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1.
Lett Appl Microbiol ; 74(6): 863-872, 2022 Jun.
Article in English | MEDLINE | ID: mdl-35148433

ABSTRACT

Flow cytometry has emerged as a promising technique for detection of SARS-CoV-2 antibodies. In this study, we developed an innovative strategy for simultaneous detection of immunoglobulin G (IgG), IgM and IgA. The SARS-CoV-2 nucleocapsid protein was covalently bound to functional beads surface applying sulpho-SMCC chemistry. BUV395 anti-IgG, BB515 anti-IgM, biotinylated anti-IgA1/IgA2 and BV421 streptavidin were used as fluorophore conjugated secondary antibodies. Serum and antibodies reaction conditions were optimized for each antibody isotype detection and a multiplexed detection assay was developed. This new cell-free assay efficiently discriminate COVID-19 negative and positive samples. The simultaneous detection of IgG, IgM and IgA showed a sensitivity of 88·5-96·2% and specificity of 100%. This novel strategy opens a new avenue for flow cytometry-based diagnosis.


Subject(s)
Antibodies, Viral , COVID-19 , SARS-CoV-2 , Antibodies, Viral/blood , COVID-19/diagnosis , Flow Cytometry , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Nucleocapsid Proteins , SARS-CoV-2/immunology , Sensitivity and Specificity
2.
Arch Gynecol Obstet ; 306(2): 295-314, 2022 08.
Article in English | MEDLINE | ID: mdl-34625835

ABSTRACT

PURPOSE: Radical hysterectomy and pelvic lymphadenectomy is the standard treatment for early cervical cancer. Studies have shown superior oncological outcome for open versus minimal invasive surgery, but peri- and postoperative complication rates were shown vice versa. This meta-analysis evaluates the peri- and postoperative morbidities and complications of robotic and laparoscopic radical hysterectomy compared to open surgery. METHODS: Embase and Ovid-Medline databases were systematically searched in June 2020 for studies comparing robotic, laparoscopic and open radical hysterectomy. There was no limitation in publication year. Inclusion criteria were set analogue to the LACC trial. Subgroup analyses were performed regarding the operative technique, the study design and the date of publication for the endpoints intra- and postoperative morbidity, estimated blood loss, hospital stay and operation time. RESULTS: 27 studies fulfilled the inclusion criteria. Five prospective, randomized-control trials were included. Meta-analysis showed no significant difference between robotic radical hysterectomy (RH) and laparoscopic hysterectomy (LH) concerning intra- and perioperative complications. Operation time was longer in both RH (mean difference 44.79 min [95% CI 38.16; 51.42]), and LH (mean difference 20.96 min; [95% CI - 1.30; 43.22]) than in open hysterectomy (AH) but did not lead to a rise of intra- and postoperative complications. Intraoperative morbidity was lower in LH than in AH (RR 0.90 [0.80; 1.02]) as well as in RH compared to AH (0.54 [0.33; 0.88]). Intraoperative morbidity showed no difference between LH and RH (RR 1.29 [0.23; 7.29]). Postoperative morbidity was not different in any approach. Estimated blood loss was lower in both LH (mean difference - 114.34 [- 122.97; - 105.71]) and RH (mean difference - 287.14 [- 392.99; - 181.28]) compared to AH, respectively. Duration of hospital stay was shorter for LH (mean difference - 3.06 [- 3.28; - 2.83]) and RH (mean difference - 3.77 [- 5.10; - 2.44]) compared to AH. CONCLUSION: Minimally invasive radical hysterectomy appears to be associated with reduced intraoperative morbidity and blood loss and improved reconvalescence after surgery. Besides oncological and surgical factors these results should be considered when counseling patients for radical hysterectomy and underscore the need for new randomized trials.


