kD Subunit Research Articles

Mammalian cleavage factor 25 targets KLF14 to inhibit hepatic stellate cell activation and liver fibrosis

Liver fibrosis is primarily caused by the activation of hepatic stellate cells (HSCs), which results from chronic liver damage. Understanding the pathogenesis of HSC activation could identify new therapeutic targets to treat liver fibrosis. In this study, we examined the protective role of the mammalian cleavage factor I 25 kD subunit (CFIm25, NUDT21) in inhibiting hepatic stellate cell activation. CFIm25 expression was measured in liver cirrhosis patients and a CCl4-induced mouse model. Adeno-associated viruses and adenoviruses were used to alter hepatic CFIm25 expression in vivo and in vitro to investigate how CFIm25 functions in liver fibrosis. The underlying mechanisms were explored using RNA-seq and co-IP assays. Here, we found that CFIm25 expression was drastically decreased in activated murine HSCs and fibrotic liver tissues. CFIm25 overexpression downregulated the expression of genes involved in liver fibrosis, inhibiting the progression of HSC activation, migration and proliferation. These effects resulted from direct activation of the KLF14/PPARγ signaling axis. KLF14 inhibition abrogated the CFIm25 overexpression-mediated reduction in antifibrotic effects. These data reveal that hepatic CFIm25 regulates HSC activation through the KLF14/PPARγ pathway as liver fibrosis progresses. CFIm25 may be a novel therapeutic target for liver fibrosis.

The role of FIP200 in Phagophore formation during Autophagy

The Moeller Molecular Modeling Team in conjunction with Dr. Jiajie Diao at the University of Cincinnati Medical College used 3D modeling and printing technology to study the vital role of the focal adhesion kinase family interacting protein of 200 kD (FIP200) in the autophagy process. The cellular process of autophagy is enabled by formation of autophagosomes, a double membraned structure that isolates substrates designated by the cell to be eliminated by autophagy. FIP200 is a 200 kD subunit of the ULK1 complex which facilitates the formation of phagophores, the first step in autophagosome formation. An ULK1 protein kinase is necessary for the activation of the ULK1 complex during autophagosome formation. The positively charged “claw domain” area at the C‐terminal region of FIP200 interacts with p62, a receptor protein, which delivers ubiquitinated cargo proteins bound to LC3B, a central regulator for autophagosome formation. After the binding of these proteins, the autophagosome forms around the ubiquitinated cargo and continues to the next step of the autophagy process.

Decreased Spikelets 4 Encoding a Novel Tetratricopeptide Repeat Domain-Containing Protein Is Involved in DNA Repair and Spikelet Number Determination in Rice.

Spikelet number per panicle is a determinative factor of rice yield. DNA repair epigenetically alters the DNA accessibility, which can eventually regulate the transcription of the target genes. However, what and how DNA repair genes are related to rice spikelet development remains unknown. Here, we report the map-based cloning of a novel spikelet number gene DES4 encoding a tetratricopeptide domain-containing protein. DES4 is a close ortholog of Arabidopsis BRU1, which is functionally related to axillary meristem development. A single base pair deletion in the last exon of DES4 caused a premature stop of the resulting protein. The des4 mutant exhibited dwarf, reduced tiller, and spikelet numbers phenotypes, as well as hypersensitivity to genotoxic stresses, suggesting its essential role in DNA repair. DES4 is predominantly expressed in young panicles and axillary meristems, and DES4 protein is localized in nucleus. A set of DNA repair genes such as cyclins, KUs (KD subunits) and recombinases were differentially regulated in des4. Meanwhile, rice spikelet number genes LAX1, LAX2, and MOC1 were significantly down-regulated in des4. In morphology, des4 showed more severe reduction of spikelet numbers than lax1, lax2, and moc1, suggesting that DES4 may work upstream of the three genes.

