RESUMEN
We present the earliest evidence for domestic cat (Felis catus L., 1758) from Kazakhstan, found as a well preserved skeleton with extensive osteological pathologies dating to 775-940 cal CE from the early medieval city of Dzhankent, Kazakhstan. This urban settlement was located on the intersection of the northern Silk Road route which linked the cities of Khorezm in the south to the trading settlements in the Volga region to the north and was known in the tenth century CE as the capital of the nomad Oghuz. The presence of this domestic cat, presented here as an osteobiography using a combination of zooarchaeological, genetic, and isotopic data, provides proxy evidence for a fundamental shift in the nature of human-animal relationships within a previously pastoral region. This illustrates the broader social, cultural, and economic changes occurring within the context of rapid urbanisation during the early medieval period along the Silk Road.
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Gatos/genética , Mascotas/historia , Alimentación Animal , Crianza de Animales Domésticos/historia , Animales , Arqueología/métodos , Huesos/fisiología , Isótopos de Carbono , Ciudades , Análisis por Conglomerados , Ecología , Variación Genética , Geografía , Historia Antigua , Kazajstán , Isótopos de Nitrógeno , FilogeniaRESUMEN
Epidural spinal cord stimulation (SCS) is a reversible but invasive procedure which should be used for neuropathic pain, e.g. complex regional pain syndrome I (CRPS) and for mostly chronic radiculopathy in connection with failed back surgery syndrome following unsuccessful conservative therapy. Epidural SCS can also successfully be used after exclusion of curative procedures and conservative therapy attempts for vascular-linked pain, such as in peripheral arterial occlusive disease stages II and III according to Fontaine and refractory angina pectoris. Clinical practice has shown which clinical symptoms cannot be successfully treated by epidural SCS, e.g. pain in complete paraplegia syndrome or atrophy/injury of the sensory pathways of the spinal cord or cancer pain. A decisive factor is a critical patient selection as well as the diagnosis. Epidural SCS should always be used within an interdisciplinary multimodal therapy concept. Implementation should only be carried out in experienced therapy centers which are in a position to deal with potential complications.
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Dolor Crónico/terapia , Terapia por Estimulación Eléctrica/métodos , Médula Espinal/fisiopatología , Angina de Pecho/fisiopatología , Angina de Pecho/terapia , Dolor Crónico/etiología , Dolor Crónico/fisiopatología , Síndromes de Dolor Regional Complejo/fisiopatología , Síndromes de Dolor Regional Complejo/terapia , Electrodos Implantados , Espacio Epidural , Medicina Basada en la Evidencia , Síndrome de Fracaso de la Cirugía Espinal Lumbar/fisiopatología , Síndrome de Fracaso de la Cirugía Espinal Lumbar/terapia , Humanos , Radiculopatía/fisiopatología , Radiculopatía/terapiaRESUMEN
AIMS: Definition of the regional pattern of dopamine transporter (DAT) dysfunction in advanced Parkinson's disease (PD) and evaluation of a potential correlation between DAT binding and symptoms; elucidation of the role of DAT imaging in the differential diagnosis of PD and multiple system atrophy (MSA); assessment and comparison of serotonin transporter (SERT) binding in PD and MSA. METHODS: [(123)I]beta-CIT SPECT was performed in 14 patients with advanced PD, 10 with moderate MSA and 20 healthy persons. Specific to nonspecific tracer binding ratios (V(3)") were calculated via ROI analysis of uptake images at 4 h (SERT binding) and 24 h (DAT binding) p. i. RESULTS: In PD bilateral reduction of striatal DAT binding (63-70%) was seen. The caudate ipsilateral to the clinically predominantly affected side showed relatively the least impairment. Significant correlations (r = -0.54 to -0.64) between DAT binding and Hoehn and Yahr stage, UPDRS-scores and duration of disease were found. In MSA DAT binding was less reduced (40-48%) targeting the putamen contralateral to the side of clinical predominance. Significantly lower SERT binding was observed in PD midbrain and MSA hypothalamus compared to controls -- and in MSA relative to PD mesial frontal cortex. CONCLUSIONS: In advanced PD striatal DAT binding is markedly reduced with the least reduction in caudate ipsilateral to the clinically predominantly affected side. In moderate MSA with asymmetrical symptoms DAT dysfunction is predominant in the contralateral putamen, a pattern seen in early PD. The reduction of SERT in the midbrain area of PD patients suggests additional tegmental degeneration while in MSA the serotonergic system seems to be more generally affected.
