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1.
J Plast Reconstr Aesthet Surg ; 72(3): 467-476, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30579912

ABSTRACT

INTRODUCTION: Composite anterolateral thigh (ALT) flap with vascularized fascia lata can reconstitute patellar tendon integrity and knee soft tissue coverage in one stage. However, long-term evidence of outcomes is lacking. This work analyzes long-term functional results, compares subtotal and total reconstruction of patellar tendon, and assesses the respective function of the extensor apparatus. PATIENTS AND METHODS: Outcomes of reconstruction using 10 ALT flaps in 9 patients (age range 21-87 years) were analyzed (mean follow-up 30 ±â€¯6 months). Knee Society Scores, isometric knee extensor strength (M1-M5), and sensory recovery were evaluated, together with active range of motion and extensor lag of the reconstructed limb, compared to contralateral. RESULTS: Ten flaps were used for tendon replacement in 9 patients. Eight (80%) free flaps and 2 (20%) propeller distally based flaps were used. Complications requiring the harvest of a second flap were seen in 2 patients. All patients could return to their daily activities without the use of walking supports. Mean active ROM was 94.4° with an extensor lag of 9.4°, without a significant difference between partial and total patellar tendon reconstruction. The mean knee and functional scores of the Knee Society were 81/100 and 77/100, respectively. CONCLUSION: Composite ALT flap with fascia lata can satisfy the twofold needs of functional restoration and soft tissue coverage, thus ensuring stable results in total and subtotal knee extensor mechanism reconstruction. Distally based flaps should be carefully considered, as they lead to higher complication rates.


Subject(s)
Fascia Lata/transplantation , Free Tissue Flaps/transplantation , Patella/surgery , Plastic Surgery Procedures/methods , Tendons/surgery , Thigh/surgery , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Plastic Surgery Procedures/adverse effects , Young Adult
2.
Cell Death Dis ; 5: e1158, 2014 Apr 10.
Article in English | MEDLINE | ID: mdl-24722283

ABSTRACT

Human diseases of impaired ribosome biogenesis resulting from disruption of rRNA biosynthesis or loss of ribosomal components are collectively described as 'ribosomopathies'. Treacher Collins syndrome (TCS), a representative human ribosomopathy with craniofacial abnormalities, is attributed to mutations in the tcof1 gene that has a homologous gene called nopp140. Previous studies demonstrated that the dao-5 (dauer and aged animal overexpression gene 5) of Caenorhabditis elegans is a member of nopp140 gene family and plays a role in nucleogenesis in the early embryo. Here, we established a C. elegans model for studying Nopp140-associated ribosomopathy. A null dao-5 mutant ok542 with a semi-infertile phenotype showed a delay in gonadogenesis, as well as a higher incidence of germline apoptosis. These phenotypes in dao-5(ok542) are likely resulted from inefficient rDNA transcription that was observed by run-on analyses and chromatin immunoprecipitation (ChIP) assays measuring the RNA Pol I occupancy on the rDNA promoter. ChIP assays further showed that the modifications of acetylated histone 4 (H4Ac) and dimethylation at the lysine 9 of histone 3 (H3K9me2) around the rDNA promoter were altered in dao-5 mutants compared with the N2 wild type. In addition, activated CEP-1 (a C. elegans p53 homolog) activity was also linked to the loss of DAO-5 in terms of the transcriptional upregulation of two CEP-1 downstream effectors, EGL-1 and CED-13. We propose that the dao-5 mutant of C. elegans can be a valuable model for studying human Nopp140-associated ribosomopathy at the cellular and molecular levels.


Subject(s)
Apoptosis , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , DNA, Ribosomal/genetics , DNA-Binding Proteins/genetics , Germ Cells/cytology , Mutation/genetics , Transcription, Genetic , Amino Acid Sequence , Animals , Caenorhabditis elegans/cytology , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/metabolism , Cell Nucleolus/metabolism , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Conserved Sequence , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Genes, Helminth , Germ Cells/metabolism , Gonads/abnormalities , Gonads/metabolism , Histones/metabolism , Humans , Models, Biological , Molecular Sequence Data , Promoter Regions, Genetic/genetics , Protein Structure, Tertiary , RNA Polymerase I/metabolism , Transcriptional Activation , Tumor Suppressor Protein p53/metabolism
3.
Cell Death Dis ; 4: e616, 2013 May 02.
Article in English | MEDLINE | ID: mdl-23640458

