scholarly journals Molecular cloning and characterization of CIDE-3, a novel member of the cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector family

2003 ◽  
Vol 370 (1) ◽  
pp. 195-203 ◽  
Author(s):  
Liang LIANG ◽  
Mujun ZHAO ◽  
Zhenhua XU ◽  
Kazunari K. YOKOYAMA ◽  
Tsaiping LI
Keyword(s):  
Amino Acids ◽  
Cell Death ◽  
Small Intestine ◽  
Dna Fragmentation ◽  
Nuclear Dna ◽  
Fluorescent Protein ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Green Fluorescent

DNA fragmentation is one of the critical steps in apoptosis, which is induced by DNA fragmentation factor (DFF). DFF is composed of two subunits, a 40kDa caspase-activated nuclease (DFF40) and a 45kDa inhibitor (DFF45). Recently a novel family of cell-death-inducing DFF45-like effectors (CIDEs) has been identified. Among CIDEs, two from human (CIDE-A and CIDE-B) and three from mouse (CIDE-A, CIDE-B and FSP27) have been reported. In this study human CIDE-3, a novel member of CIDEs, was identified upon sequence analysis of a previously unidentified cDNA that encoded a protein of 238 amino acids. It was shown to be a human homologue of mouse FSP27, and shared homology with the CIDE-N and CIDE-C domains of CIDEs. Apoptosis-inducing activity was clearly shown by DNA-fragmentation assay of the nuclear DNA of CIDE-3 transfected 293T cells. The expression pattern of CIDE-3 was different from that of CIDE-B. As shown by Northern-blot analysis, CIDE-3 was expressed mainly in human small intestine, heart, colon and stomach, while CIDE-B showed strong expression in liver and small intestine and at a lower level in colon, kidney and spleen. Green-fluorescent-protein-tagged CIDE-3 was revealed in some cytosolic corpuscles. Alternative splicing of the CIDE-3 gene was also identified by reverse transcription PCR, revealing that two transcripts, CIDE-3 and CIDE-3α, were present in HepG2 and A375 cells. CIDE-3 comprised a full-length open reading frame with 238 amino acids; in CIDE-3α exon 3 was deleted and it encoded a protein of 164 amino acids. Interestingly the CIDE-3α isoform still kept the apoptosis-inducing activity and showed the same pattern of subcellular localization as CIDE-3. Consistent with its chromosome localization at 3p25, a region associated with high frequency loss of heterozygosity in many tumours, CIDE-3 may play an important role in prevention of tumorigenesis.

One-carbon metabolism in plants: characterization of a plastid serine hydroxymethyltransferase

2010 ◽  
Vol 430 (1) ◽  
pp. 97-105 ◽  
Author(s):  
Yi Zhang ◽  
Kehan Sun ◽  
Francisco J. Sandoval ◽  
Katherine Santiago ◽  
Sanja Roje
Keyword(s):  
Fluorescent Protein ◽  
Catalytic Efficiency ◽  
Protein Characterization ◽  
Serine Hydroxymethyltransferase ◽  
Substrate Affinity ◽  
Hydroxymethyl Group ◽  
Group Transfer ◽  
Reverse Transcription Pcr ◽  
Green Fluorescent

