fourth, we demonstrated that a plant-unique Rab1/Ypt1 homolog AtRabD2a coordinates with AtSar1d to function as the molecular switch in mediating the COPII functions in the autophagy pathway. AtRabD2a appears to be essential for bridging the specific AtSar1d-positive COPII vesicles to the autophagy initiation complex and therefore contributes to autophagosome formation in plants. Taken together, we identified a plant-specific nexus of AtSar1d-AtRabD2a in regulating autophagosome biogenesis.

Autophagy is a conserved catabolic process characterized by the de novo generation of a double-membrane structure called an autophagosome with a fundamental function in the bulk turnover of cytoplasmic components, including proteins, RNAs, and organelles. Genetic studies in yeast have elucidated the molecular machinery of autophagy, whereby 42 autophagy-related (ATG) genes have been identified (1–3). These ATG genes are highly conserved among eukaryotes but often have multiple isoforms in other higher organisms, in particular in sessile plants. Albeit increasing understanding on the molecular function of Atg proteins in acting hierarchically on the phagophore assembly site (PAS) to produce autophagosomes, the origin of the autophagosomal membrane remains unclear in higher eukaryotes. Furthermore, the dedication of other membranes and machineries in the autophagy pathway remains under investigation.Plant autophagy is known to play important roles in the sessile lifestyle of plants, participating in seed germination, seedling establishment, plant development, hormone responses, lipid metabolism, and reproductive development (4). Plant autophagy research is advancing with findings not only on the counterparts of the yeast/mammalian Atg proteins but also dealing with some plant-unique factors functioning in different steps of autophagosome biogenesis, thereby uncovering novel mechanisms that might or might not be conserved in nonplant species (5). More interestingly, higher plants possess multiple protein isoforms of ATG machinery, whose functional heterogeneity in the autophagy pathway has only recently been unveiled (6).The coat protein complex II (COPII) machinery consists of five cytosolic components: the small GTPase Sar1, the inner coat protein dimer Sec23-Sec24, and the outer coat proteins Sec13-Sec31. These proteins are essential for COPII-coated vesicle formation, which buds from specialized regions of the ER, namely ER exit sites (ERESs) (7). Under nutrient-rich conditions, COPII vesicles mediate anterograde ER to Golgi transport. However, increasing evidence from yeast and mammals suggests that the COPII machinery or even COPII vesicles themselves may contribute to autophagosome formation when cells are starved for nutrients (8–16). Gene duplication events have occurred substantially in sessile plants during evolution, and the importance of distinct paralogs in environmental stress adaptation during plant development has been implied (17). Arabidopsis encodes multiple COPII paralogs in its genome, including five Sar1s, seven Sec23s, three Sec24s, two Sec13s, and two Sec31s (17). Increasing numbers of studies have pinpointed the functional diversity and importance of distinct COPII paralogs in ER protein export (18–23). Nonetheless, the mechanism by which COPII vesicles are redirected to the autophagy pathway upon nutrient starvation, and their roles in autophagosome biogenesis, remains unclear. Furthermore, the participation of specific COPII paralogs in autophagy regulation remains unknown in plants.Here, we report on a role of a specific Sar1 homolog, AtSar1d, that modulates plant autophagosome biogenesis in concert with AtRabD2a. Large-scale proteomic analysis of the ATG interactome has revealed possible mechanistic connections between the ATG machinery and specific COPII components in plants. Cellular and biochemical analyses have shown that the dominant negative (DN) mutant of AtSar1d (AtSar1dDN) specifically perturbs YFP-ATG8 vacuolar degradation upon autophagic induction. Consistently, a transfer DNA (T-DNA) insertion mutant of AtSar1d exhibited starvation-related phenotypes. Notably, AtSar1d regulates autophagosome progression through specific recognition of ATG8e by a previously uncharacterized noncanonical motif. We further identify a plant-unique Rab1/Ypt1 homolog AtRabD2a that colocalizes with AtSar1d and ATG8 upon starvation by transient expression in Arabidopsis protoplasts. A DN mutant of AtRabD2a (AtRabD2aNI) perturbs autophagy flux, while AtRabD2a is indispensable for bridging the AtSar1d-positive COPII vesicles with the ATG1 complex, thus contributing to autophagosome biogenesis in plants. Our study therefore unequivocally demonstrates that the plant-specific COPII machinery regulates autophagosome biogenesis and sheds light on the evolutionary importance of gene duplication events in the plant autophagy pathway.     