ABSTRACT

Glucose 6-phosphate dehydrogenase (G6PD) deficiency, known as favism, is classically manifested by hemolytic anemia in human. More recently, it has been shown that mild G6PD deficiency moderately affects cardiac function, whereas severe G6PD deficiency leads to embryonic lethality in mice. How G6PD deficiency affects organisms has not been fully elucidated due to the lack of a suitable animal model. In this study, G6PD-deficient Caenorhabditis elegans was established by RNA interference (RNAi) knockdown to delineate the role of G6PD in animal physiology. Upon G6PD RNAi knockdown, G6PD activity was significantly hampered in C. elegans in parallel with increased oxidative stress and DNA oxidative damage. Phenotypically, G6PD-knockdown enhanced germ cell apoptosis (2-fold increase), reduced egg production (65% of mock), and hatching (10% of mock). To determine whether oxidative stress is associated with G6PD knockdown-induced reproduction defects, C. elegans was challenged with a short-term hydrogen peroxide (H2O2). The early phase egg production of both mock and G6PD-knockdown C. elegans were significantly affected by H2O2. However, H2O2-induced germ cell apoptosis was more dramatic in mock than that in G6PD-deficient C. elegans. To investigate the signaling pathways involved in defective oogenesis and embryogenesis caused by G6PD knockdown, mutants of p53 and mitogen-activated protein kinase (MAPK) pathways were examined. Despite the upregulation of CEP-1 (p53), cep-1 mutation did not affect egg production and hatching in G6PD-deficient C. elegans. Neither pmk-1 nor mek-1 mutation significantly affected egg production, whereas sek-1 mutation further decreased egg production in G6PD-deficient C. elegans. Intriguingly, loss of function of sek-1 or mek-1 dramatically rescued defective hatching (8.3- and 9.6-fold increase, respectively) induced by G6PD knockdown. Taken together, these findings show that G6PD knockdown reduces egg production and hatching in C. elegans, which are possibly associated with enhanced oxidative stress and altered MAPK pathways, respectively.


Subject(s)
Apoptosis , Caenorhabditis elegans Proteins/metabolism , Germ Cells/metabolism , Glucosephosphate Dehydrogenase/metabolism , Amino Acid Sequence , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/antagonists & inhibitors , Caenorhabditis elegans Proteins/genetics , DNA Damage , Embryonic Development , Glucosephosphate Dehydrogenase/antagonists & inhibitors , Glucosephosphate Dehydrogenase/genetics , Glucosephosphate Dehydrogenase Deficiency/metabolism , Glucosephosphate Dehydrogenase Deficiency/pathology , Humans , Hydrogen Peroxide/toxicity , MAP Kinase Kinase 1/genetics , MAP Kinase Kinase 1/metabolism , Mice , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Molecular Sequence Data , Oxidative Stress/drug effects , RNA Interference , RNA, Small Interfering/metabolism , Sequence Alignment , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
4.
J Bone Joint Surg Br ; 94(8): 1016-23, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22844040

ABSTRACT

The current indications for functional restoration of extension of the knee following quadriceps resection or loss require reappraisal. The contribution of pedicled and free functional muscle transfer is likely to be over-emphasised in many studies, with good functional outcomes predominantly reported only in the context of cases with residual quadriceps function. In cases with total quadriceps resection or loss, all forms of reconstruction perform poorly. Furthermore, in smaller resections with loss of two or fewer components of the quadriceps, minimal impairment of function occurs in the absence of functional reconstruction, suggesting that functional restoration may not be warranted. Thus there is a paradox in the current approach to quadriceps reconstruction, in that small resections are likely to be over-treated and large resections remain under-treated. This review suggests a shift is required in the approach and rationale for reconstructing functional extension of the knee after quadriceps resection or loss. A classification based on current evidence is suggested that emphasises more clearly the indications and rationale for functional transfers.


Subject(s)
Knee Joint/surgery , Quadriceps Muscle/surgery , Humans , Knee Joint/physiopathology , Muscle, Skeletal/transplantation , Recovery of Function , Treatment Outcome
5.
J Hand Surg Br ; 30(5): 482-3, 2005 Oct.
Article in English | MEDLINE | ID: mdl-15993993

ABSTRACT

Subcutaneous emphysema in the hand is commonly associated with infection or high-pressure injection injuries, with other non-infectious causes being reported as rarities in the literature. We describe an unusual case of minor injury to the first webspace resulting in significant subcutaneous emphysema.