Binding process between E.coli SSB and ssDNA by single-molecule dynamics

Single-stranded DNA binding proteins (SSBs) widely exist in different kinds of creatures. It can bind single-stranded DNA (ssDNA) with high affinity. The binding is sequence independent. SSB can also interact with different kinds of proteins, and thus leading them to work at the special sites. It plays an essential role in cell metabolism. E.coli SSB is a representative of SSB among all kinds of SSBs, it is a homotetramer consisting of four 18.9 kD subunits, the homotetramer is stable under low concentration. E.coli SSB has different binding modes under different salt concentrations (for example NaCl). When NaCl concentration is higher than 200 mM, E.coli SSB can bind 65 nt ssDNA, when NaCl concentration is lower than 20 mM, it can bind 35 nt ssDNA, and when the NaCl concentration is between 20 mM and 200 mM, it can bind 56 nt ssDNA. The characteristics of E.coli SSB are so attractive that a large number of researches have been done to distinguish its binding process. Earlier researchers tried to use stop flow technology to study the interaction between SSB and ssDNA in bulk. However, the high affinity between SSB and ssDNA makes this interaction too rapid to be observed at all, and the dissociate interaction even could not be measured. Single molecule technology which combines with low and accurate force offers researchers another way to achieve this goal. Some researchers observed the unwrapping phenomenon in an optical tweezers pulling experiment. However, they did not find the detailed process of binding or dissociation. In our work, we use a magnetic tweezer to pull the SSB/ssDNA complex and find a special phenomenon like double-state jump. Using the single molecule dynamics to analyse the data, we find that this phenomenon is the combination and dissociation between SSB and ssDNA. After comparing the pulling curve of ssDNA only and SSB/ssDNA complex, we find that the SSB binding process consists of two stages, one is rapid combination/dissociation under the action of a critical force; the other is continuous wrapping following the reduced force. According to Bell formula and SSB/ssDNA complex binding model, we obtain the interaction rate and free energy parameters under 0 pN, and we calibrate the free energy to obtain its continuous wrapping part, so we can obtain the whole free energy landscape and understand the binding process. Our analysis way is also applicable to the case of similar interactions to obtain their interaction details and free energy characteristics.

P.31 - A diagnostic challenge of late onset adult merosinopathy

A 51-year-old caucasian woman presented with a 6 year history of, initially exercise dependent, proximal leg weakness. Her brain MRI showed extensive, predominantly supratentorial white matter changes, that could not be attributed to her known hypertension. She had a single grand mal seizure as a teenager upon photostimulation and suffers from migraine with aura. Her mother was reported to suffer from epilepsy due to traumatic brain injury and her two sisters were said to have skeletal deformities due to use of analgetics during the pregnancies. Our patient's sisters, son and daughter report no neuromuscular complaints. Her serum CK was 200 - 500 U/l, CSF analysis and lab work-up for adult white matter disorders did not reveal abnormalities, but vastus muscle biopsy showed mildly dystrophic changes with an increased fibre size variation, fibre degeneration and regeneration, some fibrosis as well as rimmed vacuoles. Immunohistochemistry showed normal reactivities, including anti-laminin α2 (NCL-MEROSIN) and -α-dystroglycan, but for anti-p62 deposition along rimmed vacuoles. However, anti-laminin α2 western blot showed loss of the 80 kDa band (Millipore MAB1922). Genetic analysis for m.A3243G and m.A8344G point mutations as well as DNAJB6 and NOTCH3 genes gave normal results. In the LAMA2 gene two variants were found, c.2171_2172del and c.2173C > A, the former a known pathogenic mutation, however, in cis. We conclude that this is a case of late onset adult merosinopathy with an affection of the second LAMA2 allele escaping detection so far. In suspected laminin α2 disorders, both, the 300 kD and 80 kD subunit, need to be examined. We did not observe a selective reduction of the 300 kD isoform that has been suggested for milder forms, and hope to be able to examine further family members to ensure that their reported symptoms are indeed unrelated.

Mitochondrial Complex I Is a Global Regulator of Secondary Metabolism, Virulence and Azole Sensitivity in Fungi.

Recent estimates of the global burden of fungal disease suggest that that their incidence has been drastically underestimated and that mortality may rival that of malaria or tuberculosis. Azoles are the principal class of antifungal drug and the only available oral treatment for fungal disease. Recent occurrence and increase in azole resistance is a major concern worldwide. Known azole resistance mechanisms include over—expression of efflux pumps and mutation of the gene encoding the target protein cyp51a, however, for one of the most important fungal pathogens of humans, Aspergillus fumigatus, much of the observed azole resistance does not appear to involve such mechanisms. Here we present evidence that azole resistance in A. fumigatus can arise through mutation of components of mitochondrial complex I. Gene deletions of the 29.9KD subunit of this complex are azole resistant, less virulent and exhibit dysregulation of secondary metabolite gene clusters in a manner analogous to deletion mutants of the secondary metabolism regulator, LaeA. Additionally we observe that a mutation leading to an E180D amino acid change in the 29.9 KD subunit is strongly associated with clinical azole resistant A. fumigatus isolates. Evidence presented in this paper suggests that complex I may play a role in the hypoxic response and that one possible mechanism for cell death during azole treatment is a dysfunctional hypoxic response that may be restored by dysregulation of complex I. Both deletion of the 29.9 KD subunit of complex I and azole treatment alone profoundly change expression of gene clusters involved in secondary metabolism and immunotoxin production raising potential concerns about long term azole therapy.