Subject(s)
Laparoscopy , Robotic Surgical Procedures , Uterine Cervical Neoplasms , Female , Humans , Hysterectomy/adverse effects , Hysterectomy/methods , Laparoscopy/methods , Morbidity , Postoperative Complications/epidemiology , Postoperative Complications/surgery , Prospective Studies , Robotic Surgical Procedures/adverse effects , Robotic Surgical Procedures/methods , Uterine Cervical Neoplasms/surgery
3.
Arch Gynecol Obstet ; 304(3): 577-587, 2021 09.
Article in English | MEDLINE | ID: mdl-34021804

ABSTRACT

PURPOSE: Radical hysterectomy with pelvic lymphadenectomy presents the standard treatment for early cervical cancer. Recently, studies have shown a superior oncological outcome for open versus minimal invasive surgery, however, the reasons remain to be speculated. This meta-analysis evaluates the outcomes of robotic and laparoscopic hysterectomy compared to open hysterectomy. Risk groups including the use of uterine manipulators or colpotomy were created. METHODS: Ovid-Medline and Embase databases were systematically searched in June 2020. No limitation in date of publication or country was made. Subgroup analyses were performed regarding the surgical approach and the endpoints OS and DFS. RESULTS: 30 studies fulfilled the inclusion criteria. Five prospective, randomized-control trials were included. Patients were analyzed concerning the surgical approach [open surgery (AH), laparoscopic surgery (LH), robotic surgery (RH)]. Additionally, three subgroups were created from the LH group: the LH high-risk group (manipulator), intermediate-risk group (no manipulator, intracorporal colpotomy) and LH low-risk group (no manipulator, vaginal colpotomy). Regarding OS, the meta-analysis showed inferiority of LH in total over AH (0.97 [0.96; 0.98]). The OS was significantly higher in LH low risk (0.96 [0.94; 0.98) compared to LH intermediate risk (0.93 [0.91; 0.94]). OS rates were comparable in AH and LH Low-risk group. DFS was higher in the AH group compared to the LH group in general (0.92 [95%-CI 0.88; 0.95] vs. 0.87 [0.82; 0.91]), whereas the application of protective measures (no uterine manipulator in combination with vaginal colpotomy) was associated with increased DFS in laparoscopy (0.91 [0.91; 0.95]). CONCLUSION: DFS and OS in laparoscopy appear to be depending on surgical technique. Protective operating techniques in laparoscopy result in improved minimal invasive survival.


Subject(s)
Carcinoma, Squamous Cell/surgery , Colpotomy/methods , Hysterectomy/methods , Laparoscopy/methods , Uterine Cervical Neoplasms/surgery , Carcinoma, Squamous Cell/mortality , Carcinoma, Squamous Cell/pathology , Colpotomy/instrumentation , Early Detection of Cancer , Female , Humans , Hysterectomy/adverse effects , Minimally Invasive Surgical Procedures , Pregnancy , Uterine Cervical Neoplasms/mortality , Uterine Cervical Neoplasms/pathology
4.
Cell Death Dis ; 6: e1994, 2015 Nov 26.
Article in English | MEDLINE | ID: mdl-26610207

ABSTRACT

We have assessed the impact of α-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinson's disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of α-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson's disease progression, particularly in the context of bioenergetic dysfunction.


Subject(s)
Induced Pluripotent Stem Cells/physiology , Neurons/pathology , Parkinson Disease/genetics , Parkinson Disease/metabolism , alpha-Synuclein/genetics , alpha-Synuclein/metabolism , Cell Differentiation/genetics , Cells, Cultured , Gene Expression Profiling , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Neurons/metabolism , Parkinson Disease/pathology
5.
J Dairy Sci ; 95(6): 3318-26, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22612965

ABSTRACT

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of intestinal disease and hemolytic uremic syndrome, a serious systemic complication that particularly affects children. Cattle are primary reservoirs for EHEC O157:H7 and the main source of infection for humans. Vaccination of cattle with different combinations of bacterial virulence factors has shown efficacy in decreasing EHEC O157:H7 shedding. It is, therefore, important to demonstrate whether vaccination of pregnant cows with EHEC O157:H7 induces high titers of transferable antibodies to avoid early colonization of calves by the bacteria. In this study we evaluated the ability of EspA, EspB, the C-terminal fragment of 280 amino acids of γ-intimin (γ-intimin C280) and inactivated Shiga toxin (Stx) 2 proteins to induce specific antibodies in colostrum and their passive transference to colostrum-fed calves. Friesian pregnant cows immunized by the intramuscular route mounted significantly high serum and colostrum IgG responses against EspB and γ-intimin C280 that were efficiently transferred to their calves. Antibodies to EspB and γ-intimin C280 were detected in milk samples of vaccinated cows at d 40 postparturition. Significant Stx2-neutralizing titers were also observed in colostrum from Stx2-vaccinated cows and sera from colostrum-fed calves. The results presented showed that bovine colostrum with increased levels of antibodies against EHEC O157:H7 may be obtained by systemic immunization of pregnant cows, and that these specific antibodies are efficiently transferred to newborn calves by feeding colostrum. Hyperimmune colostrum and milk may be an alternative to protect calves from early colonization by EHEC O157:H7 and a possible key source of antibodies to block colonization and toxic activity of this bacterium.