Cell growth defect factor 1 is crucial for the plastid import of NADPH:protochlorophyllide oxidoreductase A in Arabidopsis thaliana

Tetrapyrroles such as chlorophyll, heme, and bacteriochlorophyll play fundamental roles in the energy absorption and transduction of all photosynthetic organisms. They are synthesized via a complex pathway taking place in chloroplasts. Chlorophyll biosynthesis in angiosperms involves 16 steps of which only one is light-requiring and driven by the NADPH:protochlorophyllide oxidoreductase (POR). Three POR isoforms have been identified in Arabidopsis thaliana—designated PORA, PORB, and PORC—that are differentially expressed in etiolated, light-exposed, and light-adapted plants. All three isoforms are encoded by nuclear genes, are synthesized as larger precursors in the cytosol (pPORs), and are imported posttranslationally into the plastid compartment. Import of the precursor to the dark-specific isoform PORA (pPORA) is protochlorophyllide (Pchlide)-dependent and due to the operation of a unique translocon complex dubbed PTC (Pchlide-dependent translocon complex) in the plastid envelope. Here, we identified a ∼30-kDa protein that participates in pPORA import. The ∼30-kDa protein is identical to the previously identified CELL GROWTH DEFECT FACTOR 1 (CDF1) in Arabidopsis that is conserved in higher plants and Synechocystis. CDF1 operates in pPORA import and stabilization and hereby acts as a chaperone for PORA protein translocation. CDF1 permits tight interactions between Pchlide synthesized in the plastid envelope and the importing PORA polypeptide chain such that no photoexcitative damage occurs through the generation of singlet oxygen operating as a cell death inducer. Together, our results identify an ancient mechanism dating back to the endosymbiotic origin of chloroplasts as a key element of Pchlide-dependent pPORA import.Higher plants make use of two closely related forms of chlorophyll (Chl) for light harvesting and energy transduction, Chl a and Chl b. Both are synthesized from 5-aminolevulinic acid (5-ALA) (see refs. 1, 2 for review). Chl biosynthesis is controlled at several different levels in angiosperms, including (i) feedback inhibition by heme of the early steps leading to 5-ALA (3, 4), (ii) repression of Chl precursor accumulation at the level of protochlorophyllide (Pchlide) by the FLUORESCENT protein (5), and (iii) light activation of Pchlide to chlorophyllide (Chlide) conversion by the NADPH:Pchlide oxidoreductase (POR) (6).In Arabidopsis, three differentially expressed POR isoenzymes exist, named PORA, PORB, and PORC (summarized in ref. 6). Mutagenesis and homology modeling studies have provided important insights into the catalytic mechanism of POR (7–11). All three POR isoforms are nuclear gene products that must be imported posttranslationally into the plastids (12, 13). Components have been identified in the plastid envelope mediating this import step (14–16). Interestingly, it could be demonstrated that PORA and PORB use different pathways for import (17). Whereas transport of the pPORA is Pchlide-dependent and occurs through a unique import site, uptake of the pPORB occurs through the general import site (14–18). The pPORA and pPORB contain structurally distinct NH₂-terminal transit peptides that direct the precursors to the different protein import machineries in the chloroplast envelope (14–18).The question of how pPORA translocation is mechanistically coupled to Pchlide synthesis in the plastid envelope has not been answered yet. Here, we identified a protein interacting with pPORA during its Pchlide-dependent import and show that it is related to a previously identified cell death factor, named CELL GROWTH DEFECT FACTOR 1 (CDF1) (19). Suppression of Arabidopsis thaliana CDF1 (AtCDF1) expression led to a complete block of pPORA import into etioplasts, overaccumulation of non–protein-bound Pchlide, and light-dependent, porphyrin-sensitized cell death involving singlet oxygen. Along with biochemical data showing tight protein:protein interactions between CDF1 and PORA, we propose CDF1 to act as a POR-specific chaperone during plastid biogenesis.     