Subject(s)
Hand Injuries/complications , Subcutaneous Emphysema/etiology , Wounds, Stab/complications , Anti-Bacterial Agents/therapeutic use , Antibiotic Prophylaxis , Female , Floxacillin/therapeutic use , Hand Injuries/drug therapy , Hand Injuries/etiology , Humans , Middle Aged , Subcutaneous Emphysema/drug therapy , Tetanus Toxoid , Wounds, Stab/drug therapy
6.
J Biomed Sci ; 8(6): 492-503, 2001.
Article in English | MEDLINE | ID: mdl-11702013

ABSTRACT

Hepadnaviruses and retroviruses are evolutionarily related families because they both require a process of reverse transcription for genome replication. However, hepadnaviruses produce polymerase (pol) and core proteins separately, while retroviruses synthesize a gag-pol fusion protein that is subsequently cleaved by a virally encoded protease to release a functional polymerase. To test whether an additional sequence at the N-terminus of pol in hepatitis B virus (HBV) interferes with its function, we created two plasmids expressing core-pol fusion proteins, core144-pol and core31-pol. Secreted particles obtained from HuH-7 cells, which were cotransfected with a core-pol fusion protein-expressing plasmid and a core-expressing plasmid, showed a positive signal of HBV DNA by the endogenous polymerase assay, indicating that the core-pol fusion proteins retain DNA priming, polymerization and RNase H activities. The fusion protein was detected in the cytoplasm of transfected cells and in secreted virions by immunoprecipitation. Furthermore, we found by immunofluorescence staining that the HBV core-pol fusion protein colocalized with the hepatitis C virus (HCV) core protein in cytoplasm and in lipid droplets. Immunoprecipitation studies showed that the anti-HCV core complex contained the HBV core-pol fusion protein while the anti-HBV pol complex contained the HCV core protein, which supports the hypothesis that the HCV core protein can form a complex with the HBV core-pol fusion protein.


Subject(s)
Gene Products, pol/metabolism , Hepatitis B Core Antigens/metabolism , Recombinant Fusion Proteins/metabolism , Viral Core Proteins/metabolism , Cytoplasm/chemistry , Evolution, Molecular , Gene Products, pol/genetics , Gene Products, pol/physiology , Hepatitis B Core Antigens/genetics , Hepatitis B Core Antigens/physiology , Humans , Microscopy, Fluorescence , Precipitin Tests , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/physiology , Retroviridae , Transfection , Tumor Cells, Cultured , Virion/chemistry
7.
Virology ; 285(1): 138-52, 2001 Jun 20.
Article in English | MEDLINE | ID: mdl-11414814

ABSTRACT

Hepatitis D virus (HDV) requires the isoprenylated large delta antigen (LDAg) for interaction with hepatitis B surface antigen (HBsAg) to allow packaging and secretion out of the host cell. Phosphorylated LDAg has been found but, as yet, neither localization of LDAg within the nucleus nor any other function has been correlated with modification. In this study, we transfected HuH-7 or HeLa cells with plasmids encoding various lengths of LDAg [designated GFP-LD and GFP-LD(31-214) for full length and a deletion, respectively] or non-isoprenylated mutants of these [designated GFP-LDM and GFP-LD(31-214)M] fused to the green fluorescent protein (GFP). These fusion proteins were then characterized and it was found that: (i) the addition of the GFP did not interfere with the functioning of the full-length or N-terminally deleted LDAgs when interacting with HBsAg for secretion; (ii) the HDV small antigen (SDAg) together with the GFP-LD, but not the GFP-LD(31-214), could be cosecreted by HBsAg; and (iii) the GFP-LD, but not the GFP-LD(31-214), exerted a dominant-negative role on HDV genome replication. Analyses of transiently transfected cells and postmitotic permanent cells revealed the sequential appearance of GFP-LD in the nucleoplasm, then in the nucleolus, and finally in nuclear speckles (NS). Isoprenylation of LDAg seems to be important for targeting to and accumulating in the NS, which was evident from the dynamic and static localization of the non-isoprenylation mutant (GFP-LDM) and the distribution of wild-type (GFP-LD) when treated with an isoprenylation inhibitor, lovastatin, for more than 48 h. Permanently expressing GFP-LD cells allowed us to show the dynamic redistribution of dephosphorylated GFP-LD from the nucleolus to the SC-35 containing NS in the presence of dichlororibofuranosyl benzimidazole (DRB) and then the translocation back of the GFP-LD to the nucleolus within 2 h after removal of DRB. Our studies thus suggest that the various versions of the GFP-LD fusion protein, having the same function as their nonfusion counterparts, can be a powerful tool for the study of the dynamic localization of LDAg when correlated with the functional modification of this protein.