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Encéfalo/diagnóstico por imagen , Proteínas Portadoras/metabolismo , Cocaína/análogos & derivados , Radioisótopos de Yodo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Atrofia de Múltiples Sistemas/diagnóstico por imagen , Proteínas del Tejido Nervioso , Enfermedad de Parkinson/diagnóstico por imagen , Radiofármacos , Encéfalo/metabolismo , Cuerpo Estriado/diagnóstico por imagen , Cuerpo Estriado/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Atrofia de Múltiples Sistemas/metabolismo , Especificidad de Órganos , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Cintigrafía , Proteínas de Transporte de Serotonina en la Membrana PlasmáticaRESUMEN
The Working Group on Neuromodulation of the German Association for the Study of Pain, composed of representatives from various scientific specialty societies, met on December 9, 2000, March 24, 2001, October 5, 2001, and December 8, 2001. As a result of these discussions grounded in current knowledge, the following guidelines were formulated for the standardization of invasive techniques of neuromodulation intended to serve as a systematic aid in decision-making and to provide recommendations for practice-oriented methods. The guidelines were based on both the clinical and practical experience of the group participants (see information box on the next page) as well as on the current scientific literature and guidance from the consensus report of the European Federation of IASP Chapters (EFIC) [23]. The guidelines serve the purpose of orientation and have no effect on either assumption of liability or discharge from liability. The guidelines were conceived for use by physicians in private practice,doctors in hospitals,and nonmedical personnel concerned with the care of chronic pain patients. The Working Group consists of unsalaried volunteers. The participants received no honorarium and were only reimbursed for normal travel expenses in accordance with customary directives. The guidelines will be revised should new scientific results become available, at the latest in 2 years. The plan exists to further develop the guidelines to stages II and III (AWMF). The Steering Committee of the DGSS appraised the guidelines and authorized the guidelines before publication.
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Analgésicos/uso terapéutico , Neurotransmisores/fisiología , Dolor Intratable/tratamiento farmacológico , Guías de Práctica Clínica como Asunto/normas , Analgésicos/administración & dosificación , Europa (Continente) , Humanos , Inyecciones Espinales , Dolor Intratable/fisiopatología , Dolor Intratable/psicología , Garantía de la Calidad de Atención de SaludRESUMEN
Adult respiratory distress syndrome (ARDS) has a high mortality. Its only effective treatment is respiratory therapy. If this fails mortality is probably 100 per cent. No other treatment for ARDS has proved effective including "magic bullets." Twenty patients suffering from ARDS secondary to trauma and/or sepsis failed to respond to treatment with mechanical ventilation and positive end-expiratory pressure. On the assumption that disseminated intravascular coagulation initiates ARDS by occluding the pulmonary microcirculation with microclots, the patients were treated with plasminogen activators. The patients responded with significant improvement in partial pressure of oxygen in arterial blood. No bleeding occurred and clotting parameters remained normal. We conclude that ARDS can be safely treated with plasminogen activator.
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Activadores Plasminogénicos/uso terapéutico , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Estreptoquinasa/uso terapéutico , Activador de Plasminógeno de Tipo Uroquinasa/uso terapéutico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Análisis de los Gases de la Sangre , Niño , Preescolar , Terapia Combinada , Femenino , Humanos , Masculino , Persona de Mediana Edad , Traumatismo Múltiple/complicaciones , Oxígeno/sangre , Activadores Plasminogénicos/farmacología , Respiración Artificial , Síndrome de Dificultad Respiratoria/complicaciones , Síndrome de Dificultad Respiratoria/metabolismo , Síndrome de Dificultad Respiratoria/mortalidad , Sepsis/complicaciones , Índice de Severidad de la Enfermedad , Estreptoquinasa/farmacología , Análisis de Supervivencia , Resultado del Tratamiento , Activador de Plasminógeno de Tipo Uroquinasa/farmacologíaRESUMEN
Forty-three patients with peripheral neuropathic pain, exclusively pain reduced by spinal cord stimulation (SCS), were switched into a painful state after SCS inactivation. This mode was used to assess the pain-relieving effect of carbamazepine (CMZ) and opioids in a double-blinded, placebo-controlled trial. In Phase 1, the patients were randomly allocated to receive either CMZ (600 mg/d) or placebo during an SCS-free period of 8 days. In Phase 2, after a CMZ elimination interval of 7 days, 38 patients received either sustained-release morphine (90 mg/d) or placebo for 8 days. In cases of intolerable pain, the patients were authorized to reactivate their SCS. The pain intensity was rated on a numeric analog scale. In 38 patients who completed Phase 1, significant delay in pain increase was observed in the CMZ group as compared with placebo (P = 0.038). In Phase 2, the trend observed with morphine was insignificant (P = 0.41). Two CMZ patients and one morphine patient showed complete pain relief and preferred to continue the medication. Thirty-five patients returned to SCS. We conclude that CMZ is effective in peripheral neuropathic pain. Morphine obviously requires larger individually titrated dosages than those used in this study for results to be adequately interpreted.