The unstructured linker of Mlh1 contains a motif required for endonuclease function which is mutated in cancers

Eukaryotic DNA mismatch repair (MMR) depends on recruitment of the Mlh1-Pms1 endonuclease (human MLH1-PMS2) to mispaired DNA. Both Mlh1 and Pms1 contain a long unstructured linker that connects the N- and carboxyl-terminal domains. Here, we demonstrated the Mlh1 linker contains a conserved motif (Saccharomyces cerevisiae residues 391–415) required for MMR. The Mlh1-R401A,D403A-Pms1 linker motif mutant protein was defective for MMR and endonuclease activity in vitro, even though the conserved motif could be >750 Å from the carboxyl-terminal endonuclease active site or the N-terminal adenosine triphosphate (ATP)-binding site. Peptides encoding this motif inhibited wild-type Mlh1-Pms1 endonuclease activity. The motif functioned in vivo at different sites within the Mlh1 linker and within the Pms1 linker. Motif mutations in human cancers caused a loss-of-function phenotype when modeled in S. cerevisiae. These results suggest that the Mlh1 motif promotes the PCNA-activated endonuclease activity of Mlh1-Pms1 via interactions with DNA, PCNA, RFC, or other domains of the Mlh1-Pms1 complex.

DNA mismatch repair (MMR) acts on mispairs arising from DNA-replication errors, formation of homologous recombination intermediates, and some chemically modified DNA bases (1–3). During MMR, mispair recognition by MutS homologs, primarily Msh2-Msh6 and Msh2-Msh3 in eukaryotes (4–8), is required to recruit MutL homologs to mispaired DNA, primarily Mlh1-Pms1 in eukaryotes (called MLH1-PMS2 in humans) (1–3, 9). In organisms other than Escherichia coli and related bacteria (10), the MutL homologs have an endonuclease activity that specifically nicks double-stranded DNA on strands containing pre-existing nicks (11–13). Nicking by Mlh1-Pms1 in vitro is required for Exo1-mediated repair on substrates with a nick 3′ to the mispair, as formation of a strand-specific nick 5′ to the mispair allows the 5′–3′ exonuclease activity of Exo1 to excise the mispair (11–14). The absolute requirement of this Mlh1-Pms1 nicking activity in vivo is not well understood, as both 5′ and 3′ nicks relative to mispairs are likely already present on newly synthesized DNA strands (15, 16). One proposal suggests that Mlh1-Pms1 activity maintains single-stranded discontinuities, which appear to identify the newly synthesized strand, even in the presence of the competing activities, like DNA ligation and gap filling by DNA polymerases (15, 17).MutL homologs are comprised of an N-terminal GHKL family adenosine triphosphatase (ATPase) domain, a carboxyl-terminal dimerization domain, and a predicted unstructured linker domain that connects the folded N- and carboxyl-terminal domains (18–21). In Saccharomyces cerevisiae, the unstructured linkers of Mlh1 and Pms1 are ∼150 and 250 amino acids long, respectively (22). These linkers have a biased sequence composition with reduced hydrophobic amino acids, like the large (>50 amino acid) intrinsically disordered regions (IDRs) present in many proteins (23–25). IDRs often mediate intermolecular interactions, play functional roles, and sometimes become ordered when bound to partners (23–25).MutL homologs, including Mlh1-Pms1, form DNA-bound rings called sliding clamps following