RESUMO
The challenge in treating traumatic hemipelvectomy is the dynamics of the complex and life-threatening consequences of the injury. These include skin and soft tissue defects, osseous, neural and vascular injuries as well as the subsequent hemostatic derangement and organ dysfunction as part of the shock process. The treatment requires rapid and targeted decisions to save the patient's life. In this particular case a 34-year-old farmer was trapped between a wheeled loader and a stationary trailer. Upon arrival at the hospital the patient was in a state of hemorrhagic shock with accompanying acute traumatic coagulopathy and a grade III open pelvic trauma with complete ischemia of the left leg and a bladder injury. After performing emergency surgery and a two-stage approach for pelvic stabilization the patient's condition deteriorated up to multiorgan failure, necessitating left-sided hemipelvectomy as an immediate life-saving salvage procedure. In the further course multiple revision surgeries and plastic reconstructions due to wound infections and the presence of skin and soft tissue damage were required. Due to the rare confrontation with this type of injury in everyday practice and the absence of a universal treatment algorithm, the following case report is intended to contribute to a better understanding of the treatment and to illustrate the coherent interactions of the individual organ systems affected.
Assuntos
Hemipelvectomia , Humanos , Adulto , Masculino , Hemipelvectomia/métodos , Traumatismo Múltiplo/cirurgia , Resultado do Tratamento , Ossos Pélvicos/lesões , Ossos Pélvicos/cirurgia , Traumatismos da Perna/cirurgia , Cirurgia de Cuidados CríticosRESUMO
Softening neural implants that change their elastic modulus under physiological conditions are promising candidates to mitigate neuroinflammatory response due to the reduced mechanical mismatch between the artificial interface and the brain tissue. Intracortical neural probes have been used to demonstrate the viability of this material engineering approach. In our paper, we present a robust technology of softening neural microelectrode and demonstrate its recording performance in the hippocampus of rat subjects. The 5 mm long, single shank, multi-channel probes are composed of a custom thiol-ene/acrylate thermoset polymer substrate, and were micromachined by standard MEMS processes. A special packaging technique is also developed, which guarantees the stable functionality and longevity of the device, which were tested under in vitro conditions prior to animal studies. The 60 micron thick device was successfully implanted to 4.5 mm deep in the hippocampus without the aid of any insertion shuttle. Spike amplitudes of 84 µV peak-to-peak and signal-to-noise ratio of 6.24 were achieved in acute experiments. Our study demonstrates that softening neural probes may be used to investigate deep layers of the rat brain.
Assuntos
Eletrodos Implantados , Hipocampo/fisiologia , Potenciais de Ação/fisiologia , Animais , Impedância Elétrica , Polímeros/química , Ratos Wistar , Processamento de Sinais Assistido por Computador , TemperaturaRESUMO
Several events of insertional mutagenesis in pre-clinical and clinical gene therapy studies have created intense interest in assessing the genomic insertion profiles of gene therapy vectors. For the construction of such profiles, vector-flanking sequences detected by inverse PCR, linear amplification-mediated-PCR or ligation-mediated-PCR need to be mapped to the host cell's genome and compared to a reference set. Although remarkable progress has been achieved in mapping gene therapy vector insertion sites, public reference sets are lacking, as are the possibilities to quickly detect non-random patterns in experimental data. We developed a tool termed QuickMap, which uniformly maps and analyzes human and murine vector-flanking sequences within seconds (available at www.gtsg.org). Besides information about hits in chromosomes and fragile sites, QuickMap automatically determines insertion frequencies in +/- 250 kb adjacency to genes, cancer genes, pseudogenes, transcription factor and (post-transcriptional) miRNA binding sites, CpG islands and repetitive elements (short interspersed nuclear elements (SINE), long interspersed nuclear elements (LINE), Type II elements and LTR elements). Additionally, all experimental frequencies are compared with the data obtained from a reference set, containing 1 000 000 random integrations ('random set'). Thus, for the first time a tool allowing high-throughput profiling of gene therapy vector insertion sites is available. It provides a basis for large-scale insertion site analyses, which is now urgently needed to discover novel gene therapy vectors with 'safe' insertion profiles.