CPSF30 at the Interface of Alternative Polyadenylation and Cellular Signaling in Plants.

Post-transcriptional processing, involving cleavage of precursor messenger RNA (pre mRNA), and further incorporation of poly(A) tail to the 3' end is a key step in the expression of genetic information. Alternative polyadenylation (APA) serves as an important check point for the regulation of gene expression. Recent studies have shown widespread prevalence of APA in diverse systems. A considerable amount of research has been done in characterizing different subunits of so-called Cleavage and Polyadenylation Specificity Factor (CPSF). In plants, CPSF30, an ortholog of the 30 kD subunit of mammalian CPSF is a key polyadenylation factor. CPSF30 in the model plant Arabidopsis thaliana was reported to possess unique biochemical properties. It was also demonstrated that poly(A) site choice in a vast majority of genes in Arabidopsis are CPSF30 dependent, suggesting a pivotal role of this gene in APA and subsequent regulation of gene expression. There are also indications of this gene being involved in oxidative stress and defense responses and in cellular signaling, suggesting a role of CPSF30 in connecting physiological processes and APA. This review will summarize the biochemical features of CPSF30, its role in regulating APA, and possible links with cellular signaling and stress response modules.

Simian virus Large T antigen interacts with the N-terminal domain of the 70 kD subunit of Replication Protein A in the same mode as multiple DNA damage response factors.

Simian virus 40 (SV40) serves as an important model organism for studying eukaryotic DNA replication. Its helicase, Large T-antigen (Tag), is a multi-functional protein that interacts with multiple host proteins, including the ubiquitous ssDNA binding protein Replication Protein A (RPA). Tag recruits RPA, actively loads it onto the unwound DNA, and together they promote priming of the template. Although interactions of Tag with RPA have been mapped, no interaction between Tag and the N-terminal protein interaction domain of the RPA 70kDa subunit (RPA70N) has been reported. Here we provide evidence of direct physical interaction of Tag with RPA70N and map the binding sites using a series of pull-down and mutational experiments. In addition, a monoclonal anti-Tag antibody, the epitope of which overlaps with the binding site, blocks the binding of Tag to RPA70N. We use NMR chemical shift perturbation analysis to show that Tag uses the same basic cleft in RPA70N as multiple of DNA damage response proteins. Mutations in the binding sites of both RPA70N and Tag demonstrate that specific charge reversal substitutions in either binding partner strongly diminish the interaction. These results expand the known repertoire of contacts between Tag and RPA, which mediate the many critical roles of Tag in viral replication.

Integration of developmental and environmental signals via a polyadenylation factor in Arabidopsis.

The ability to integrate environmental and developmental signals with physiological responses is critical for plant survival. How this integration is done, particularly through posttranscriptional control of gene expression, is poorly understood. Previously, it was found that the 30 kD subunit of Arabidopsis cleavage and polyadenylation specificity factor (AtCPSF30) is a calmodulin-regulated RNA-binding protein. Here we demonstrated that mutant plants (oxt6) deficient in AtCPSF30 possess a novel range of phenotypes – reduced fertility, reduced lateral root formation, and altered sensitivities to oxidative stress and a number of plant hormones (auxin, cytokinin, gibberellic acid, and ACC). While the wild-type AtCPSF30 (C30G) was able to restore normal growth and responses, a mutant AtCPSF30 protein incapable of interacting with calmodulin (C30GM) could only restore wild-type fertility and responses to oxidative stress and ACC. Thus, the interaction with calmodulin is important for part of AtCPSF30 functions in the plant. Global poly(A) site analysis showed that the C30G and C30GM proteins can restore wild-type poly(A) site choice to the oxt6 mutant. Genes associated with hormone metabolism and auxin responses are also affected by the oxt6 mutation. Moreover, 19 genes that are linked with calmodulin-dependent CPSF30 functions, were identified through genome-wide expression analysis. These data, in conjunction with previous results from the analysis of the oxt6 mutant, indicate that the polyadenylation factor AtCPSF30 is a regulatory hub where different signaling cues are transduced, presumably via differential mRNA 3′ end formation or alternative polyadenylation, into specified phenotypic outcomes. Our results suggest a novel function of a polyadenylation factor in environmental and developmental signal integration.