loading by MutS homologs, ATP binding, and dimerization of the N-terminal ATPase domains; these rings rapidly diffuse along the DNA axis (26–30). The extended length of the unstructured interdomain linkers has been suggested to allow these MutL homolog clamps to migrate past protein–DNA complexes, which are normally a barrier to MutS homolog clamps, although Msh2-Msh3 clamps appear to be able to open and close on encountering a protein–DNA complex and hop over it (26–29, 31, 32). Remarkably, cleavage of the S. cerevisiae Mlh1 linker in vivo causes increased mutation rates, suggesting that intact sliding clamps are important for MMR (22). The importance of the combined lengths of the Mlh1 and Pms1 linkers in vivo is suggested by the synergistic increases in mutation rate that have been observed when combining S. cerevisiae mlh1 and pms1 mutations that shorten the linkers (26). In contrast, some linker missense mutations, which do not alter linker lengths, cause MMR defects (22, 33–35). Moreover, deletions within the S. cerevisiae Mlh1 linker tend to cause MMR defects, whereas deletions in the S. cerevisiae Pms1 linker tend not to, except for the pms1-Δ390–610 deletion that eliminates almost the entire Pms1 linker, resulting in a mutant complex that cannot be recruited by Msh2–Msh6 to mispair-containing DNA and fails to bind to DNA under low ionic strength conditions (22). Together, the data suggest that length is only one requirement for the Mlh1 and Pms1 linkers and that the Mlh1 and Pms1 linkers differ in importance for MMR.Here, we have identified a motif in the Mlh1 linker, which spans residues 391–415, that is conserved from S. cerevisiae to humans and is required for MMR. Mutation of two of the residues in this motif, R401 and I409, to alanine caused an MMR defect, as did short deletions affecting other partially conserved residues within the motif. We found that the motif was functional when moved to different positions on the Mlh1 linker and when the distances between motif and the folded N- and carboxyl-terminal domains were altered. Moreover, moving a copy of the motif to the Pms1 subunit complemented the MMR defect caused by loss of the motif in Mlh1; in addition, swapping the Mlh1 linker with the Pms1 linker supported MMR. Mutant Mlh1-Pms1 complexes with amino acid substitutions in the conserved Mlh1 motif could not support reconstituted MMR reactions in vitro and were defective for Mlh1-Pms1 endonuclease activity but were recruited to mispair-containing DNA by Msh2-Msh6. Peptides encoding the conserved motif, but not control peptides, inhibited wild-type Mlh1-Pms1 endonuclease activity. Consistent with these observations, increased levels of Pms1-4GFP foci, which are MMR intermediates (36), were caused by mutations disrupting the conserved Mlh1 motif, similar to other mutations that reduce Mlh1-Pms1 endonuclease activity (36–38). Mutations of the motif were observed in human cancers, and these mutations disrupted MMR in vivo when modeled in the S. cerevisiae MLH1 gene. Taken together, these data are consistent with a requirement of the Mlh1 linker motif for Mlh1-Pms1 endonuclease activity in MMR, which could be due to an interaction of the motif with the DNA substrate, with the endonuclease active site, and/or with the endonuclease-activating PCNA.     