Subject(s)
Hepatitis Antigens/metabolism , Hepatitis Delta Virus/immunology , Luminescent Proteins/metabolism , Ribonucleoproteins , Cell Nucleolus/metabolism , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Dichlororibofuranosylbenzimidazole/pharmacology , Enzyme Inhibitors/pharmacology , Green Fluorescent Proteins , HeLa Cells , Hepatitis Antigens/genetics , Hepatitis B Surface Antigens/metabolism , Hepatitis Delta Virus/genetics , Hepatitis delta Antigens , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Indicators and Reagents , Lovastatin/pharmacology , Mutagenesis, Site-Directed , Mutation , Nuclear Proteins , RNA, Viral/biosynthesis , Recombinant Fusion Proteins/metabolism , Serine-Arginine Splicing Factors , Time Factors , Transfection
8.
Biochem J ; 357(Pt 1): 57-64, 2001 Jul 01.
Article in English | MEDLINE | ID: mdl-11415436

ABSTRACT

Rhodostomin (RHO), a disintegrin isolated from snake venom, has been demonstrated to inhibit platelet aggregation through interaction with integrin alphaIIbbeta3, but there is a lack of direct evidence for RHO-integrin alphaIIbbeta3 binding. In addition, no study on the length of Arg(49)-Gly(50)-Asp(51) (RGD) loop of RHO influencing on its binding to integrin alphaIIbbeta3 has been reported. In the present study we have developed a highly sensitive dot-blot and glutathione S-transferase-RHO pull-down assays; the latter was coupled with a biotin-avidin-horseradish peroxidase enhanced-chemiluminescence detection system. These were able to demonstrate the direct binding of RHO to integrin alphaIIbbeta3. The pull-down assay further showed that four alanine-insertion mutants upstream of the RGD motif and three insertions downstream of the RGD were able to decrease integrin alphaIIbbeta3 binding activity to only a limited extent. By contrast, two insertions immediately next to RGD and one insertion in front of the Cys(57) caused almost complete loss of binding activity to alphaIIbbeta3. The results of the platelet-aggregation-inhibition assay and platelet-adhesion assay for the insertion mutants were consistent with results of the pull-down assay. It is thus concluded that, although an insertion of a single alanine residue in many positions of the RGD loop has only minor effects on RHO binding to integrin alphaIIbbeta3, the specific position of Pro(53) residue adjacent to the RGD sequence is important for RHO binding to platelet integrin alphaIIbbeta3.


Subject(s)
Blood Platelets/physiology , Oligopeptides/chemistry , Peptides/chemistry , Peptides/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/chemistry , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Proline , Alanine , Amino Acid Sequence , Binding Sites , Cloning, Molecular , Escherichia coli , Glutathione Transferase/metabolism , Humans , Kinetics , Luminescent Measurements , Molecular Sequence Data , Mutagenesis, Insertional , Peptides/genetics , Peptides/pharmacology , Platelet Adhesiveness/drug effects , Platelet Adhesiveness/physiology , Platelet Aggregation/drug effects , Platelet Aggregation/physiology , Platelet Aggregation Inhibitors/chemistry , Platelet Aggregation Inhibitors/pharmacology , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , Sequence Alignment , Thrombin/pharmacology
9.
Proteins ; 43(4): 499-508, 2001 Jun 01.
Article in English | MEDLINE | ID: mdl-11340665

ABSTRACT

Rhodostomin (Rho) is a snake venom protein isolated from Calloselasma rhodostoma. Rho is a disintegrin that inhibits platelet aggregation by blocking the binding of fibrinogen to the integrin alpha(IIb)beta3 of platelets. Rho produced in Escherichia coli inhibited platelet aggregation with a K(I) value of 263 nM. Although functional, Rho produced in E. coli is misfolded based on our 2D and 3D NMR studies. In order to correct the folding problem, Rho was expressed in Pichia pastoris. The recombinant Rho expressed in P. pastoris inhibited platelet aggregation with a resulting K(I) value of 70 nM. This is the same potency as that of native Rho. CD analysis showed that the secondary structures of Rho are pH-independent and contain 3.5-7.9% alpha-helix, 48.2-50.5% beta-structures, and 42.3-47% coil. The sequential assignment and structure analysis of Rho were obtained using 2D and 3D 15N-edited NMR spectra. These results provide the first direct evidence that highly disulfide-bonded disintegrin can be expressed in P. pastoris with the correct fold. This evidence may serve as the basis for exploring the structure and function relationships as well as the dynamics of disintegrin and its variants.