SHMT (serine hydroxymethyltransferase; EC 2.1.2.1) catalyses reversible hydroxymethyl group transfer from serine to H4PteGlun (tetrahydrofolate), yielding glycine and 5,10-methylenetetrahydrofolate. In plastids, SHMTs are thought to catalytically direct the hydroxymethyl moiety of serine into the metabolic network of H4PteGlun-bound one-carbon units. Genes encoding putative plastid SHMTs were found in the genomes of various plant species. SHMT activity was detected in chloroplasts in pea (Pisum sativum) and barley (Hordeum vulgare), suggesting that plastid SHMTs exist in all flowering plants. The Arabidopsis thaliana genome encodes one putative plastid SHMT (AtSHMT3). Its cDNA was cloned by reverse transcription–PCR and the encoded recombinant protein was produced in Escherichia coli. Evidence that AtSHMT3 is targeted to plastids was found by confocal microscopy of A. thaliana protoplasts transformed with proteins fused to enhanced green fluorescent protein. Characterization of recombinant AtSHMT3 revealed that substrate affinity for and the catalytic efficiency of H4PteGlu1-8 increase with n, and that H4PteGlu1-8 inhibit AtSHMT3. 5-Methyltetrahydrofolate and 5-formyltetrahydrofolate with one and five glutamate residues inhibited AtSHMT3-catalysed hydroxymethyl group transfer from serine to H4PteGlu6, with the pentaglutamylated inhibitors being more effective. Calculations revealed inhibition with 5-methyltetrahydrofolate or 5-formyltetrahydrofolate resulting in little reduction in AtSHMT3 activity under folate concentrations estimated for plastids.

scholarly journals Expression of the nfa1 Gene Cloned from Pathogenic Naegleria fowleri in Nonpathogenic N. gruberi Enhances Cytotoxicity against CHO Target Cells In Vitro

2005 ◽  
Vol 73 (7) ◽  
pp. 4098-4105 ◽  
Author(s):  
Seok-Ryoul Jeong ◽  
Sang-Chul Lee ◽  
Kyoung-Ju Song ◽  
Sun Park ◽  
Kyongmin Kim ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Target Cells ◽  
Naegleria Fowleri ◽  
Specific Primers ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Nægleria Fowleri ◽  
Green Fluorescent ◽  
Naegleria Gruberi

ABSTRACT The pathogenic amoeba Naegleria fowleri has a 360-bp nfa1 gene that encodes the Nfa1 protein (13.1 kDa), which is located in the pseudopodia of the amoeba, and an anti-Nfa1 antibody reduces N. fowleri-induced mammalian-cell cytotoxicity in vitro. In contrast, an anti-Nfa1 antibody cannot detect Nfa1 protein expression in the nonpathogenic amoeba Naegleria gruberi, which also possesses the nfa1 gene. In the present study, the nfa1 gene cloned from pathogenic N. fowleri was transfected into nonpathogenic N. gruberi to determine whether it was related to pathogenicity. The nfa1 gene was initially inserted into a eukaryotic transfection vector, pEGFP-C2, containing a cytomegalovirus promoter and the green fluorescent protein (GFP) gene, and was designed as pEGFP-C2/nfa1UTR (nfa1UTR contains 5′ upstream regions, the nfa1 open reading frame, and 3′ downstream regions). After transfection, the green fluorescence was observed in the cytoplasm of N. gruberi trophozoites. These transfectants were preserved for more than 9 months after selection. The transfected nfa1 gene was observed by PCR using nfa1- and vector-specific primers in the genomic DNA of N. gruberi transfected with the pEGFP-C2/nfa1UTR vector. In addition, the nfa1 and GFP genes were identified by reverse transcription-PCR in transgenic N. gruberi. The Nfa1 protein expressed in transgenic N. gruberi was identified as a 13.1-kDa band by Western blotting using an anti-Nfa1 antibody. Finally, N. gruberi transfected with the pEGFP-C2/nfa1UTR vector was found to have enhanced cytotoxicity against CHO cells compared with naïve N. gruberi.

scholarly journals Yeast Mitochondrial Initiator tRNA Is Methylated at Guanosine 37 by the Trm5-encoded tRNA (Guanine-N1-)-methyltransferase

2007 ◽  
Vol 282 (38) ◽  
pp. 27744-27753 ◽  
Author(s):  
Changkeun Lee ◽  
Gisela Kramer ◽  
David E. Graham ◽  
Dean R. Appling
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Fluorescent Protein ◽  
Mitochondrial Protein ◽  
Subcellular Fractionation ◽  
Mitochondrial Protein Synthesis ◽  
Reading Frame ◽  
Initiator Trna ◽  
Chromatography Analysis ◽  
Green Fluorescent