C-terminal processing of reaction center protein D1 is essential for the function and assembly of photosystem II in Arabidopsis

Photosystem II (PSII) reaction center protein D1 is synthesized as a precursor (pD1) with a short C-terminal extension. The pD1 is processed to mature D1 by carboxyl-terminal peptidase A to remove the C-terminal extension and form active protein. Here we report functional characterization of the Arabidopsis gene encoding D1 C-terminal processing enzyme (AtCtpA) in the chloroplast thylakoid lumen. Recombinant AtCtpA converted pD1 to mature D1 and a mutant lacking AtCtpA retained all D1 in precursor form, confirming that AtCtpA is solely responsible for processing. As with cyanobacterial ctpa, a knockout Arabidopsis atctpa mutant was lethal under normal growth conditions but was viable with sucrose under low-light conditions. Viable plants, however, showed deficiencies in PSII and thylakoid stacking. Surprisingly, unlike its cyanobacterial counterpart, the Arabidopsis mutant retained both monomer and dimer forms of the PSII complexes that, although nonfunctional, contained both the core and extrinsic subunits. This mutant was also essentially devoid of PSII supercomplexes, providing an unexpected link between D1 maturation and supercomplex assembly. A knock-down mutant expressing about 2% wild-type level of AtCtpA showed normal growth under low light but was stunted and accumulated pD1 under high light, indicative of delayed C-terminal processing. Although demonstrating the functional significance of C-terminal D1 processing in PSII biogenesis, our study reveals an unsuspected link between D1 maturation and PSII supercomplex assembly in land plants, opening an avenue for exploring the mechanism for the association of light-harvesting complexes with the PSII core complexes.Photosystem II (PSII) consists of more than 20 subunits. Assembly of this photosystem is a multistep process that functions in a highly coordinated fashion (1–3). The process starts with PSII initiation complexes (D2, PsbE, PsbF, and PsbI), and then D1 and CP47 are sequentially recruited to form CP47-RC complexes, followed by addition of PsbH, PsbM, PsbTc, and PsbR subunits. Next, CP43 and other subunits are added to generate PSII monomers, which develop into PSII dimers. Finally, light-harvesting complex (LHC) II is attached to form PSII supercomplexes. The D1 protein of PSII is prone to photodamage under excessive light conditions (4). To sustain photosynthesis, damaged D1 protein is degraded and replaced with a newly synthesized copy via PSII repair—a highly complex and critical process whose mechanism remains unclear (3, 4).In most oxygen-evolving photosynthetic organisms, D1 protein is synthesized as a precursor (pD1) with a C-terminal tail. The pD1 protein is integrated into the thylakoid membrane and forms the initial PSII reaction center combined with other PSII subunits. The C-terminal tail of pD1 must be cleaved by an endopeptidase named the carboxyl terminal peptidase (Ctp) to produce mature D1, the functional form (5). In the cyanobacterium Synechocystis PCC 6803, there are three Ctp homologs (CtpA, CtpB, and CtpC), but only one, CtpA, is responsible for cleavage of the pD1 C-terminal extension (5). Disruption of CtpA leads to a loss of PSII activity and oxygen evolution from failure to form the manganese cluster (4, 6). The processing of pD1 is also critical for the association of extrinsic proteins on the luminal side to stabilize the PSII complexes (6, 7).In contrast to cyanobacteria, our knowledge of the significance of Ctp enzymes and D1 C-terminal processing is limited in land plants. Previous researchers reported the purification of CtpA-like protein from pea (8) and spinach (9). The spinach study further showed that the recombinant Ctp protein expressed from Escherichia coli displays activity against pD1 (9). However, because we lack a genetic approach, the functional significance of CtpA and C-terminal processing remains unknown in those and other land plants. In this study, we applied genetics to identify a gene (At4g17740) encoding a CtpA enzyme in Arabidopsis and showed that it is required for PSII function and chloroplast development. We found that Arabidopsis CtpA is essential for assembling functional PSII core complexes, dimers, and PSII supercomplexes. The enzyme is also critical for the PSII damage–repair cycle during the photoinhibition process.     

Usher type 1G protein sans is a critical component of the tip-link complex, a structure controlling actin polymerization in stereocilia

The mechanotransducer channels of auditory hair cells are gated by tip-links, oblique filaments that interconnect the stereocilia of the hair bundle. Tip-links stretch from the tips of stereocilia in the short and middle rows to the sides of neighboring, taller stereocilia. They are made of cadherin-23 and protocadherin-15, products of the Usher syndrome type 1 genes USH1D and USH1F, respectively. In this study we address the role of sans, a putative scaffold protein and product of the USH1G gene. In Ush1g(-/-) mice, the cohesion of stereocilia is disrupted, and both the amplitude and the sensitivity of the transduction currents are reduced. In Ush1g(fl/fl)Myo15-cre(+/-) mice, the loss of sans occurs postnatally and the stereocilia remain cohesive. In these mice, there is a decrease in the amplitude of the total transducer current with no loss in sensitivity, and the tips of the stereocilia in the short and middle rows lose their prolate shape, features that can be attributed to the loss of tip-links. Furthermore, stereocilia from these rows undergo a dramatic reduction in length, suggesting that the mechanotransduction machinery has a positive effect on F-actin polymerization. Sans interacts with the cytoplasmic domains of cadherin-23 and protocadherin-15 in vitro and is absent from the hair bundle in mice defective for either of the two cadherins. Because sans localizes mainly to the tips of short- and middle-row stereocilia in vivo, we conclude that it belongs to a molecular complex at the lower end of the tip-link and plays a critical role in the maintenance of this link.     