Requirements for 5′dRP/AP lyase activity in Ku

The non-homologous end joining (NHEJ) pathway is used in diverse species to repair chromosome breaks, and is defined in part by a requirement for Ku. We previously demonstrated mammalian Ku has intrinsic 5′ deoxyribosephosphate (5′dRP) and apurinic/apyrimidinic (AP) lyase activity, and showed this activity is important for excising abasic site damage from ends. Here we employ systematic mutagenesis to clarify the protein requirements for this activity. We identify lysine 31 in the 70 kD subunit (Ku70 K31) as the primary candidate nucleophile required for catalysis, but additional mutation of Ku70 K160 and six other lysines within Ku80 were required to eliminate all activity. Ku from Saccharomyces cerevisiae also possesses 5′dRP/AP lyase activity, and robust activity was also reliant on lysines in Ku70 analogous to K31 and K160. By comparison, these lysines are not conserved in Xenopus laevis Ku, and Ku from this species has negligible activity. A role for residues flanking Ku70 K31 in expanding the range of abasic site contexts that can be used as substrate was also identified. Our results suggest an active site well located to provide the substrate specificity required for its biological role.

CHBP in Preservation Solution and Autologous Blood Perfusate Protects Isolated Ischemic Porcine Kidneys.

Introduction. There is an imperative need to develop new approaches to prevent ischemia reperfusion injury (IRI) during organ cold storage (CS) and reperfusion. A novel cyclic helix B peptide (CHBP) derived from erythropoietin was newly synthesized and its renoprotective roles were evaluated in an ex vivo renal model. Methods. Porcine kidneys subjected to 20 min warm ischemia were retrieved and flushed with 500 ml hyperosmolar citrate. The kidneys, as well as autologous blood, were cold preserved with or without 10.56 nmol/L CHBP for 18 hr. The kidneys were then reperfused with oxygenated autologous blood for 3 hr on an isolated organ perfusion system to assess renal function and structure, caspase-3 expression, apoptosis and inflammation. Results. CHBP significantly increased renal blood flow during 2 to 3 h reperfusion, with increased oxygen consumption and urine output, but decreased serum potassium. 32 kD precursor, as well as 12 and 17 kD active subunits of caspase-3 were all down-regulated by CHBP in after 3 h reperfusion, while only the 12 kD subunit was decreased post CS. In addition, apoptotic cells were significantly decreased in tubular areas, but increased in lumens and interstitial areas in post CS and reperfusion kidneys. Heat shock protein 70 was up-regulated; myeloperoxidase+ cells was reduced and renal tissue damage was also ameliorated by CHBP. Conclusions. The administration of CHBP during cold preservation of kidneys as well as autologous blood ameliorated IRI after hemoreperfusion with better renal blood flow, oxygenation, tubular function and tissue structure, which might be due to decreased caspase-3 protein, apoptosis and inflammation.Figure: No Caption available.

NFκB Signaling Is Essential for the Lipopolysaccharide-Induced Increase of Type 2 Deiodinase in Tanycytes

The enzyme type 2 deiodinase (D2) is a major determinant of T₃ production in the central nervous system. It is highly expressed in tanycytes, a specialized cell type lining the wall of the third ventricle. During acute inflammation, the expression of D2 in tanycytes is up-regulated by a mechanism that is poorly understood at present, but we hypothesized that cJun N-terminal kinase 1 (JNK1) and v-rel avian reticuloendotheliosis viral oncogene homolog A (RelA) (the 65 kD subunit of NFκB) inflammatory signal transduction pathways are involved. In a mouse model for acute inflammation, we studied the effects of lipopolysaccharide (LPS) on mRNA expression of D2, JNK1, and RelA in the periventricular area (PE) and the arcuate nucleus-median eminence of the hypothalamus. We next investigated LPS-induced D2 expression in primary tanycyte cell cultures. In the PE, the expression of D2 was increased by LPS. In the arcuate nucleus, but not in the PE, we found increased RelA mRNA expression. Likewise, LPS increased D2 and RelA mRNA expression in primary tanycyte cell cultures, whereas JNK1 mRNA expression did not change. Phosphorylation of RelA and JNK1 was increased in tanycyte cell cultures 15-60 minutes after LPS stimulation, confirming activation of these pathways. Finally, inhibition of RelA with the chemical inhibitors sulfasalazine and 4-Methyl-N¹-(3-phenylpropyl)benzene-1,2-diamine (JSH-23) in tanycyte cell cultures prevented the LPS-induced D2 increase. We conclude that NFκB signaling is essential for the up-regulation of D2 in tanycytes during inflammation.