The TRM5 gene encodes a tRNA (guanine-N1-)-methyltransferase (Trm5p) that methylates guanosine at position 37 (m1G37) in cytoplasmic tRNAs in Saccharomyces cerevisiae. Here we show that Trm5p is also responsible for m1G37 methylation of mitochondrial tRNAs. The TRM5 open reading frame encodes 499 amino acids containing four potential initiator codons within the first 48 codons. Full-length Trm5p, purified as a fusion protein with maltose-binding protein, exhibited robust methyltransferase activity with tRNA isolated from a Δtrm5 mutant strain, as well as with a synthetic mitochondrial initiator tRNA (tRNAMetf). Primer extension demonstrated that the site of methylation was guanosine 37 in both mitochondrial tRNAMetf and tRNAPhe. High pressure liquid chromatography analysis showed the methylated product to be m1G. Subcellular fractionation and immunoblotting of a strain expressing a green fluorescent protein-tagged version of the TRM5 gene revealed that the enzyme was localized to both cytoplasm and mitochondria. The slightly larger mitochondrial form was protected from protease digestion, indicating a matrix localization. Analysis of N-terminal truncation mutants revealed that a Trm5p active in the cytoplasm could be obtained with a construct lacking amino acids 1–33 (Δ1–33), whereas production of a Trm5p active in the mitochondria required these first 33 amino acids. Yeast expressing the Δ1–33 construct exhibited a significantly lower rate of oxygen consumption, indicating that efficiency or accuracy of mitochondrial protein synthesis is decreased in cells lacking m1G37 methylation of mitochondrial tRNAs. These data suggest that this tRNA modification plays an important role in reading frame maintenance in mitochondrial protein synthesis.

scholarly journals An Acidic Cluster of Human Cytomegalovirus UL99 Tegument Protein Is Required for Trafficking and Function

2004 ◽  
Vol 78 (3) ◽  
pp. 1488-1502 ◽  
Author(s):  
Thomas R. Jones ◽  
Shi-Wu Lee
Keyword(s):  
Amino Acids ◽  
Human Cytomegalovirus ◽  
Fluorescent Protein ◽  
Amino Acid Sequences ◽  
Cytoplasmic Vacuole ◽  
Viral Growth ◽  
Reading Frame ◽  
Tegument Proteins ◽  
Green Fluorescent ◽  
And Function

ABSTRACT The human cytomegalovirus (HCMV) virion is comprised of a linear double-stranded DNA genome, proteinaceous capsid and tegument, and a lipid envelope containing virus-encoded glycoproteins. Of these components, the tegument is the least well defined in terms of both protein content and function. Several of the major tegument proteins are phosphoproteins (pp), including pp150, pp71, pp65, and pp28. pp28, encoded by the UL99 open reading frame (ORF), traffics to vacuole-like cytoplasmic structures and was shown recently to be essential for envelopment. To elucidate the UL99 amino acid sequences necessary for its trafficking and function in the HCMV replication cycle, two types of viral mutants were analyzed. Using a series of recombinant viruses expressing various UL99-green fluorescent protein fusions, we demonstrate that myristoylation at glycine 2 and an acidic cluster (AC; amino acids 44 to 57) are required for the punctate perinuclear and cytoplasmic (vacuole-like) localization observed for wild-type pp28. A second approach involving the generation of several UL99 deletion mutants indicated that at least the C-terminal two-thirds of this ORF is nonessential for viral growth. Furthermore, the data suggest that an N-terminal region of UL99 containing the AC is required for viral growth. Regarding virion incorporation or UL99-encoded proteins, we provide evidence that suggests that a hypophosphorylated form of pp28 is incorporated, myristoylation is required, and sequences within the first 57 amino acids are sufficient.