Subject(s)
Adhesins, Bacterial/pharmacology , Antibodies, Bacterial/immunology , Bacterial Outer Membrane Proteins/pharmacology , Cattle/immunology , Colostrum/immunology , Escherichia coli O157/immunology , Escherichia coli Proteins/pharmacology , Immunity, Maternally-Acquired/immunology , Shiga Toxin 2/pharmacology , Vaccination/veterinary , Adhesins, Bacterial/immunology , Animals , Animals, Newborn/immunology , Bacterial Outer Membrane Proteins/immunology , Escherichia coli Proteins/immunology , Female , Pregnancy , Shiga Toxin 2/immunology
6.
Vet Microbiol ; 156(3-4): 336-42, 2012 May 04.
Article in English | MEDLINE | ID: mdl-22119188

ABSTRACT

Pets can be reservoirs of Shiga toxin-producing Escherichia coli (STEC) strains. The aim of this study was to examine nine strains belonging to several serotypes (O91:H21, O91:H16, O178:H19, O8:H19, O22:H8, O22:HNT, ONT:H8), previously recovered from cats or dogs. To this end, we assessed a set of additional virulence genes (stx(2) subtype, subAB, ehxA, eae and saa), cytotoxic activity, and genetic relationships with strains isolated from cattle, meat and humans using pulsed-field gel electrophoresis (PFGE). Most of the isolates carried the stx(2) and/or stx(2vh-b) sequences, while only the O91:H21 isolate presented the mucus-activatable stx(2d) variant, as confirmed by sequencing the genes of subunits A and B. All the strains showed cytotoxic activity in cultured cells. One of the two O178:H19, selected for its high level of cytotoxicity in Vero cells, showed the ability to cause functional alterations in the human colon mucosa in vitro. None of the strains possessed the subAB, eae or saa genes and only the strains belonging to serotype O8:H19 carried the ehxA gene. The isolates shared 90-100% similarity by PFGE to epidemiologically unrelated strains of the corresponding serotypes recovered from cattle, meat or humans. Our results demonstrate that dogs and cats may have a role in the infection of humans by STEC, probably serving as a vehicle for bovine strains in the cycle of human infection, and thus emphasize the health risks for owners and their families.


Subject(s)
Cats/microbiology , Dogs/microbiology , Shiga-Toxigenic Escherichia coli/classification , Amino Acid Sequence , Animals , Argentina , Cattle/microbiology , Chlorocebus aethiops , Electrophoresis, Gel, Pulsed-Field , Escherichia coli Proteins/genetics , Feces/microbiology , Humans , Meat/microbiology , Molecular Sequence Data , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Serotyping , Shiga-Toxigenic Escherichia coli/pathogenicity , Vero Cells , Virulence Factors/genetics
7.
Am J Nephrol ; 32(4): 340-6, 2010.
Article in English | MEDLINE | ID: mdl-20733289

ABSTRACT

BACKGROUND/AIMS: In Argentina, hemolytic uremic syndrome (HUS) constitutes the most frequent cause of acute renal failure in children. The aim of our study was to analyze the early tubular response under the effect of Shiga toxin type 2 (Stx2) in a rat experimental model of HUS. METHODS: Adult male Sprague-Dawley rats were injected intraperitoneally with culture supernatant from recombinant Escherichia coli expressing Stx2. Functional, histological, immunohistochemical and Western blot studies were performed at 48 h postinoculation. RESULTS: Renal tubules showed the loss of the epithelial markers E-cadherin and ß-catenin, and an increase in transforming growth factor-ß1 expression. We detected the expression of α-smooth muscle actin in the interstitium and fibrosis in the periglomerular areas. CONCLUSION: Our results indicate that the early tubular response to the effects of Stx2 is related to an immunophenotype change of tubular cells and the presence of mild fibrosis in the interstitium.