Assuntos
Mapeamento Cromossômico/métodos , Processamento Eletrônico de Dados/métodos , Vetores Genéticos/uso terapêutico , Mutagênese Insercional/genética , Retroviridae/genética , Software , Acesso à Informação , Animais , Sequência de Bases/genética , Sítios de Ligação , Sítios Frágeis do Cromossomo , Biologia Computacional/métodos , Ilhas de CpG , Bases de Dados Genéticas , Terapia Genética/métodos , Terapia Genética/normas , Vetores Genéticos/efeitos adversos , Genoma Humano/genética , Humanos , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Sequências Repetitivas de Ácido Nucleico , Segurança , Análise de Sequência de DNA/métodosRESUMO
This prospective consecutive study judges the effectiveness of pulsed low-intensity ultrasound for treatment of boney healing disorders. 86 out of 100 treatments were successful. Although in these 100 cases 64 delayed unions and 36 nonunions were enrolled ultrasound therapy was performed as an alternative to the indicated operation in every case. Excluding ten cases of ununited fractures of the scaphoid no additional therapy was performed in any case. Stratifying the data no significant differences in healing rate and treatment time were observed between delayed unions and non-unions and between atrophic and hypertrophic healing disorders. Judging the healing rate of 86% one has to take into consideration that according to our in- and excluding criteria we had a preselected sample of patients. Nevertheless the effectiveness of pulsed low-intensity ultrasound for treatment of disorders of the fracture repair process is evident.
Assuntos
Fraturas não Consolidadas/terapia , Terapia por Ultrassom/instrumentação , Adulto , Feminino , Seguimentos , Consolidação da Fratura/fisiologia , Fraturas não Consolidadas/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Radiografia , Resultado do TratamentoRESUMO
The objective of this study design is to describe the diagnosis and successful treatment of a pediatric patient with an osteochondroma of the thoracic spine. An osteochondroma is a bone tumor that rarely occurs in the thoracic spine, especially in the pediatric population. A simple painless mass may be the only presenting symptom. The laboratory findings are usually nonspecific. Radiographs may be nondiagnostic in certain cases, and computed tomography is the imaging modality of choice. The diagnosis, treatment, and outcome of a pediatric patient with an osteochondroma of the thoracic spine, including a possible genetic predisposition, are reviewed, along with a complete review of the literature. Anteroposterior and lateral plain radiographs illustrated a well-defined solid mass arising from the posterior elements of the tenth thoracic vertebrae. A computed tomography (CT) scan further delineated that the mass arose from the spinous process with no obvious impingement of the nerve roots. After excision of the lesion, gross pathological and histologic evaluation was consistent with an osteochondroma. The use of CT allowed accurate diagnosis of the osteochondroma. This led to appropriate surgical intervention, resulting in definitive treatment.
Assuntos
Osteocondroma , Neoplasias da Coluna Vertebral , Vértebras Torácicas , Fatores Etários , Criança , Feminino , Seguimentos , Humanos , Osteocondroma/diagnóstico , Osteocondroma/patologia , Osteocondroma/cirurgia , Neoplasias da Coluna Vertebral/diagnóstico , Neoplasias da Coluna Vertebral/patologia , Neoplasias da Coluna Vertebral/cirurgia , Vértebras Torácicas/patologia , Vértebras Torácicas/cirurgia , Fatores de Tempo , Tomografia Computadorizada por Raios XRESUMO
Continuous intraoperative monitoring of spinal cord function using somatosensory evoked potentials (SSEP) has gained nearly universal acceptance as a reliable and sensitive method for detecting and possibly preventing neurologic injury during surgical correction of spinal deformities. In several reports, spinal cord injury was identified successfully based on changes in SSEP response characteristics, specifically amplitude and latency. Less well documented and used, however, is monitoring of peripheral nerve function with SSEPs to identify and prevent the neurologic sequelae of prolonged prone positioning on a spinal frame. The authors describe a patient who underwent surgical removal of spinal instrumentation but was not monitored. A brachial plexopathy developed in this patient from pressure on the axilla exerted by a Relton-Hall positioning frame during spinal surgery. In addition, data are presented from 15 of 500 consecutive pediatric patients who underwent surgical correction of scoliosis between 1993 and 1997 with whom intermittent monitoring of ulnar nerve SSEPs was used successfully to identify impending brachial plexopathy, a complication of prone positioning. A statistically significant reduction in ulnar nerve SSEP amplitude was observed in 18 limbs of the 500 patients (3.6%) reviewed. Repositioning the arm(s) or shoulders resulted in nearly immediate improvement of SSEP amplitude, and all awoke without signs of brachial plexopathy. This complication can be avoided by monitoring SSEPs to ulnar nerve stimulation for patients placed in the prone position during spinal surgery.