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Analgésicos no Narcóticos/uso terapéutico , Analgésicos Opioides/administración & dosificación , Carbamazepina/uso terapéutico , Síndromes de Dolor Regional Complejo/tratamiento farmacológico , Terapia por Estimulación Eléctrica , Morfina/administración & dosificación , Dolor/tratamiento farmacológico , Médula Espinal/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Preparaciones de Acción Retardada , Método Doble Ciego , Humanos , Persona de Mediana EdadRESUMEN
Developmental dislocation and/or dysplasia of the hip (DDH), formerly referred to as congenital dislocation of the hip, is a frequent problem of the neonatal and infant hip. At that age, the femoral head and acetabulum consist of cartilage components that are not visible on plain radiography but readily identified by ultrasound. Realtime sonography allows for assessment of the hip in multiple planes, both at rest and with movement. Ultrasound can replace radiographic studies and thereby reduce radiation exposure to the young infant.
RESUMEN
Equipment and software used for dual energy x-ray absorptiometry (DXA) was developed for adults. As use of the method grows in pediatrics, understanding technical issues is important for those performing and interpreting DXA studies in infants, children, and adolescents. Detection of very low bone mineral density requires alteration of automated scan techniques. Normal values based on chronologic age have been published. The Z score becomes the more meaningful statistic as opposed to the T score used in reporting adult DXA measurement of bone mineral density.
RESUMEN
The safety and tolerance of neuroleptanaesthesia (NLA), balanced anaesthesia (BAL), and intravenous anaesthesia with propofol (IVA) were analysed for the first time in a prospective, randomised clinical trial. METHODS. In all, 1318 surgical patients received either NLA, BAL, or IVA. Patients who had regional anaesthesia, were aged under 18 years, or were non-cooperative or vitally threatened (ASA class i.v. to V) did not participate in the study. Premedication and anaesthetic course were set up at a standard of 30% oxygen and 70% nitrous oxide. Incidents, events, and complications due to anaesthesia were obtained (IEC key of the German Society of Anaesthesia and Critical Care Medicine, DGAI). Furthermore, postanaesthetic alertness based on specific recovery tests and the quality of anaesthesia from the patient's viewpoint, rated by patient questionnaires from the DGAI were evaluated. All parameters were calculated and checked for statistical significance using the chi-square test. RESULTS AND DISCUSSION. The groups were broadly comparable with respect to age (P = 0.91), ASA class (P = 0.42), preoperative blood pressure (P = 0.36), and length of anaesthesia (P = 0.82). The anaesthesia, which averaged 103 min, comprised the following regimens: (1) NLA: 7.1 mg droperidol and 0.008 mg/kg body weight fentanyl, (2) IVA: 493.4 mg propofol and 0.004 mg/kg body weight fentanyl, and (3) BAL: 2.6 mg droperidol and 0.004 mg/kg body weight fentanyl with 0.4 vol.% isoflurane. With respect to anaesthetic risk, the following reactions were observed: the use of NLA led to a high incidence of tachycardia (P = 0.001), arrhythmias (P = 0.05), and hypertensive reactions (P = 0.001), whereas in the IVA group only hypotension (P = 0.0001) occurred. However, after the use of BAL none of the aforementioned complications were detectable to any considerable degree. Similarly, patients who had cardiac disease showed greater IEC changes after the use of NLA than after BAL or IVA (P = 0.02) (Tables 1 and 2). The heart rates and blood pressures during BAL and IVA were extremely stable, and therefore, vasoactive therapy was required considerably less in comparison to NLA (P = 0.001) (Table 4). Recovery after the use of IVA was strikingly rapid: the patient's responsiveness, orientation, and ability to concentrate was significantly better than after the other anaesthetic regimen (P = 0.01) (Table 5). With regard to the typical discomforts after anaesthesia, IVA was highly superior to BAL and NLA: nausea (P = 0.0003) and retching (P = 0.03) hardly ever occurred (Table 6). Due to the tolerable manner of waking up and rapid return of orientation and the ability to concentrate, IVA was highly favoured by the patients (P = < 0.01) (Table 7). CONCLUSION. The present results show clear clinical advantages of BAL and IVA in contrast to neuroleptanaesthesia. Due to the very low incidence of side effects such as nausea and vomiting IVA was highly recommended by the patients, at least in part because of the rapid recovery time.