Subject(s)
Hemolytic-Uremic Syndrome/pathology , Hemolytic-Uremic Syndrome/physiopathology , Kidney Tubules/pathology , Actins/analysis , Animals , Cadherins/analysis , Epithelial Cells/chemistry , Epithelial Cells/drug effects , Immunohistochemistry , Kidney Tubules/chemistry , Male , Models, Animal , Rats , Rats, Sprague-Dawley , Shiga Toxin 2 , Transforming Growth Factor beta1/analysis , beta Catenin/analysis
8.
Cell Death Differ ; 11(7): 760-70, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15017383

ABSTRACT

Potassium withdrawal is commonly used to induce caspase-mediated apoptosis in cerebellar granule neurons in vitro. However, the underlying and cell death-initiating mechanisms are unknown. We firstly investigated potassium efflux through the outward delayed rectifier K+ current (Ik) as a potential mediator. However, tetraethylammoniumchloride, an inhibitor of Ik, was ineffective to block apoptosis after potassium withdrawal. Since potassium withdrawal reduced intracellular pH (pHi) from 7.4 to 7.2, we secondly investigated the effects of intracellular acidosis. To study intracellular acidosis in cerebellar granule neurons, we inhibited the Na+/H+ exchanger (NHE) with 4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate (HOE 642) and 5-(N-ethyl-N-isopropyl)-amiloride. Both inhibitors concentration-dependently induced cell death and potentiated cell death after potassium withdrawal. Although inhibition of the NHE induced cell death with morphological criteria of apoptosis in light and electron microscopy including chromatin condensation, positive TUNEL staining and cell shrinkage, no internucleosomal DNA cleavage or activation of caspases was detected. In contrast to potassium withdrawal-induced apoptosis, cell death induced by intracellular acidification was not prevented by insulin-like growth factor-1, cyclo-adenosine-monophosphate, caspase inhibitors and transfection with an adenovirus expressing Bcl-XL. However, cycloheximide protected cerebellar granule neurons from death induced by potassium withdrawal as well as from death after treatment with HOE 642. Therefore, the molecular mechanisms leading to cell death after acidification appear to be different from the mechanisms after potassium withdrawal and resemble the biochemical but not the morphological characteristics of paraptosis.


Subject(s)
Acidosis/etiology , Amiloride/analogs & derivatives , Apoptosis/drug effects , Caspases/physiology , Cerebellum/cytology , Neurons/physiology , Sodium-Hydrogen Exchangers/antagonists & inhibitors , Acidosis/metabolism , Adenoviridae/genetics , Amiloride/pharmacology , Animals , Cell Survival , Cells, Cultured , Cycloheximide/pharmacology , Dose-Response Relationship, Drug , Guanidines/pharmacology , Hydrogen-Ion Concentration/drug effects , Neurons/drug effects , Neurons/ultrastructure , Potassium/metabolism , Protein Synthesis Inhibitors/pharmacology , Rats , Rats, Sprague-Dawley , Sodium-Hydrogen Exchangers/drug effects , Sulfones/pharmacology
9.
Mol Cell Neurosci ; 17(4): 717-31, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11312607

ABSTRACT

Cerebellar granule neurons (CGN) cultured in the presence of serum and depolarizing potassium concentrations undergo apoptosis when switched to serum-free medium containing physiological potassium concentrations. Here we show that processing of the key protease, caspase-3, depends on the activation of caspase-9, but not of caspase-8. Selective peptide inhibitors of caspase-9 block processing of caspase-3 and caspase-8 and inhibit apoptosis, whereas a selective inhibitor of caspase-8 blocks neither processing of caspase-3 nor cell death. The data obtained with peptide inhibitors were confirmed by adenovirally mediated ectopic expression of the cytokine response modifier A (crmA), the baculovirus protein p35, and the X chromosome-linked inhibitor of apoptosis (XIAP). Further, caspase-8-activating death receptors do not mediate apoptosis in CGN and potassium withdrawal-induced apoptosis evolves unaltered in gld or lpr mice, which harbor mutations in the CD95/CD95 ligand system. Thus, neuronal apoptosis triggered by potassium deprivation is death receptor-independent but involves the mitochondrial pathway of caspase activation.