Subject(s)
Circular Dichroism , Disulfides/chemistry , Escherichia coli/genetics , Magnetic Resonance Spectroscopy , Peptides/chemistry , Peptides/metabolism , Pichia/metabolism , Recombinant Fusion Proteins/isolation & purification , Amino Acid Sequence , Animals , Escherichia coli/metabolism , Gene Expression , Peptides/genetics , Pichia/genetics , Platelet Aggregation/drug effects , Polymerase Chain Reaction , Protein Folding , Protein Structure, Secondary , Recombinant Fusion Proteins/genetics , Sequence Homology, Amino Acid , Solubility
10.
J Virol Methods ; 92(1): 83-90, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11164921

ABSTRACT

Although hepatitis B virus (HBV) DNA has been detected in the human hepatoma cell line, HAGS 2.1, viral and cellular junction sequences have not been investigated fully. To facilitate the analysis of HBV DNA integration sites in HAGS 2.1 cells, a combination of conventional polymerase chain reaction (PCR) and inverse PCR (IPCR) was carried out to identify the junction between the viral and the cellular gene. The HBV integrant and its cellular counterpart sequence were cloned and analyzed. The sequencing data indicated that the breakpoints on the HBV integrant are at nucleotide 2111 of the C gene and nucleotide 1558 of the X gene. The length of the integrated HBV DNA in HAGS 2.1 was approximately 2.6 kb, which includes partial C, P, and X genes and an intact S gene. The cellular sequence flanking the integrated HBV gene was very similar to a human satellite III repetitive sequence with 43 and 56 of GGAAT repeats on the left- and right-hand side, respectively. Although the findings on the viral-cellular junction in HAGS 2.1 cells cannot explain the liver tumorigenesis, the current study shows that by choosing the nearest restriction site, which can be determined by conventional PCR rather than using a unique site within the integrated viral sequence to do IPCR, gives a higher successful rate for cloning and the subsequent analysis of the viral-cellular junctions.


Subject(s)
DNA, Viral/genetics , Hepatitis B virus/genetics , Polymerase Chain Reaction , Virus Integration , Cloning, Molecular , DNA, Satellite/drug effects , DNA, Viral/chemistry , Humans , Sequence Analysis, DNA , Tumor Cells, Cultured
11.
Virology ; 275(2): 398-410, 2000 Sep 30.
Article in English | MEDLINE | ID: mdl-10998339

ABSTRACT

The hepatitis B virus (HBV) genome is known to contain four conserved and overlapped open reading frames (ORFs) encoding the viral core, polymerase (P), surface (S), and X proteins. Whether HBV encodes other proteins has long been a major interest in the field. Using (32)P-labeling of an introduced protein kinase A site attached to the N- or C-terminus of the HBV polymerase gene, a 43-kDa P-S fusion protein was detected in cell lysate, secreted virions, and 22-nm subviral particles. Immunobiochemical studies showed that the 43-kDa protein contains the epitopes of the N-terminus of polymerase and most parts of the surface proteins. This 43-kDa protein was shown to be a glycoprotein, similar to the surface protein. RT-PCR and sequence analyses identified a spliced mRNA which was derived from pregenomic RNA with a deletion of 454 nucleotides (nt) from nt 2447 to 2902. This splice event creates a P-S fusion ORF. This finding is consistent with the result obtained from an immunobiochemical study. Mutations at the splice donor or acceptor site on the HBV genome abrogated the production of the 43-kDa protein. These mutants had no effect on viral replication in transfected HuH-7 cells. However, this P-S fusion protein is able to substitute for the LS protein in virion maturation. On the basis of these results, we conclude that the 43-kDa protein is a polymerase-surface fusion protein encoded by a spliced RNA. Similar to the LS protein, the 43-kDa P-S fusion protein is a structural protein of HBV and might play a role in the HBV life cycle.


Subject(s)
Hepatitis B virus/genetics , RNA Splicing , Viral Fusion Proteins/genetics , Viral Structural Proteins/genetics , Aspartic Acid Endopeptidases/genetics , Cell Line , Electrophoresis, Polyacrylamide Gel , Epitopes , Gene Products, pol/genetics , Glycoproteins/genetics , Glycoproteins/metabolism , Glycosylation , Hepatitis B Surface Antigens/genetics , Hepatitis B virus/metabolism , Humans , Molecular Weight , Open Reading Frames , Precipitin Tests , Reverse Transcriptase Polymerase Chain Reaction , Viral Fusion Proteins/metabolism , Viral Structural Proteins/metabolism
12.
J Biotechnol ; 78(2): 115-22, 2000 Mar 10.
Article in English | MEDLINE | ID: mdl-10725535

ABSTRACT

We have previously developed the TraT display system to express the preS1 peptide of human hepatitis B virus (HBV) and the snake venom rhodostomin (RHO) on the surface of Escherichia coli. In this study, we modified the pT2 vector by adding a thrombin cutting site and a phosphorylation tag of protein kinase A before the multiple restriction enzyme sites. The modified vector allowed us to label the TraT fusion protein (TraT-RHO) with [32P] and to increase the detection sensitivity of TraT-RHO expression bacteria binding to and being internalized into BHK-21 cells. After the thrombin cleavage, the isotope labeled RHO could be detected in a free form. We therefore suggest that the new version of pT2 vector, pT2-KL, will facilitate to identify the counterpart of displayed peptide.