Molecular characterization of hepatopancreas vitellogenin and its expression during the ovarian development by in situ hybridization in the banana shrimp Fenneropenaeus merguiensis

A complete cDNA sequence of vitellogenin (Vg-H) was cloned from the hepatopancreas of Fenneropenaeus merguiensis (De Man, 1888). The full-length Vg-H gene consists of 7958 bp and contains an ORF of 7761 bp. It encodes a polypeptide of 2587 amino acids, comprising a predicted molecular mass of 283 kD and the theoretical pI is 6.5. The amino acid sequences of the mature vitellogenin (Vg) composed of a signal peptide, three possible O-glycosylation sites, three phosphorylation sites and putative possessing sites (R-X-K/R-R) recognized by subtilisin-like endoproteases. Cleavage at residues 725 to 728 will produce two Vg subunits (78 and 200 kD), which the N-terminal amino acid sequence of 78 kD subunit is identical to that of vitellin purified from the ovary. These properties suggest that Vg protein encoding by Vg-H undergoes processing at its synthetic sites prior to being transported to developing oöcytes. The deduced primary structure of Vg-H showed the highest identity (98.8%) to the Vg-O previously cloned from the ovary of the same species. It is more related to the penaeid Vg sequences than that of nonpenaeids. In situ hybridization revealed that mRNA encoding Vg was expressed both in follicle cells in the ovary and parenchyma cells in the hepatopancreas. In the developing ovary, highest levels were detected during the early vitellogenic stage 2, declining thereafter. In the hepatopancreas, Vg mRNA levels reached a maximum at stage 3 of ovarian development. Profiles of Vg mRNA expression in the ovary and hepatopancreas suggest that the contribution of these tissues to vitellogenesis is inversely related during gonad maturation. Thus, Vg gene sequences expressed in the ovary and hepatopancreas are most likely identical and both tissues are responded for yolk protein synthesis in F. merguiensis.

The p110alpha and p110delta Isoforms of PI3 Kinase Are Dispensable for Hematopoietic Stem Cell Self-Renewal but Have Redundant Roles in B Cell Differentiation.