Subject(s)
Apoptosis/physiology , Caspases/metabolism , Cerebellum/cytology , Neurons/cytology , Neurons/enzymology , Potassium/pharmacology , Animals , Apoptosis/drug effects , Caspase 3 , Caspase 8 , Caspase 9 , Caspase Inhibitors , Cell Survival/physiology , Cells, Cultured , Coumarins/pharmacology , Enzyme Inhibitors/pharmacology , Enzyme Precursors/antagonists & inhibitors , Enzyme Precursors/metabolism , Gene Expression Regulation, Viral , Inhibitor of Apoptosis Proteins , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred MRL lpr , Oligopeptides/pharmacology , Proteins/genetics , Rats , Rats, Sprague-Dawley , Serpins/genetics , Specific Pathogen-Free Organisms , Transgenes/physiology , Viral Proteins/genetics , X-Linked Inhibitor of Apoptosis Protein
10.
Mol Cell Neurosci ; 17(1): 78-96, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11161471

ABSTRACT

In order to achieve neuron-restricted expression of antiapoptotic proteins, cellular promoters were investigated for their expression profiles in the context of adenoviral vectors. Both the synapsin 1 gene and the tubulin alpha1 gene promoters were strictly neuron specific in cocultures of primary neurons with their essential feeder cells. The neuron-specific enolase gene promoter exhibited only weak activity in cultured hippocampal neurons and was not neuron specific in preparations of cerebellar granule cells. By attaining virtually 100% transduction efficiency we were able to generate "quasi-transgenic" primary neuron cultures using both differentiated and completely undifferentiated hippocampal neurons. In a functional assay, we used the synapsin promoter to evaluate the effect of Bcl-X(L) overexpression on potassium-withdrawal-induced apoptosis of cerebellar granule neurons. We found nearly complete inhibition of caspase-9 and -3 activation and apoptosis, indicating a major role for mitochondrial pathways in this paradigm of neuronal cell death. The excellent suitability of the synapsin promoter as a strong panneuronal promoter was further demonstrated by its restricted neuronal activity in various brain regions of adult rats in vivo.


Subject(s)
Adenoviridae/genetics , Genetic Vectors/genetics , Neurons/metabolism , Promoter Regions, Genetic/genetics , Synapsins/genetics , Animals , Apoptosis/drug effects , Brain/drug effects , Brain/metabolism , Caspase 3 , Caspase 9 , Caspase Inhibitors , Caspases/metabolism , Cell Differentiation/genetics , Cells, Cultured , Cerebellum/cytology , Cerebellum/drug effects , Cerebellum/metabolism , Coculture Techniques , Cytomegalovirus/genetics , Gene Expression , Genetic Vectors/metabolism , Hippocampus/cytology , Hippocampus/drug effects , Hippocampus/embryology , Hippocampus/metabolism , Mitochondria/metabolism , Neuroglia/cytology , Neurons/cytology , Neurons/drug effects , Potassium/metabolism , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/pharmacology , Rats , Rats, Sprague-Dawley , Transgenes , Tubulin/genetics , bcl-X Protein
11.
J Neurosci ; 20(24): 9126-34, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11124990

ABSTRACT

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces clinical, biochemical, and neuropathological changes reminiscent of those occurring in idiopathic Parkinson's disease (PD). Here we show that a peptide caspase inhibitor, N-benzyloxy-carbonyl-val-ala-asp-fluoromethyl ketone, or adenoviral gene transfer (AdV) of a protein caspase inhibitor, X-chromosome-linked inhibitor of apoptosis (XIAP), prevent cell death of dopaminergic substantia nigra pars compacta (SNpc) neurons induced by MPTP or its active metabolite 1-methyl-4-phenylpyridinium in vitro and in vivo. Because the MPTP-induced decrease in striatal concentrations of dopamine and its metabolites does not differ between AdV-XIAP- and control vector-treated mice, this protection is not associated with a preservation of nigrostriatal terminals. In contrast, the combination of adenoviral gene transfer of XIAP and of the glial cell line-derived neurotrophic factor to the striatum provides synergistic effects, rescuing dopaminergic SNpc neurons from cell death and maintaining their nigrostriatal terminals. These data suggest that a combination of a caspase inhibitor, which blocks death, and a neurotrophic factor, which promotes the specific function of the rescued neurons, may be a promising strategy for the treatment of PD.