Subject(s)
Bacterial Outer Membrane Proteins/genetics , Cyclic AMP-Dependent Protein Kinases/genetics , Escherichia coli Proteins , Escherichia coli/genetics , Genetic Vectors , Amino Acid Sequence , Bacterial Outer Membrane Proteins/chemistry , Base Sequence , Binding Sites , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Peptides/genetics , Phosphorus Radioisotopes , Phosphorylation , Recombinant Fusion Proteins , Thrombin/metabolism
13.
J Biomed Sci ; 7(1): 42-50, 2000.
Article in English | MEDLINE | ID: mdl-10644888

ABSTRACT

Previously, we developed a TraT display system to express snake venom rhodostomin (RHO), a disintegrin, on the external surface of Escherichia coli [J Biomed Sci 6:64-70;1999]. To show a new potential use of the TraT display system, we employed a biotin labeling technique coupled with SDS-PAGE and flow cytometry analyses to further demonstrate and confirm the expression of TraT-RHO on the E. coli surface. We also showed that the expression of TraT-RHO on the cell surface not only facilitated the bacteria adhesion to BHK-21 cells but also induced bacterial internalization into BHK-21 cells. This feature allowed us to enrich the TraT-RHO expression bacteria about 10,000-fold starting with a mixture of TraT-RHO bacteria with beta-galactosidase-positive bacteria in a ratio of 10(2):10(7) through four cycles of BHK-21 cell endocytosis and replating of engulfed bacteria on agar plates. We therefore suggest that the TraT display system can be applied to select out bacteria expressing a specific peptide sequence from a large population of display library through the process of receptor-mediated endocytosis and reamplification cycles.


Subject(s)
Cloning, Molecular/methods , Endocytosis/drug effects , Peptides/metabolism , Platelet Aggregation Inhibitors/metabolism , Receptors, Cell Surface/physiology , Animals , Cell Adhesion , Cell Line/metabolism , Cricetinae , Escherichia coli/chemistry , Escherichia coli/genetics , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression , Membrane Proteins/biosynthesis , Mesocricetus , Peptide Library , Phenotype , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
14.
Exp Cell Res ; 250(2): 387-400, 1999 Aug 01.
Article in English | MEDLINE | ID: mdl-10413593

ABSTRACT

Platelet activation has been a focus of numerous studies in normal and abnormal states. Morphological changes and calcium signals found with activated platelets in vitro have been well characterized. However, the rate of cell spreading on substrates and the frequency of calcium oscillation within individual platelets upon activation have not yet been reported. In this study, we first examined the ability of a recombinant fusion protein of rhodostomin (GST-rhodostomin), a snake disintegrin containing an Arg-Gly-Asp (RGD) motif, to activate platelets when GST-rhodostomin served as a substrate. Four aspects of platelet activities induced by immobilized GST-rhodostomin and fibrinogen were analyzed in parallel. Examinations of (1) translocation of P-selectin from intracellular compartments to the plasma membrane, (2) platelet adhesion to and spreading on substrates, (3) platelet contact pattern on substrates, and (4) the degree of phosphorylation of focal adhesion kinase in platelets indicated that GST-rhodostomin was a better substrate for platelet activation than fibrinogen. Analysis of the rate of platelet spreading on GST-rhodostomin was examined by time-lapsed video microscopy. The spreading rate averaged 0.43 micrometer/minute, while cell spreading averaged 0.22 micrometer/minute when platelets were plated on fibrinogen and treated with thrombin. A newly developed method, using time-lapsed microscopy and the Metamorph program, was used to analyze calcium signals within platelets. We found that platelets on GST-rhodostomin evoked calcium oscillation at a frequency of 4.77 spike/cell/minute vs 2.76 spike/cell/minute on fibrinogen. The results of cell spreading and calcium oscillation were consistent with the results of microscopic and biochemical assays. We therefore conclude that the determination of the rate of platelet spreading and the frequency of calcium oscillation within platelets performed in this study provides more quantitative parameters for measuring platelet activities. Our results also suggest that GST-rhodostomin might potentially be used as a probe to dissect the molecular mechanisms underlying the kinetic processes of platelet activation.