Galectin-3 is modulated in pancreatic cancer cells under hypoxia and nutrient deprivation

2020 ◽  
Vol 401 (10) ◽  
pp. 1153-1165 ◽  
Author(s):  
Antônio F. da Silva Filho ◽  
Lucas B. Tavares ◽  
Maira G. R. Pitta ◽  
Eduardo I. C. Beltrão ◽  
Moacyr J. B. M. Rêgo
Keyword(s):  
Pancreatic Cancer ◽  
Cell Death ◽  
Mrna Expression ◽  
Cancer Cells ◽  
Ductal Adenocarcinoma ◽  
Reverse Transcription Pcr ◽  
Pancreatic Cancer Cells ◽  
Through Flow ◽  
Galectin 3

AbstractPancreatic ductal adenocarcinoma is one of the most aggressive tumors with a microenvironment marked by hypoxia and starvation. Galectin-3 has been evaluated in solid tumors and seems to present both pro/anti-tumor effects. So, this study aims to characterize the expression of Galectin-3 from pancreatic tumor cells and analyze its influence for cell survive and motility in mimetic microenvironment. For this, cell cycle and cell death were accessed through flow cytometry. Characterization of inside and outside Galectin-3 was performed through Real-Time Quantitative Reverse Transcription PCR (qRT-PCR), immunofluorescence, Western blot, and ELISA. Consequences of Galectin-3 extracellular inhibition were investigated using cell death and scratch assays. PANC-1 showed increased Galectin-3 mRNA expression when cultivated in hypoxia for 24 and 48 h. After 24 h in simultaneously hypoxic/deprived incubation, PANC-1 shows increased Galectin-3 protein and secreted levels. For Mia PaCa-2, cultivation in deprivation was determinant for the increasing in Galectin-3 mRNA expression. When cultivated in simultaneously hypoxic/deprived condition, Mia PaCa-2 also presented increasing for the Galectin-3 secreted levels. Treatment of PANC-1 cells with lactose increased the death rate when cells were incubated simultaneously hypoxic/deprived condition. Therefore, it is possible to conclude that the microenvironmental conditions modulate the Galectin-3 expression on the transcriptional and translational levels for pancreatic cancer cells.

scholarly journals Rotavirus Virus-Like Particles as Surrogates in Environmental Persistence and Inactivation Studies

2004 ◽  
Vol 70 (7) ◽  
pp. 3904-3909 ◽  
Author(s):  
Santiago Caballero ◽  
F. Xavier Abad ◽  
Fabienne Loisy ◽  
Françoise S. Le Guyader ◽  
Jean Cohen ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Uv Light ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Virus Removal ◽  
Virus Like Particles ◽  
Reverse Transcription Pcr ◽  
Actual Field ◽  
Uv Dose ◽  
Green Fluorescent

ABSTRACT Virus-like particles (VLPs) with the full-length VP2 and VP6 rotavirus capsid proteins, produced in the baculovirus expression system, have been evaluated as surrogates of human rotavirus in different environmental scenarios. Green fluorescent protein-labeled VLPs (GFP-VLPs) and particles enclosing a heterologous RNA (pseudoviruses), whose stability may be monitored by flow cytometry and antigen capture reverse transcription-PCR, respectively, were used. After 1 month in seawater at 20°C, no significant differences were observed between the behaviors of GFP-VLPs and of infectious rotavirus, whereas pseudovirus particles showed a higher decay rate. In the presence of 1 mg of free chlorine (FC)/liter both tracers persisted longer in freshwater at 20°C than infectious viruses, whereas in the presence of 0.2 mg of FC/liter no differences were observed between tracers and infectious rotavirus at short contact times. However, from 30 min of contact with FC onward, the decay of infectious rotavirus was higher than that of recombinant particles. The predicted Ct value for a 90% reduction of GFP-VLPs or pseudoviruses induces a 99.99% inactivation of infectious rotavirus. Both tracers were more resistant to UV light irradiation than infectious rotavirus in fresh and marine water. The effect of UV exposure was more pronounced on pseudovirus than in GFP-VLPs. In all types of water, the UV dose to induce a 90% reduction of pseudovirus ensures a 99.99% inactivation of infectious rotavirus. Recombinant virus surrogates open new possibilities for the systematic validation of virus removal practices in actual field situations where pathogenic agents cannot be introduced.

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