Assuntos
Plexo Braquial/fisiopatologia , Plexo Braquial/cirurgia , Escoliose/cirurgia , Fusão Vertebral/efeitos adversos , Adulto , Potenciais Somatossensoriais Evocados/fisiologia , Humanos , Masculino , Monitorização Fisiológica , Escoliose/fisiopatologiaRESUMO
SUMMARY: The authors report on 41 patients with acute or subacute knee pain and early or midstage degenerative arthrosis with osteonecrotic lesions in the subchondral and metaphyseal region of the medial proximal tibia. Each lesion was identified only on magnetic resonance images (MRI). These MRI changes are classified and the clinical course is defined during a follow-up period averaging 4.5 years. Radiographically, 22 patients had minimal degenerative changes; 12 had moderate arthritis; and 7 were normal. With MRI, 3 distinct types of lesions were identified. Type A lesions had localized areas of decreased signal in the subchondral area. Type B lesions had diffuse signal changes with extension into the metaphysis. Type C lesions had metaphyseal involvement as well as a marginated serpentine subchondral rim usually associated with advanced osteonecrosis. There were 9 type A lesions, 23 type B, and 9 type C. At the end of 1 year, 33 patients (80%) had no or mild symptoms, and 8 (20%) had persistent moderate pain. At 4.5-year follow-up, most patients had symptoms consistent with progressive osteoarthrosis, 12 patients had severe symptoms (29%), 17 (41%) had mild or moderate symptoms, and only 12 (29%) were asymptomatic or had minimal symptoms. The type of MRI change seen initially was predictive of prognosis. Only 6 (19%) of the 32 patients with type A or B findings had severe symptoms at last follow-up. Six (66%) of the 9 patients with a type C MRI lesion had severe symptoms or had an operation by last follow-up. Twelve patients had follow-up MRI at a mean 15 months (range, 12 to 18 months) after the initial evaluation. The type A and B changes were either absent or significantly reduced. The type C subchondral marginated rim changes remained but metaphyseal involvement was reduced. There appears to be a spectrum of tibial subchondral MRI changes associated with sudden onset of medial knee pain in patients with early osteoarthritis of the knee. These changes may be indicative of osteonecrosis. The initial MRI classification is useful in predicting prognosis. Recognition of this problem may avoid unnecessary intra-articular surgery.
Assuntos
Cartilagem Articular/patologia , Articulação do Joelho/patologia , Imageamento por Ressonância Magnética , Osteoartrite/diagnóstico , Osteonecrose/complicações , Tíbia/patologia , Idoso , Idoso de 80 Anos ou mais , Artrografia , Cartilagem Articular/diagnóstico por imagem , Feminino , Humanos , Articulação do Joelho/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Osteoartrite/etiologia , Osteonecrose/diagnóstico , Prognóstico , Estudos Retrospectivos , Tíbia/diagnóstico por imagemRESUMO
We have conducted a DNA immunization study to evaluate how the immune response is influenced by the physical structure and secretion of the expressed Ag. For this purpose, we used a series of plasmid constructs encoding different forms of the envelope glycoprotein E of the flavivirus tick-borne encephalitis virus. These included a secreted recombinant subviral particle, a secreted carboxyl-terminally truncated soluble homodimer, a nonsecreted full-length form, and an inefficiently secreted truncated form. Mice were immunized using both i.m. injection and Gene Gun-mediated application of plasmids. The functional immune response was evaluated by determining specific neutralizing and hemagglutination-inhibiting Ab activities and by challenging the mice with a lethal dose of the virus. As a measure for the induction of a Th1 and/or Th2 response, we determined specific IgG subclasses and examined IFN-gamma, Il-4, and Il-5 induction. The plasmid construct encoding a secreted subviral particle, which carries multiple copies of the protective Ag on its surface, was superior to the other constructs in terms of extent and functionality of the Ab response as well as protection against virus challenge. As expected, the type of Th response was largely dependent on the mode of application (i.m. vs Gene Gun), but our data show that it was also strongly influenced by the properties of the Ag. Most significantly, the plasmid encoding the particulate form was able to partially overcome the Th2 bias imposed by the Gene Gun, resulting in a balanced Th1/Th2 response.