Subject(s)
Adenoviridae/genetics , Genetic Therapy/methods , Nerve Growth Factors , Nerve Tissue Proteins/genetics , Parkinson Disease, Secondary/therapy , Proteins/genetics , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine , Animals , Apoptosis/drug effects , Caspase Inhibitors , Cells, Cultured , Dopamine/metabolism , Drug Synergism , Enzyme Inhibitors/pharmacology , Gene Transfer Techniques , Genetic Vectors/genetics , Genetic Vectors/pharmacology , Glial Cell Line-Derived Neurotrophic Factor , Humans , Male , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/pharmacology , Parkinson Disease, Secondary/chemically induced , Parkinson Disease, Secondary/metabolism , Presynaptic Terminals/drug effects , Presynaptic Terminals/metabolism , Proteins/metabolism , Proteins/pharmacology , Rats , Rats, Sprague-Dawley , Substantia Nigra/drug effects , Substantia Nigra/metabolism , Substantia Nigra/pathology , X-Linked Inhibitor of Apoptosis Protein
12.
Biochemistry ; 37(16): 5394-406, 1998 Apr 21.
Article in English | MEDLINE | ID: mdl-9548921

ABSTRACT

Crystal structures of wild-type tryptophan synthase alpha2beta2 complexes from Salmonella typhimurium were determined to investigate the mechanism of allosteric activation of the alpha-reaction by the aminoacrylate intermediate formed at the beta-active site. Using a flow cell, the aminoacrylate (A-A) intermediate of the beta-reaction () was generated in the crystal under steady state conditions in the presence of serine and the alpha-site inhibitor 5-fluoroindole propanol phosphate (F-IPP). A model for the conformation of the Schiff base between the aminoacrylate and the beta-subunit cofactor pyridoxal phosphate (PLP) is presented. The structure is compared with structures of the enzyme determined in the absence (TRPS) and presence (TRPSF-IPP) of F-IPP. A detailed model for binding of F-IPP to the alpha-subunit is presented. In contrast to findings by Hyde et al. [(1988) J. Biol. Chem. 263,17857-17871] and Rhee et al. [(1997) Biochemistry 36, 7664-7680], we find that the presence of an alpha-site alone ligand is sufficient for loop alphaL6 closure atop the alpha-active site. Part of this loop, alphaThr183, is important not only for positioning the catalytic alphaAsp60 but also for coordinating the concomitant ordering of loop alphaL2 upon F-IPP binding. On the basis of the three structures, a pathway for communication between the alpha- and beta-active sites has been established. The central element of this pathway is a newly defined rigid, but movable, domain that on one side interacts with the alpha-subunit via loop alphaL2 and on the other side with the beta-active site. These findings provide a structural basis for understanding the allosteric properties of tryptophan synthase.


Subject(s)
Tryptophan Synthase/chemistry , Alanine/analogs & derivatives , Alanine/metabolism , Allosteric Site , Crystallography, X-Ray , Enzyme Activation , Ligands , Models, Molecular , Protein Structure, Secondary , Protein Structure, Tertiary , Salmonella typhimurium/enzymology , Serine/metabolism , Structure-Activity Relationship
13.
Virology ; 218(2): 396-9, 1996 Apr 15.
Article in English | MEDLINE | ID: mdl-8610467

ABSTRACT

The large surface protein (L) of the enveloped hepatitis B virus (HBV) is myristylated at glycine 2. To investigate whether this fatty acid moiety is required for HBV infectivity we made use of a point mutant in which the myristyl acceptor was mutated to a nonfunctional alanine. The mutant virus and a wild-type control were expressed in a human hepatoma cell line by transfection of genomic DNA constructs. Comparable amounts of virions were secreted by both strains as measured by the endogenous polymerase activity of immunoprecipitated virions. The presentation of an N-terminal epitope of L on the virion surface was not influenced by the mutation. To test the infectivity of this mutant virus primary human hepatocytes were incubated with the media of transfected cells. The covalently circular closed HBV DNA molecules generated after infection were discriminated from the open circular DNA genomes of inoculated virions by a sensitive PCR-based technique. The experiments demonstrated that the wild type was infectious but not the myristate negative mutant. This reflects the phenotype of an homologous duck hepatitis B virus mutant although the N-terminal L protein domains of this virus and of HBV show no primary sequence homology.