Abstract 2322PI3 kinase (PI3K) is a key transducer of signals by hematopoietic growth factors, which control hematopoietic stem cell (HSC) proliferation, differentiation, self-renewal, and survival. Therefore, it can be expected that pharmacologic targeting of the PI3K pathway in malignancy could affect HSC function, and potentially result in myelotoxicity. The class IA PI3Ks are heterodimers, consisting of a catalytic 110-kd subunit termed p110, which has 3 isoforms in hematopoietic cells (α, β, and δ), and a regulatory subunit, which has 5 isoforms (p85α, p55α, p50α, p85β, or p55γ). Germline deletion of p85α and p85β causes a reduction in the number of fetal liver progenitors and impaired competitive repopulating ability of fetal liver HSCs (Haneline et al, Blood, 2006. 107(4):1375–82). This suggests that Class IA PI3K is an important regulator of HSC function. However it is unclear whether PI3K signaling is essential for adult hematopoiesis, and the roles of the individual PI3K isoforms in HSCs are unknown. In fibroblasts, the deletion of p110α impairs growth factor signaling and transformation by oncogenic signals. P110αflox/flox;Mx1-Cre mice have anemia, but HSC number and function are unchanged (Gritsman et al, Blood, ASH Annual Meeting Abstracts, Nov 2009; 114: 3620). While germline deletion of p110δ affects B and T cell development and function, p110δ−/− offspring have normal blood counts, suggesting a minimal role for p110δ in HSCs (Jou et al, MCB, 2002. 22(24):8580–91).To determine whether there is redundancy between p110α and p110δ in HSCs, we generated mice with the genotype p110δ−/−;p110αflox/flox;Mx1-Cre (“PDFPA;Mx1-Cre”). After PolyI-PolyC (PIPC) injection to delete p110α in HSCs, PDFPA;Mx1-Cre mice develop leukopenia and anemia, decreased bone marrow (BM) cell counts and spleen weight, but survive for at least 8 months with no signs of disease. The B cell lineage is the most severely affected, with a 4-fold decrease in B220+ B cells in the BM of PDFPA;Mx1-Cre mice compared with WT controls, and a 3-fold decrease compared with p110δ−/−;p110αflox/flox (PDFPA) controls. Multiparameter flow cytometry on the BM revealed a decrease in the frequency of LSK cells (Lin−c-kit+Sca1+ cells) in PDFPA;Mx1-Cre BM compared with WT;Mx1-Cre BM. While there was no effect on the frequency of long-term HSCs (CD150+CD48- LSK), there was a 5-fold decrease in the frequency of short-term HSCs (CD150-CD48-Flk2- LSK), multipotential progenitors (CD150-CD48-Flk2+ LSK), and common lymphoid progenitors (CLPs, Lin-IL7R+ckitloSca1loFlk2+), and a 2-fold decrease in the frequency of common myeloid progenitors (Lin-ckit+Sca1-FCRgmidCD34hi). We observed a similar trend in p110δ−/− BM.To determine whether p110δ and p110α are required for HSC self-renewal, we performed competitive and non-competitive repopulation assays. PDFPA;Mx1-Cre cells are capable of long-term multilineage reconstitution in a non-competitive setting. In the competitive setting (1:1 donor:competitor ratio), p110δ−/− BM cells were capable of normal trilineage long-term reconstitution, compared with WT BM. However, in recipients of PDFPA;Mx1-Cre BM, we observed a significant decrease in donor chimerism in the peripheral blood starting at 4 weeks after PIPC, with a complete lack of B cell donor reconstitution, compared with PDFPA controls. In contrast, myeloid and T cell reconstitution was detectable up to 20 weeks. There was a significant decrease in CLP donor chimerism in recipients of PDFPA;Mx1-Cre BM, while LT-HSC chimerism was unchanged. This suggests that p110α and p110δ have important redundant roles in HSC proliferation and differentiation along the lymphoid lineage, in addition to their known redundant roles in B cell development and BCR signaling (Ramadani etal, Science Signaling 2010. 3(134):p.1–10). Phospho-flow cytometry reveals significantly decreased Akt signaling in PDFPA;Mx1-Cre LSK cells in response to SCF.In summary, we have uncovered important and partially redundant roles for p110α and p110δ in HSC proliferation and lymphoid differentiation, which most significantly impact early B cell development. Remarkably, despite being required for growth factor-induced Akt signaling in HSCs, these PI3K isoforms are not essential for HSC self-renewal in vivo. This work will have important implications for targeting the PI3K pathway in cancer treatment, and particularly in B cell malignancies. Disclosures:Gilliland:Merck Research Laboratories: Employment. Roberts:Novartis Pharmaceuticals Corporation: Consultancy. Zhao:Novartis Pharmaceuticals Corporation: Consultancy.

ZINGIPAIN, A CYSTEINE PROTEASE FROM Zingiber ottensii VALETON RHIZOMES WITH ANTIPROLIFERATIVE ACTIVITIES AGAINST FUNGI AND HUMAN MALIGNANT CELL LINES

The objective of this study was to investigate the activity of a protein identified as cysteine protease, purified from Zingiber ottensii Valeton rhizomes, in terms of antiproliferation against fungi, bacteria, and human malignant cell lines. By means of buffer extraction followed by (NH4)2SO4 precipitation and ion-exchange chromatography, the obtained dominant protein (designated F50) was submitted to non-denaturing and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), where a single band and three bands were revealed from eletrophoretic patterns, respectively. It could be concluded at this point that the F50 was potentially a heterotrimer or heterodimer composed of either two small (∼13.8 and ∼15.2 kD) subunits or these two together with a larger (∼32.5 kD) one. In-gel digestion was carried out for the most intense band from reducing SDS-PAGE, and to the resulting material was applied liquid chromatography (LC)–mass spectroscopy (MS)/MS. The main F50 subunit was found to contain fragments with 100% similarity to zingipain-1, a cysteine protease first discovered in Zingiber officinale. The activity corresponding to the identified data, cysteine protease, was then confirmed in the F50 by azocasein assay and a positive result was obtained. The F50 then was further investigated for antiproliferation against three plant pathogenic fungi species by disk diffusion test, four bacterial species by direct exposure in liquid culture and dish diffusion tests, and five human malignant cell lines by tissue culture assay. It was found that a dose of 23.6 µg F50/0.3 cm2 of paper disk exhibited the best inhibitory effect against Collectotrichum cassiicola, while lesser effects were found in Exserohilum turicicum and Fusarium oxysporum, respectively. No inhibitory effect against bacterial proliferation was detected in all studied bacterial strains. However, relatively strong antiproliferative effects were found against five human cell lines, with IC50 values ranging from 1.13 µg/mL (hepatoma cancer; HEP-G2) to 5.37 µg/mL (colon cancer; SW620). By periodic acid–Schiff's staining and phenol–sulfuric acid assay, the F50 was determined as a glycoprotein containing 26.30 ± 1.01% (by weight) of carbohydrate. Thus, a new glycoprotein with protease activity was successfully identified in Zingiber ottensii rhizome. The glycoprotein also contained antiproliferative activity against some plant pathogenic fungi and human cancer cell lines.