Assuntos
Antígenos Virais/imunologia , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Plasmídeos/imunologia , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/biossíntese , Antígenos Virais/administração & dosagem , Antígenos Virais/biossíntese , Antígenos Virais/genética , Biolística , Vírus da Encefalite Transmitidos por Carrapatos/genética , Feminino , Isotipos de Imunoglobulinas/biossíntese , Injeções Intramusculares , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos BALB C , Modelos Imunológicos , Plasmídeos/administração & dosagem , Plasmídeos/síntese química , Isoformas de Proteínas/administração & dosagem , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Células Th1/imunologia , Células Th2/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/síntese química , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/síntese químicaRESUMO
To study the epidemiology of hantavirus infections in Austria, 1215 humans and 596 rodents of different species were tested for the presence of antibodies against Puumala and Hantaan virus. Direct virus identification by polymerase chain reaction in lung tissue of serologically positive rodents was performed to verify antibody results and to determine the genetic identity of viral RNA by phylogenetic analysis of a part of the hantavirus M segment. For 32 of the 37 cases of nephropathia epidemica diagnosed in Austria, the location where transmission took place could be traced to specific areas in the Austrian federal states of Carinthia and Styria. The overall seroprevalence in humans was 1.2% and ranged from 0.02% in Villach, Carinthia, to 0.8% in Korneuburg, Lower Austria, and 1.8% in Wolfsberg, Carinthia. Virus RNA could be amplified from three Clethrionomys glareolus voles collected in Klippitztörl, Carinthia, and from one collected in Ernstbrunn, Lower Austria. The sequences were all identified as Puumala virus by phylogenetic analysis and were found to be most closely related to the western European Puumala viruses from Germany and France. No evidence of the existence of Hantaan-like infections and viruses in Austria was found.
Assuntos
Infecções por Hantavirus/epidemiologia , Orthohantavírus/classificação , Animais , Áustria/epidemiologia , DNA Viral/análise , Genótipo , Orthohantavírus/genética , Orthohantavírus/imunologia , Infecções por Hantavirus/imunologia , Infecções por Hantavirus/virologia , Humanos , Muridae/virologia , Filogenia , Roedores/virologia , Análise de Sequência de DNA , Estudos SoroepidemiológicosRESUMO
The incorporation of radioactive orthophosphate into the cell walls of Saccharomyces cerevisiae was studied. 33P-labeled cell walls were extensively extracted with hot sodium dodecyl sulfate (SDS). Of the remaining insoluble radioactivity more than 90% could be released by laminarinase. This radioactive material stayed in the stacking gel during SDS-polyacrylamide gel electrophoresis but entered the separating gel upon treatment with N-glycosidase F, indicating that phosphate was linked directly or indirectly to N-mannosylated glycoproteins. The phosphate was bound to covalently linked cell wall proteins as mannose-6-phosphate, the same type of linkage shown previously for soluble mannoproteins (L. Ballou, L. M. Hernandez, E. Alvarado, and C. E. Ballou, Proc. Natl. Acad. Sci. USA 87:3368-3372, 1990). From the phosphate-labeled glycoprotein fraction released by laminarinase, three cell wall mannoproteins, Ccw12p, Ccw13p and Ccw14p, were isolated and identified by N-terminal sequencing. For Ccw13p (encoded by DAN1 [also called TIR3]) and Ccw12p the association with the cell wall has not been described before; Ccw14p is identical with cell wall protein Icwp (I. Moukadiri, J. Armero, A. Abad, R. Sentandreu, and J. Zueco, J. Bacteriol. 179:2154-2162, 1997). In ccw12, ccw13, or ccw14 single or double mutants neither the amount of radioactive phosphate incorporated into cell wall proteins nor its position in the stacking gel was changed. However, the triple mutant brought about a shift of the 33P-labeled glycoprotein components from the stacking gel into the separating gel. The disruption of CCW12 results in a pronounced sensitivity of the cells to calcofluor white and Congo red. In addition, the ccw12 mutant shows a decrease in mating efficiency and a defect in agglutination.