Subject(s)
Hepatitis B virus/metabolism , Myristic Acids/metabolism , Viral Envelope Proteins/metabolism , Base Sequence , Carcinoma, Hepatocellular , Cells, Cultured , DNA, Viral/analysis , DNA, Viral/chemistry , DNA, Viral/genetics , Epitopes/analysis , Hepatitis B Surface Antigens/analysis , Hepatitis B virus/pathogenicity , Humans , Liver/cytology , Liver/virology , Molecular Sequence Data , Myristic Acid , Nucleic Acid Conformation , Point Mutation , Protein Precursors/analysis , Transfection , Tumor Cells, Cultured , Virion/chemistry
14.
Intervirology ; 39(1-2): 23-31, 1996.
Article in English | MEDLINE | ID: mdl-8957666

ABSTRACT

The hepatitis B virus (HBV) envelope and the subviral lipoprotein particles contain three viral surface proteins (L, M, and S) which are expressed from one open reading frame by the usage of three start codons and a common stop codon. The largest surface protein L has some unusual properties. It adopts two different transmembrane topologies due to a posttranslational switch of the folding in approximately half of the L proteins. L molecules which expose their N-terminal preS1 domain on the viral particle surface are probably ligands for a putative virus receptor and determine the species specificity and liver tropism of this virus. L chains with internal preS1 domains are required in virion morphogenesis and mediate the contact to the nucleocapsid like a matrix protein. Overexpression of this form of the L protein is also responsible for the inhibition of viral particle release. This short review summarizes our knowledge on the biosynthesis and maturation of the HBV surface proteins and their functions in viral particle morphogenesis with special emphasis on the L protein.


Subject(s)
Hepatitis B virus/physiology , Viral Envelope Proteins/physiology , Virus Assembly/physiology , Animals , Genetic Vectors , Genome , Hepatitis B Surface Antigens , Hepatitis B Vaccines , Humans , Morphogenesis , Nucleocapsid , Receptors, Virus , Viral Envelope Proteins/metabolism , Virion/physiology
15.
J Virol ; 69(2): 1201-8, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7815496

ABSTRACT

The virus family Hepadnaviridae comprises two genera: orthohepadnaviruses isolated from humans (hepatitis B virus [HBV]) and rodents (e.g., woodchuck hepatitis virus [WHV]) and avihepadnaviruses isolated from birds (e.g., duck hepatitis B virus [DHBV]). They carry in their envelopes two (DHBV) or three (HBV and WHV) coterminal proteins referred to as small (S), middle (M), or large (L) surface protein. These proteins are also secreted from infected cells as subviral particles consisting of surface protein and lipid (e.g., 20-nm hepatitis B surface antigen for HBV). To investigate the assembly of these proteins, we asked whether surface proteins from different hepadnaviruses are able to mix phenotypically with each other. By coexpression and coimmunoprecipitation with species-specific antibodies, we could show the formation of mixed subviral particles and disulfide-linked heterodimers between the WHV S and HBV M proteins whereas the DHBV and HBV surface proteins did not coassemble. Complementation of HBV genomes defective in expressing the S or L protein and therefore incompetent to form virions was possible with the closely related WHV S protein or a WHV pre-S-HBV S chimera, respectively, but not with the less related DHBV S or L protein or with a DHBV L-HBV S chimera. The results suggest that the assembly of HBV subviral particles and virion envelopes requires relatively precise molecular interactions of their surface proteins, which are not conserved between the two hepadnavirus genera. This contrasts with the ability of, e.g., rhabdoviruses or retroviruses, to incorporate envelope proteins even from unrelated viruses.


Subject(s)
Hepatitis B Surface Antigens/metabolism , Hepatitis B Virus, Duck/metabolism , Hepatitis B Virus, Woodchuck/metabolism , Hepatitis B virus/metabolism , Viral Envelope Proteins/metabolism , Base Sequence , Humans , Molecular Sequence Data , Phenotype , Protein Precursors/metabolism
16.
J Biol Chem ; 267(12): 8254-9, 1992 Apr 25.
Article in English | MEDLINE | ID: mdl-1569079