Epstein-Barr virus-encoded EBNA1 enhances RNA polymerase III-dependent EBER expression through induction of EBER-associated cellular transcription factors

BackgroundEpstein-Barr Virus (EBV)-encoded RNAs (EBERs) are non-polyadenylated RNA molecules transcribed from the EBV genome by RNA polymerase III (pol III). EBERs are the most abundant viral latent gene products, although the precise mechanisms by which EBV is able to achieve such high levels of EBER expression are not fully understood. Previously EBV has been demonstrated to induce transcription factors associated with EBER expression, including pol III transcription factors and ATF-2. We have recently demonstrated that EBV-encoded nuclear antigen-1 (EBNA1) induces cellular transcription factors, and given these findings, we investigated the role of EBNA1 in induction of EBER-associated transcription factors.ResultsOur data confirm that in epithelial cells EBNA1 can enhance cellular pol III transcription. Transient expression of EBNA1 in Ad/AH cells stably expressing the EBERs led to induction of both EBER1 and EBER2 and conversely, expression of a dominant negative EBNA1 led to reduced EBER expression in EBV-infected Ad/AH cells. EBNA1 can induce transcription factors used by EBER genes, including TFIIIC, ATF-2 and c-Myc. A variant chromatin precipitation procedure showed that EBNA1 is associated with the promoters of these genes but not with the promoters of pol III-transcribed genes, including the EBERs themselves. Using shRNA knock-down, we confirm the significance of both ATF-2 and c-Myc in EBER expression. Further, functional induction of a c-Myc fusion protein led to increased EBER expression, providing c-Myc binding sites upstream of EBER1 were intact. In vivo studies confirm elevated levels of the 102 kD subunit of TFIIIC in the tumour cells of EBV-positive nasopharyngeal carcinoma biopsies.ConclusionsOur findings reveal that EBNA1 is able to enhance EBER expression through induction of cellular transcription factors and add to the repertoire of EBNA1's transcription-regulatory properties.

Apoptosis inducing effect of ponicidin in leukemia K562 cells and its mechanisms of action

To investigate the apoptosis inducing effects of ponicidin (PON) on leukemic K562 cells and its mechanisms of action. K562 cells in culture medium in vitro were given different concentrations of PON (10-50 micromol x L(-1)) for 24, 48 and 72 h. The inhibitory rate of the cells was measured by MTT assay, cell apoptotic rates were detected by flow cytometry (FCM) using Annexin V staining after K562 cells were treated with different concentrations of PON for 72 hours, and cell morphology was observed by Wright-Giemsa staining. Western blot was used to detect caspase-3 and poly(ADP-ribose) polymerase (PARP) expression, and the protein levels in mitogen-activated protein kinase signaling pathways (MAPKs, p-P38, p-ERK and p-JNK) as well as p-AKT and p-P85 in PI3K/AKT signaling pathways were also detected. PON (over 30 micromol x L(-1)) could inhibit the growth of K562 cells in both time- and dose-dependent manner. FCM analysis revealed that apoptotic cells were gradually increased in a dose-dependent manner after treatment for 72 hours, and that marked morphological changes of cell apoptosis such as condensation of chromatin was clearly observed by Wright-Giemsa staining after treatment by 50 micromol x L(-1) PON. Western blot showed cleavage of the caspase-3 zymogen protein (32 kD), with the appearance of its 17 kD subunit, and a cleaved 89 kD fragment of 116 kD PARP was also found. Furthermore, Western blotting also showed that expression of p-AKT and p-P85 in PI3K/AKT signaling pathways was downregulated dramatically whereas the expression of p-P38 as well as p-ERK and p-JNK remained unchanged after the cells were treated by PON for 48 h. The results demonstrate that PON exhibits in vitro anti-leukemia effect by induction of apoptosis in K562 cells, and that PON induced apoptosis in K562 cells mainly related to activation of caspase-3 as well as inactivation of PI3K/AKT signaling pathway via down regulation of the expression of p-AKT and p-P85 protein levels. These results provide strong laboratory evidence for further anti-leukemia trials of PON.