Inhibition of the RhoA/Rho-associated,coiled-coil-containing protein kinase-1 pathway is involved in the therapeutic effects of simvastatin on pulmonary arterial hypertension

Background: Recent research has shown that statins improve pulmonary arterial hypertension (PAH), but their mechanisms of action are not fully understood. This study aimed to investigate the role of RhoA/ROCK1 regulation in the therapeutic effects of simvastatin on PAH. Methods: For in vivo experiments, rats (N = 40) were randomly assigned to four groups: control, simvastatin, monocrotaline (MCT), and MCT + simvastatin. The MCT group and MCT + simvastatin groups received proline dithiocarbamate (50 mg/kg, i.p.) on the first day of the study. The MCT + simvastatin group received simvastatin (2 mg/kg) daily for 4 weeks, after which pulmonary arterial pressure was measured by right heart catheterization. The protein and mRNA levels of Rho and ROCK1 were measured by immunohistochemistry, Western blot, and PCR. For in vitro experiments, human pulmonary endothelial cells were divided into seven groups: control, simvastatin, monocrotaline pyrrole (MCTP), MCTP + simvastatin, MCTP + simvastatin + mevalonate, MCTP + simvastatin + farnesyl pyrophosphate (FPP), and MCTP + simvastatin + FPP + geranylgeranyl pyrophosphate (GGPP). After 72 h exposed to the drugs, the protein and mRNA levels of RhoA and ROCK1 were measured by Western blot and PCR. Results: The MCT group showed increased mean pulmonary arterial pressure, marked vascular remodeling, and increased protein and mRNA levels of RhoA and ROCK1 compared to the other groups (P < 0.05). In vitro, the MCTP group showed a marked proliferation of vascular endothelial cells, as well as increased protein and mRNA levels of RhoA and ROCK1 compared to the MCTP + simvastatin group. The MCTP + simvastatin + mevalonate group, MCTP + simvastatin+ FPP group, and MCTP + simvastatin + FPP + GGPP group showed increased mRNA levels of RhoA and ROCK1, as well as increased protein levels of RhoA, compared to the MCTP + simvastatin group. Conclusions: Simvastatin improved vascular remodeling and inhibited the development of PAH. The effects of simvastatin were mediated by inhibition of RhoA/ROCK1. Simvastatin decreased RhoA/ROCK1 overexpression by inhibition of mevalonate, FPP, and GGPP synthesis.     

Aberrant protein aggregation is essential for a mutant desmin to impair the proteolytic function of the ubiquitin-proteasome system in cardiomyocytes

Aberrant protein aggregates in cardiomyocytes are frequently observed in many forms of cardiomyopathies and are often associated with impairment of proteolytic function of the ubiquitin-proteasome system (UPS). However, a causal relationship between mutant desmin (MT-des) induced aberrant protein aggregation and UPS impairment has not been established. The present study has tested the causal relationship. In cultured neonatal rat ventricular myocytes, modest overexpression of a human (cardio)myopathy-linked MT-des protein led to formation of desmin-positive aggregates and inhibited UPS proteolytic function in cardiomyocytes in a dose-dependent manner. Prevention or reduction of aberrant protein aggregation by co-expression of a heat shock protein (Hsp), alphaB-crystallin or inducible Hsp70, or by treatment of Congo red prevented and/or significantly attenuated the induction of UPS malfunction by MT-des. These findings prove for the first time that aberrant protein aggregation is not only sufficient but also required for MT-des to impair UPS proteolytic function in cardiomyocytes.     

真核绿色荧光蛋白表达载体pEGFP-C1/PAK-1的构建及其在结直肠癌SW480细胞内的表达

目的:构建重组p21-activated kinase-1(PAK1)基因绿色荧光蛋白表达载体pEGFP-C1/PAK1,并转染入结直肠癌细胞SW480中表达.方法:在南方医科大学附属南方医院消化研究所实验室,从人类结直肠癌细胞株SW620细胞提取总RNA,经逆转录聚合酶链式反应获得人PAK1 cDNA片段,经过限制性内切酶进行酶切,T4连接酶进行连接,将目的基因克隆至真核绿色荧光蛋白表达载体pEGFP-C1上,然后转染结直肠癌细胞株SW480,观察其在细胞中表达.结果:重组载体经限制性内切酶酶切鉴定和DNA序列分析验证,显示插入载体的序列与目的基因一致,而且该重组载体能够在SW480细胞中表达.结论:成功构建了真核绿色荧光蛋白表达载体pEGFP-C1/PAK1,为研究PAK1在结直肠癌中的生物学功能奠定了基础.     