ABSTRACT

We have analyzed the developmental pattern of expression of the chicken transferrin receptor (CTR) gene in various chick embryonic tissues. Northern analyses of RNA from embryonic tissues at different stages of development and cultured chick embryonic fibroblasts (CEFs) show that CTR is hyperexpressed in differentiating erythroid cells such that the steady-state level of CTR mRNA in these cells could be 200 or more times higher than in nonerythroid cells. In vitro nuclear transcription assays using nuclei from embryonic erythroid and brain cells, as well as CEFs, demonstrate that the vast differences in CTR mRNA levels in these cells are reflected in their respective CTR gene transcriptional activities. During development, the steady-state level of CTR mRNA declines in all tissues and, in erythroid cells, this pattern is accompanied by a similar decline in beta-globin mRNA levels. These changes are concurrent with the decreases in CTR and beta-globin mRNA transcriptional activities during erythroid maturation. Taken together, our results indicate that the hyperexpression of the CTR gene in differentiating erythroid cells is regulated to a significant degree at the transcriptional level. We also demonstrate that, in erythroid cells, neither CTR gene transcription nor CTR mRNA stability is regulated by intracellular iron levels.


Subject(s)
Cell Differentiation , Erythrocytes/cytology , Gene Expression , Receptors, Transferrin/genetics , Transcription, Genetic , Animals , Blotting, Northern , Chick Embryo , Erythrocytes/metabolism , Fibroblasts/metabolism , Globins/genetics , RNA, Messenger/metabolism , Tissue Distribution
17.
Przegl Lek ; 49(1-2): 67-8, 1992.
Article in Polish | MEDLINE | ID: mdl-1333622

ABSTRACT

The prevalence of anti-HCV antibodies, HBs antigenemia and transaminase level were examined in the 136 patients with chronic renal failure on maintenance hemodialysis, treated in dialytic centers of South Poland. In this group anti-HBs and anti-HBc antibodies were also investigated in 60 and 42 patients respectively. Anti-HCV antibodies were present in 16.7-60.0% (mean = 44.1%) of patients depending on the center. These values considerably exceed the percentage of antigen HBs positive patients but they are lower that the percentage of anti-HBs and anti-HBc positive patients.


Subject(s)
Anemia/therapy , Blood Donors , Hepacivirus/immunology , Hepatitis Antibodies/immunology , Hepatitis C/immunology , Kidney Failure, Chronic/therapy , Transfusion Reaction , Adolescent , Adult , Aged , Anemia/etiology , Anemia/immunology , Female , Hepatitis Antibodies/analysis , Hepatitis C/etiology , Hepatitis C/transmission , Humans , Kidney Failure, Chronic/blood , Kidney Failure, Chronic/complications , Kidney Failure, Chronic/immunology , Long-Term Care , Male , Middle Aged , Renal Dialysis
18.
Gene ; 102(2): 249-54, 1991 Jun 30.
Article in English | MEDLINE | ID: mdl-1874449

ABSTRACT

Recombinant cDNA clones encoding the chicken transferrin receptor (cTR) have been isolated and sequenced. Comparison of the deduced primary structure of cTR with those of the human transferrin receptor (hTR) and mouse transferrin receptor (mTR) shows that their size, hydropathy profile, location of sites for posttranslational modifications, and domain organization are highly similar. The cytoplasmic domain of cTR contains the motif Tyr-Xaa-Arg-Phe (YXRF) that is the recognition signal for high-efficiency endocytosis of hTR. The cTR has several highly conserved regions within its extracellular domain, including those flanking the putative N-glycosylation sites. Overall, however, the extracellular domain of cTR is only 53% identical to the extracellular domains of hTR and mTR. The cTR also lacks three of the six Cys residues found in the extracellular domains of the mammalian TRs. These differences can account for functional and structural properties that distinguish cTR and mammalian TRs.


Subject(s)
Receptors, Transferrin/genetics , Amino Acid Sequence , Animals , Base Sequence , Chickens , DNA , Exons , Humans , Molecular Sequence Data , Sequence Alignment
19.
Acta Anat (Basel) ; 141(2): 132-8, 1991.
Article in English | MEDLINE | ID: mdl-1746221

ABSTRACT

To avoid artifacts, native nerve fibers were investigated by phase contrast light microscopy. It was shown that Ranvier's nodes cannot be seen. At the sites at which they can be produced by thermal and chemical effects, a tight joint of the tube ends of the 'internodes' inverted to the inside are to be found. Some structures of Ranvier's nodes do not exist in intact living nerves. This fact can also be proved by electron micrography.


Subject(s)
Artifacts , Ranvier's Nodes/ultrastructure , Animals , Birds , Cells, Cultured , Cricetinae , Guinea Pigs , Mice , Microscopy, Electron , Microscopy, Phase-Contrast , Rana esculenta , Rats
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