Protein Targeting to the Albino3 Insertase

The chloroplast thylakoid membrane is the site of photosynthetic light capture and oxygenic electron transport. Nuclear encoded light harvesting chlorophyll‐binding proteins (LHCPs), which bind chlorophyll a/b, provide much of the light absorbing capacity in plants. Following their import into the chloroplast, LHCPs are routed to the thylakoid where they are inserted into the lipid bilayer by the Albino3 (Alb3) insertase. Intra‐chloroplast targeting of LHCPs to Alb3 is accomplished by a chloroplast signal recognition particle (cpSRP), which binds LHCPs in the soluble stroma and directs them to the membrane where they are thought to be passed to Alb3 for post‐translational insertion. CpSRP is composed of a conserved 54 kD subunit (cpSRP54) and a chloroplast‐specific 43 kD subunit (cpSRP43). Data from protein localization and protein interaction assays shows that cpSRP43 binding to Alb3 is critical for LHCP localization. Alb3 is also thought to function in the co‐translational insertion of chloroplast synthesized proteins by a co‐translational targeting/insertion mechanism. This presents the possibility that the post‐ and co‐translational targeting pathways converge at Alb3 for insertion into the membrane. Results of studies presented here may provide an ability to understand how the relative level of post‐ and co‐translational insertion activity by Alb3 is regulated during thylakoid biogenesis.

Exogenously expressed human Ku70 stabilizes Ku80 in Xenopus oocytes and induces heterologous DNA-PK catalytic activity

The Ku protein is a heterodimer composed of 70 kD (Ku70) and 80 kD (Ku80) subunits. Ku is the regulatory component of the DNA-dependent protein kinase (DNA-PK) that has a catalytic subunit of approximately 460 kD (DNA-PK(cs)). In this study, the two polypeptides (Ku80/Ku70) of the human Ku were expressed in Xenopus oocytes in order to investigate their over-expression, sub-cellular localization, and functional interaction with the Xenopus DNA-PK(cs). In vitro-transcribed mRNAs for Ku70 and Ku80 were obtained from the respective plasmid constructs. The exogenously expressed proteins from the injected mRNAs were immunoprecipitated using a specific anti-T7 Tag antibody. The T7 Tag epitope is present in the vector at the amino-terminus and is in-frame with the Ku cDNA sequences. While injected Ku70 mRNA translated to a full-length Ku70 polypeptide that translocated to the nucleus, injected Ku80 mRNA resulted in the expression of a truncated product that was retained in the cytoplasm. Although Ku80 mRNA was stable for a period of 18 h in the oocytes post-microinjection, the protein was only stabilized when co-expressed with Ku70, suggesting that Ku80 is susceptible to proteolytic degradation when not dimerized with Ku70. Furthermore, the immunocomplex was capable of phosphorylating the DNA-PK-specific substrate thereby indicating that the holoenzyme could functionally reconstitute in vivo in the oocytes by heterologous subunits thus demonstrating evolutionary conservation of the enzyme subunit structure and function among diverse species.

Expression and purification of his-tagged recombinant mouse ζ-crystallin

ζ-Crystallin is an NADPH-binding protein consisting of four identical 35 kD subunits. The protein possesses quinone oxidoreductase activity, and is present in large amounts in the lenses of camelids, certain hystricomorphic rodents, and the Japanese tree frog, and in lower catalytic amounts in certain tissues of various species. In this study, recombinant methods were used to produce substantial quantities of his-tagged recombinant mouse ζ-crystallin, which was then purified to homogeneity. The yield of pure recombinant mouse ζ-crystallin was five times that obtained previously for purification of recombinant guinea pig ζ-crystallin. The quinone oxidoreductase activity of purified his-tagged recombinant mouse ζ-crystallin was comparable to that of purified native guinea pig lens ζ-crystallin, and to that previously reported for recombinant guinea pig ζ-crystallin. The method permits production of substantial amounts of recombinant ζ-crystallin for conducting studies on the biological role of this interesting protein, which exists in such high concentration in the lenses of certain species.