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Anestesia Intravenosa , Anestésicos Intravenosos , Neuroleptanalgesia , Propofol , Adulto , Anestesia Intravenosa/efectos adversos , Anestésicos Intravenosos/administración & dosificación , Presión Sanguínea/efectos de los fármacos , Método Doble Ciego , Fentanilo , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Complicaciones Intraoperatorias , Persona de Mediana Edad , Neuroleptanalgesia/efectos adversos , Propofol/administración & dosificación , Estudios ProspectivosRESUMEN
The choice of appropriate anaesthesia in a more or less seriously ill patient requires detailed information on the risk and tolerance of each specific anaesthetic regimen. The objective of this prospective, randomised clinical trial was to test the hypothesis that three regimens of general anaesthesia--neurolept-(NLA), balanced (BAL), and intravenous propofol anaesthesia (IVA)--differ with regard to safety and comfort. The criteria for the intraoperative safety and postoperative comfort of the patients were the incidents, events and complications (IEC) that required medical treatment as well as the evaluation of postoperative complaints by the patients according to the IEC list and patient questionnaires of the German Society of Anaesthesia and Critical Care Medicine (DGAI). METHODS. The study duration was about 4 months, from January to April 1992. During this period the patients of all nine operative departments of the hospital received strictly randomised NLA, BAL, or IVA. Patients who had regional anaesthesia or were not capable of understanding the German language, were nonco-operative, or were seriously ill (ASA class IV to V) as well as children under 18 years of age did not participate in the study. All eligible patients provided their informed consent. ANAESTHESIA. For premedication 10 mg chlorazepate was administered the night before and on the day of surgery. Anaesthesia was conducted under normoventilation using a mixture of 70% nitrous oxide and 30% oxygen. NLA patients were induced intravenously with 0.2 mg/kg body weight etomidate and received 0.005 mg/kg fentanyl and 0.07 mg/kg droperidol before the start of surgery. The repetition dose was 0.2 mg fentanyl and 2.5 mg droperidol. In the BAL patients the dose of fentanyl and droperidol was reduced to 50% due to the addition of isoflurane up to 1 vol. %. IVA patients received 2 mg/kg propofol over 3 min followed by an infusion of 3-5 mg/kg per hour together with 0.2 mg fentanyl/h. Neuromuscular blockade was accomplished with vecuronium 0.1 mg/kg. If the blood pressure and heart rate increased by more than 20% of preoperative values, analgesia was reinforced by an additional fentanyl dose. Anaesthesia was subsequently enhanced by increasing the neurolept/propofol/isoflurane dose by up to 50%. DATA COLLECTION. The following parameters were registered: patients' personal data and physical condition according to ASA classification; the grade of risk according to the Munich risk checklist; the frequency of IEC during surgery; the patients' permanent medications; postanaesthetic vigilance and recovery; the acceptance of the assigned anaesthetic by the physician; the cost of the anaesthetic used; and pre- and post-operative complaints as well as the assessment of anaesthesia by the patient. The statistical evaluation was performed using the chi-square test. RESULTS. A total of 1,346 patients were enrolled in the study; 28 (2%) were excluded because the treatment protocol was changed by the anaesthesiologist. Seventy per cent were recruited from general, gynaecologic, or otorhinolaryngologic surgery. The three anaesthetic regimens (NLA, BAL, and IVA) were used in other departments with the same frequency with the exception of ophthalmology and urology (P > 0.1) (Fig. 1). Of the 1,318 eligible patients, 443 received NLA, 443 BAL, and 432 IVA (P = 0.8). The distribution of the various parameters was surprisingly similar among the three groups: the average age was 50 years (P = 0.91), body weight 71 kg (P = 0.33), reference or initial blood pressure 130/80 mm Hg (P = 0.36), average time of anaesthesia 103 min (P = 0.82), and all had the same risk score (P = 0.42). Sixty per cent were female. An average of 85% of the 18- to 89-year-old patients were considered to be healthy according to the ASA risk classification (P = 0.42). However, on applying the Munich risk checklist the average number of healthy individuals was 5% to 10% lower than that of the ASA risk classification.