Subject(s)
Blood Platelets/drug effects , Calcium Signaling/drug effects , Calcium/metabolism , Peptides/pharmacology , Platelet Activation/drug effects , Actins/metabolism , Blood Platelets/cytology , Blood Platelets/metabolism , Blood Platelets/ultrastructure , Cell Adhesion Molecules/metabolism , Cell Size/drug effects , Disintegrins/metabolism , Disintegrins/pharmacology , Fibrinogen/metabolism , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Humans , Kinetics , Microscopy, Electron, Scanning , Microscopy, Video , P-Selectin/metabolism , Peptides/metabolism , Phosphorylation , Platelet Aggregation/drug effects , Protein-Tyrosine Kinases/metabolism , Pseudopodia/drug effects , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , Solubility , Thrombin/metabolism
15.
J Biomed Sci ; 6(1): 64-70, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9933744

ABSTRACT

The traT gene is one of the F factor transfer genes and encodes an outer membrane protein which is involved in interactions between an Escherichia coli and its surroundings. This protein was altered so as to permit the expression of foreign proteins on the outer membrane of E. coli in this study. A 729-bp DNA fragment, including the leader and entire structural gene sequence of traT, was amplified and obtained by PCR. This sequence was then subcloned downstream of the tac promoter of pDR540, resulting in a TraT expression vector, pT2. Here, we report that the expression of TraT protein, fused either with a partial pre-S antigen of hepatitis B virus (60 and 98 amino acids, respectively) or with the snake venom rhodostomin (72 amino acids), was successfully achieved on the outer membrane of E. coli, using the pT2 plasmid. This result was demonstrated using dot blot and immunofluorescence analysis. This finding supports the notion that the pT2 plasmid can be used as an E. coli display system. This system can detect a foreign peptide of about 100 amino acid residues in length on the bacterial surface.


Subject(s)
Bacterial Outer Membrane Proteins/genetics , Escherichia coli Proteins , Escherichia coli/genetics , Hepatitis B Surface Antigens/genetics , Peptides/genetics , Protein Precursors/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Bacterial Outer Membrane Proteins/metabolism , Epitopes , Hepatitis B Surface Antigens/immunology , Hepatitis B Surface Antigens/metabolism , Peptides/immunology , Peptides/metabolism , Plasmids/genetics , Protein Precursors/immunology , Protein Precursors/metabolism
16.
Toxicon ; 36(8): 1087-99, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9690777

ABSTRACT

We have previously reported that non-activated platelets can be induced by morphological changes from the recombinant fusion protein of GST-rhodostomin [GST-RHO(RGD)], a member of disintegrin with an arginine-glycine-aspartic acid (RGD) motif. In this study, we further characterized the factors involved in platelet shape changes induced by rhodostomin. From less to full-spreading, four cell spreading indexes, p1, p2, s1 and s2, were designated to the platelet shape based on the scanning electron micrographs. Results of peptide competition and antibody blocking confirmed that interaction between the RGD of rhodostomin and the alpha(IIb)beta3 integrins of platelets was required for induction of a higher percentage of s2 cells. When platelets were pretreated with calphostin C, herbimycin A and cytochalasin B, respectively, the percentage of p1 and p2 cells on rhodostomin-coated plates was increased and, concomitantly, the percentage of s1 and s2 cells was decreased. Biochemical analyses indicated that the focal adhesion kinase (FAK or pp125FAK) in platelets that adhered to GST-RHO(RGD) was phosphorylated in contrast to little or no phosphorylation of FAK in cells adhered to fibrinogen or non-activated cells. Furthermore, the degree of FAK phosphorylation was consistently correlated with morphological changes in platelets treated with various drugs. Taking all the results together, we suggested that rhodostomin could directly bind to integrins of platelets and then trigger signal transduction leading to FAK phosphorylation and actin polymerization and finally resulting in platelet full-spreading.


Subject(s)
Blood Platelets/drug effects , Cell Adhesion Molecules/metabolism , Peptides/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Protein-Tyrosine Kinases/metabolism , Blood Platelets/cytology , Blood Platelets/enzymology , Cell Adhesion , Cell Movement/drug effects , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Glutathione Transferase/metabolism , Humans , Peptides/metabolism , Phosphorylation , Platelet Aggregation Inhibitors/metabolism , Protein Binding , Protein Kinase C/metabolism , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Signal Transduction
17.
Virus Genes ; 16(2): 147-8, 1998.
Article in English | MEDLINE | ID: mdl-9608658

ABSTRACT

The latency associated transcript (LAT) of herpes simplex virus (HSV) appears to exist as an RNA molecule only. This phenomenon is consistent with the concept of functioning at an RNA level, and several lines of evidence suggest that the LAT may be a ribozyme. This provides an insight into understanding the role of LAT during HSV reactivation.