Humoral responses to a secretory Onchocerca volvulus protein: differences in the pattern of antibody isotypes to recombinant Ov20/OvS1 in generalized and hyperreactive onchocerciasis

The Onchocerca volvulus secretory protein Ov20/OvS1 represents a dominant antigen expressed in the infective larvae, microfilariae and adult stages of the parasite. The humoral responses to this protein have not yet been analysed in the polar clinical and immunological forms of onchocerciasis. Analysis by ELISA of class and subclass antibodies to Ov20/OvS1 in persons with the generalized or the hyperreactive form of onchocerciasis revealed similar strong responses of IgG1, IgG4 and IgM antibody levels in both forms of onchocerciasis and significant differences were observed in the IgE and IgA antibody classes. Computation of the ratios of antibodies showed that persons with the generalized form exhibited significantly higher ratios of IgG4 to IgG1, IgG4 to IgE, and IgM to IgE than patients with the hyperreactive form. To investigate the isotype recognition of antigenic sites on Ov20/OvS1 protein, three recombinantly expressed fragments (F1-3) of Ov20/OvS1 were probed using sera which strongly reacted with intact recombinant Ov20/OvS1. Epitope(s) on F1 comprising amino acid residues 1-63 were significantly recognized by IgG1 and IgE, while IgM recognized epitopes on all three fragments. The strongest reaction of IgM occurred with epitope(s) formed by residues 108-171 (F3). In contrast, IgG4 type antibodies were not reactive with either of the three OvS1 fragments, but they reacted with intact Ov20/OvS1 protein. Generalized onchocerciasis, unable to eliminate microfilariae, and hyperreactive onchocerciasis, with a high potency to eliminate or to reduce parasite loads, can be distinguished by a distinct pattern of isotype responses to Ov20/OvS1.     

FEV1/FEV6 is effective as a surrogate for FEV1/FVC in the diagnosis of chronic obstructive pulmonary disease

Background and objectiveChronic Obstructive Pulmonary Disease (COPD) causes substantial morbidity and mortality across the globe. Diagnosis of COPD requires post-bronchodilator FEV1/FVC <0.70 as per GOLD Guidelines. FVC maneuver requires a minimum of 6 seconds of forceful expiration with no flow for 1 second for an accepted effort, which lacks any fixed cut-off point. This leads to discomfort, especially in advanced COPD and old aged population. We conducted this study to find the utility of FEV1/FEV6 as a surrogate for FEV1/FVC, the correlation between the two ratios, and the fixed cut-off value of FEV1/FEV6 for COPD diagnosis.MethodsThis was a prospective, cross-sectional study approved by the institutional ethics committee conducted from January 2017 to November 2018. Consented patients above 18 years suspected of COPD underwent Spirometry as per ATS guidelines. FEV1, FEV6, FEV1/FEV6 and FEV1/FVC ratios were recorded from the best acceptable maneuver.ResultsOut of 560 screened patients, 122 diagnosed as COPD. The correlation coefficient between the post-bronchodilator FEV1/FVC ratio and FEV1/FEV6 ratio was 0.972 (p < 0.01). The relationship between the post-bronchodilator FEV1/FVC ratio and FEV1/FEV6 ratio (linear regression analysis) was found out as: FEV1/FVC = ?1.845 + 1.009(FEV1/FEV6). Using this formula, the post-bronchodilator FEV1/FEV6 value of 71.845 was obtained corresponding to the post-bronchodilator FEV1/FVC value of 70.00.ConclusionWe found a positive correlation coefficient (r = 0.972, p < 0.001) between the FEV1/FEV6 and FEV1/FVC ratios and the cut off value of 71.845 (p < 0.01) for the post-bronchodilator FEV1/FEV6 ratio for the diagnosis of COPD. Thus FEV1/FEV6 should be used as a surrogate for FEV1/FVC for the diagnosis of COPD.