Assuntos
Parede Celular/metabolismo , Proteínas Fúngicas/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Deleção de Sequência , Sequência de Aminoácidos , Benzenossulfonatos/farmacologia , Cromatografia Líquida , Vermelho Congo/farmacologia , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Glicoproteínas/isolamento & purificação , Espectrometria de Massas , Metilação , Mutação , Oligossacarídeos/análise , Oligossacarídeos/isolamento & purificação , Oligossacarídeos/metabolismo , Fenótipo , Fosfatos/metabolismo , Fosforilação , Polissacarídeos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Análise de SequênciaRESUMO
Despite of the good results of operative treatment disorders of fracture healing are still a challenge for surgeons. In some cases all endeavours are frustrating and bone does not heal. Low intensity pulsed ultrasound (frequency: 1.5 MHz, pulsed by 1 KHz, signal burst width: 200 musec, intensity: 30 mW/cm2) offers the opportunity of an alternative treatment. In contrast to magnetic field therapy, electric stimulation or use of extracorporal shock waves this therapy is judged positively all over the literature. Within an own series of 76 cases at present time we can show a 86% healing rate under defined conditions. Within this paper we present three case reports, which clearly show the efficiency of low intensity pulsed ultrasound.
Assuntos
Fraturas não Consolidadas/terapia , Terapia por Ultrassom , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
At our hospital, closed dislocations of the elbow without neurovascular or osseous lesion are treated conservatively after successful reduction in the absence of a tendency to redislocation. Indications for surgery are irreducible dislocations and those with a tendency to redislocation as well as open soft tissue injuries, neurovascular and osseous concomitant injuries. During a period of 10 years 113 patients were treated according to this strategy. Of these patients, 46 had simple dislocations of the elbow and 67 suffered from dislocations with osseous lesions. In 95 patients follow-ups could be performed after a mean period of 55 months. In more than 75% of cases good or excellent functional results were documented; unfavourable results were seen only in patients with osseous lesions. Nearly all of the patients had a stable elbow joint at the end of therapy.
Assuntos
Lesões no Cotovelo , Fraturas Ósseas/cirurgia , Luxações Articulares/cirurgia , Instabilidade Articular/cirurgia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Articulação do Cotovelo/cirurgia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/etiologia , Amplitude de Movimento Articular/fisiologia , Resultado do TratamentoRESUMO
The epidemiology of tick-borne encephalitis virus was investigated by comparative sequence analysis of virus strains isolated in endemic areas of Europe and Asia. Phylogenetic relationships were determined from the nucleotide and amino acid sequences of the major envelope (E) protein of 16 newly sequenced strains and nine previously published sequences. Three genetic lineages could be clearly distinguished, corresponding to a European, a Far Eastern and a Siberian subtype. Amino acids characteristic for each of the subtypes ('signature' amino a cids) were identified and their location in the atomic structure of protein E was determined. The degree of variation between strains within subtypes was low and exhibited a maximum of only 2.2% at the amino acid level. A maximum difference of 5.6% was found between the three subtypes, which is in the range of variation reported for other flaviviruses.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/genética , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Ásia/epidemiologia , Sequência de Bases , DNA Viral , Encefalite Transmitida por Carrapatos/virologia , Europa (Continente)/epidemiologia , Variação Genética , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNARESUMO
Recent years have seen the advent of new cardiac markers. These have been shown to be valuable in large controlled clinical trials. The breakthrough of the troponins world-wide for detection of myocardial lesions has meant that the Cardiac Troponin T parameter has been available in our hospital since August 1996. Analysis of the samples is carried out on the Elecsys, an automated immunology analyzer from Roche Diagnostics, Austria. The purpose of the present paper is to investigate whether study results on the diagnostic utility, sensitivity and specificity of troponin T can be confirmed in multimorbid patients in routine clinical practice, who are otherwise excluded from such trials. Troponin T was investigated in five cases including patients presenting with myocarditis and parallel damage of the skeletal muscles, as well as in haemodialysis patients. Cardiac troponin T has proved to be a very reliable parameter in all of these patients due to its high sensitivity and specificity, where other conventional diagnostic procedures were not helpful due to co-morbidity and confounding factors. New clinically relevant information can be obtained by both individual as well as serial measurements. Cardiac troponin T determination is a powerful new tool and should be considered seriously, regardless of other underlying diseases: it is, therefore, not only a diagnostic but also a management tool.