Subject(s)
Herpesvirus 1, Human/physiology , Herpesvirus 2, Human/physiology , RNA, Catalytic/physiology , Virus Activation/physiology , Virus Latency/physiology , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/growth & development , Herpesvirus 2, Human/genetics , Herpesvirus 2, Human/growth & development , Humans , RNA, Viral/physiology
18.
Cell Mol Life Sci ; 54(12): 1403-11, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9893713

ABSTRACT

Many virus and transposon DNAs can integrate into the host genome. In this review, techniques, including inverse polymerase chain reaction (IPCR), novel Alu-PCR and vectorette- or splinkerette-PCR are introduced as possible strategies for cloning flanking DNA regions of the integrants. Targeted gene-walking PCR, restriction-site PCR, capture PCR, and panhandle PCR and boomerang DNA amplification are also described. The principles, advantages and limitations of each approach are discussed.


Subject(s)
Cloning, Molecular/methods , DNA, Viral/genetics , Animals , DNA, Viral/analysis , Humans , Polymerase Chain Reaction/methods , Sequence Analysis, DNA
19.
Toxicon ; 35(2): 195-204, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9080576

ABSTRACT

Rhodostomin (RHO) from Agkistrodon rhodostoma venom, consisting of 68 amino acids with an arginine-glycine-aspartic acid (RGD) sequence and 12 cysteine residues, is a potent inhibitor of platelet aggregation. We previously demonstrated that cell culture plates coated with the bacterially produced fusion protein of glutathione S-transferase-RHO [GST-RHO(RGD)] can facilitate human hepatoma cell attachment via intergrin interaction within 15 min. In this study, we further characterized the effect of RHO fusion protein on platelet cells by creating two other related fusion proteins, GST-RHO(RGE) and GST-(PS)RHO. The former was a single amino acid-substituted mutant, in which the aspartic acid residue of RGD was replaced by glutamic acid, and the latter was an insertion mutant, in which a pentapeptide of protein kinase A phosphorylation site was inserted between GST and RHO. These two mutant proteins together with a wild-type of GST-RHO(RGD) and native form of RHO were used to study effects on the inhibition of ADP-induced platelet aggregation. Results indicated that GST-RHO(RGD) inhibited platelet aggregation as potently as the native RHO, while the two other mutants were inactive. Furthermore, when unactivated platelet cells attached on the GST-RHO(RGD)-coated plate, they became a flattened pancake shape. From the results of facilitation of cell attachment on fusion protein-coated plates, we concluded that: (1) the GST-RHO(RGD) fusion protein is equally functional in inhibition of platelet aggregation and facilitation of cell attachment, which is through the interaction of RGD and integrins on the cell membrane; (2) the GST-RHO(RGE) mutant protein is unable to bind with integrins and results in loss of function; (3) the insertion mutant of GST-(PS)RHO may disrupt a proper conformation of RHO and also results in loss of function; (4) the bacterially produced fusion protein GST-RHO(RGD) can be properly used as an antithrombotic agent and an extracellular matrix.


Subject(s)
Blood Platelets/drug effects , Glutathione Transferase/pharmacology , Peptides/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Animals , Base Sequence , Humans , Molecular Sequence Data , Platelet Aggregation/drug effects , Rabbits
20.
J Biomed Sci ; 4(5): 235-243, 1997.
Article in English | MEDLINE | ID: mdl-12386385

ABSTRACT

Rhodostomin from venom of Agkistrodon rhodostoma (also called Calloselasma rhodostoma) contains 68 amino acid residues including 6 pairs of disulfide bonds and an arginine-glycine-aspartic acid (RGD) sequence at positions 49-51. It has been known as one of the strongest antagonists to platelet aggregation among the family termed disintegrin. In this review paper, in addition to introducing the characteristics of disintegrin and its related molecules, the advantages of using recombinant DNA technology to produce rhodostomin are described. The recombinant rhodostomin has been demonstrated to facilitate cell adhesion via interaction between the RGD motif of rhodostomin and integrins on the cell surface. This property allowed us to use the recombinant rhodostomin as an extracellular matrix to study cell adhesion and to distinguish attachment efficiency between two melanoma cell lines B16-F1 and B16-F10, the former is a low metastasis cell while the latter is a high metastasis cell. Furthermore, by using the recombinant rhodostomin as a substrate, osteoprogenitor-like cells are able to be selected and enriched within 3 days from rat bone marrow which contains a heterogeneous cell population. Finally, we show that the recombinant rhodostomin can be immobilized on beads and which serve as an affinity column to dissect cell-surface protein(s) binding to the RGD motif of rhodostomin.

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