Assuntos
Falência Renal Crônica/diagnóstico , Miocardite/diagnóstico , Troponina T/sangue , Idoso , Feminino , Humanos , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Diálise Renal , Sensibilidade e EspecificidadeRESUMO
Infectious cDNA clones of two strains of tick-borne encephalitis (TBE) virus, i.e. European subtype prototypic strain Neudoerfl and the closely related but more virulent strain Hypr, were constructed. The recombinant constructs consisted of cDNAs stably inserted into the bacterial plasmid pBR322 under the control of T7 promoter elements. The genome of TBE virus strain Neudoerfl was successfully cloned, both as a full-length cDNA and as two partial cDNAs. In the case of strain Hypr, the genome is represented by two cDNA clones corresponding to the 5'- and 3'-terminal halves of the genome. Highly infectious RNAs can be produced from the full-length cDNA clone or from the partial clones ligated in vitro to form full-length cDNA templates prior to T7 transcription. The biological properties of the recombinant progeny viruses, including virulence characteristics, were indistinguishable from the corresponding parent virus strains. Thus, the described infectious cDNA clones represent a useful and reliable experimental system for the specific mutagenesis of TBE virus.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/genética , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Cricetinae , DNA Complementar , DNA Viral , Vírus da Encefalite Transmitidos por Carrapatos/classificação , Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Europa (Continente) , Camundongos , Dados de Sequência Molecular , Plasmídeos , RNA Viral/biossíntese , Recombinação Genética , Especificidade da Espécie , VirulênciaRESUMO
Escape mutants of tick-borne encephalitis (TBE) virus were selected using neutralizing monoclonal antibodies (MAbs) that react with three different and previously unrecognized epitopes in the envelope protein E of TBE virus. Two of these variants (V-IC3 and V-IE3) exhibited a significantly reduced reactivity with their selecting MAbs, as determined by ELISA, whereas with one variant (V-IO3), reactivity was completely unchanged. Comparative sequence analyses demonstrated that each of the variants differed from the wild-type virus by a single amino acid substitution located at exposed positions within domains I, II and III of protein E. In the mouse model, all three mutants were still neuro-virulent but exhibited a significantly reduced neuro-invasiveness after subcutaneous inoculation. Virus replication, however, was sufficient to induce a specific antibody response. The observed alterations in virulence properties were not associated with reduced growth rates in vertebrate cell cultures, but one variant (V-IE3) exhibited a small plaque phenotype. The mutation of variant V-IO3 resulted in a temperature-sensitive phenotype and a significant elevation of the pH-threshold of the conformational change necessary for fusion activity.
Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais/biossíntese , Vírus da Encefalite Transmitidos por Carrapatos/genética , Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Sistema Nervoso/virologia , Mutação Puntual , Proteínas do Envelope Viral/genética , Animais , Anticorpos Antivirais/sangue , Sequência de Bases , Embrião de Galinha , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Ensaio de Imunoadsorção Enzimática , Variação Genética , Camundongos , Dados de Sequência Molecular , Testes de Neutralização , Fenótipo , RNA Viral/química , RNA Viral/isolamento & purificação , Temperatura , Proteínas do Envelope Viral/análise , Proteínas do Envelope Viral/imunologia , Ensaio de Placa Viral , VirulênciaRESUMO
In higher plants, sugars are required not only to sustain heterotrophic growth but also to regulate the expression of a variety of genes. Environmental stresses, such as pathogen infection and wounding, activate a cascade of defense responses and may also affect carbohydrate metabolism. In this study, the relationship between sugar- and stress-activated signal transduction pathways and the underlying regulatory mechanism was analyzed. Photoautotrophically growing suspension culture cells of Chenopodium rubrum were used as a model system to study the effects of the metabolic regulator D-glucose and of different stress-related stimuli on photosynthesis, sink metabolism, and defense response by analyzing the regulation of mRNAs for representative enzymes of these pathways. Glucose as well as the fungal elicitor chitosan, the phosphatase inhibitor endothall, and benzoic acid were shown to result in a coordinated regulatory mechanism. The mRNAs for phenylalanine ammonia-lyase, a key enzyme of defense response, and for the sink-specific extracellular invertase were induced. In contrast, the mRNA for the Calvin cycle enzyme ribulose bisphosphate carboxylase was repressed. This inverse regulatory pattern was also observed in experiments with wounded leaves of C. rubrum plants. The differential effect of the protein kinase inhibitor staurosporine on mRNA regulation demonstrates that the carbohydrate signal and the stress-related stimuli independently activate different intracellular signaling pathways that ultimately are integrated to coordinately regulate source and sink metabolism and activate defense responses. The various stimuli triggered the transient and rapid activation of protein kinases that phosphorylate the myelin basic protein. The involvement of phosphorylation in signal transduction is further supported by the effect of the protein kinase inhibitor staurosporine on mRNA levels.