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Anestesia Intravenosa , Neuroleptanalgesia , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anestesia Intravenosa/efectos adversos , Anestesia Intravenosa/economía , Femenino , Humanos , Complicaciones Intraoperatorias , Masculino , Persona de Mediana Edad , Neuroleptanalgesia/efectos adversos , Neuroleptanalgesia/economía , Medicación Preanestésica , Estudios Prospectivos , RiesgoRESUMEN
Phenoxypropanols have been used for some time as ingredients in surface and instrument disinfectants. Since the residual solutions of these preparations are discharged into the waste-water, the biodegradability of the ingredients used is of great importance. Tests by means of the Zahn-Wellens test in accordance with OECD guidelines have shown that phenoxypropanols are biodegradable. In further studies it was investigated whether unwanted intermediate products are formed in considerable amounts. Of particular interest was the question of the possible development of phenol outside the bacterial cells. To test the biodegradability of the aromatic alcohols and to ascertain whether there is extracellular formation of phenol, microorganisms were isolated from the municipal sewage plant at Stellinger Moor, Hamburg, and from water from the River Elbe. Phenoxypropanol-containing solutions were then inoculated with the bacteria and incubated at 30 degrees C. At regular intervals both the concentration of the alcohols was determined and the test for phenol was performed by means of liquid chromatography on reverse phase material (RP 18) and detection with a photodiode array detector. The breakdown of the phenoxypropanols was monitored for a period of up to 4 weeks. During this period a clear breakdown of the aromatic alcohols was observed. No phenol was detected.
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Bacterias/metabolismo , Desinfectantes/metabolismo , Glicoles de Propileno/metabolismo , Microbiología del Agua , Biodegradación Ambiental , Biomasa , Cromatografía Líquida de Alta Presión , Agua Dulce , Alemania , Fenol , Fenoles/análisis , Aguas del AlcantarilladoRESUMEN
New specifications for quality control of nuclear medical instrumentation are given in the German "Richtlinie Strahlenschutz in der Medizin", published in 1993. These specifications include the corresponding DIN-Normen, the German standards. In both papers the description of the various test procedures is given in a very abbreviated form, so that many of the people having to perform these test procedures are more or less puzzled. This paper will provide for a better understanding of what is meant and will also give many useful hints in performing the test procedures. A discussion of the necessary test phantoms and auxiliary devices completes this paper.
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Medicina Nuclear/instrumentación , Garantía de la Calidad de Atención de Salud , Alemania , Agencias Gubernamentales , HumanosRESUMEN
Nineteen patients suffering from adult respiratory distress syndrome (ARDS) secondary to trauma or sepsis, or both, failed to respond to treatment with mechanical ventilation with oxygen and positive end-expiratory pressure. On the premise that ARDS may be caused by the microclots of disseminated intravascular coagulation obstructing the pulmonary microcirculation, the patients were treated with either streptokinase or urokinase. Eighteen patients responded with significant improvement in PaO 2 value. No bleeding occurred and clotting parameters remained normal.
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Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Estreptoquinasa/uso terapéutico , Activador de Plasminógeno de Tipo Uroquinasa/uso terapéutico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oxígeno/sangre , Síndrome de Dificultad Respiratoria/etiología , Síndrome de Dificultad Respiratoria/patología , Resultado del TratamientoRESUMEN
Spinal cord stimulation (SCS) has routinely been used since the beginning of the 1970s. The initial indications for stimulation were the so-called deafferentation or neurogenic pain. Further work has confirmed that neurostimulation is useful in severe peripheral vascular disease in relieving pain and increasing capillary blood flow and oxygen tension. The effects are similar to those of sympathectomy. In 1964 Apthorp et al. discovered that sympathectomy relieved angina in about 75% of patients. The use of SCS to treat angina follows logically from its use in peripheral vascular disease. METHODS. The pain-relieving effect of SCS was investigated in two patients, 54 and 69 years old, who were hospitalised for 8 and 28 days. Both patients had severe angina pectoris (duration 2 and 15 years, New York Heart Association class III and II), related to three-vessel disease, and one of them had previously undergone his third bypass operation. The other patient was not considered suitable for surgery. The antianginal treatment (long-acting nitrates, beta-blockers, calcium antagonists) was regarded as optimal and was not changed during the observation period (Table 1). SURGICAL TECHNIQUE AND STIMULATION EQUIPMENT. We used the commercially available Medtronic SCS system. The operation was performed under local anaesthesia to allow the patient to answer questions during the intraoperative stimulation. The epidural space was punctured at the level of T7-T8 in one case and T11-T12 in the other. The electrode tip was positioned in the midline or a few millimetres to the left at the T1-T2 level (Figs. 1, 2), so that the patient felt a prickling sensation in the precordial area and into the arms. The distal end of the electrode was sutured to the fascia and connected via a tunnelled extension lead to the external pulse generator. The pulse width was 200 microseconds, frequency 80 Hz. An appropriate amplitude (usually 8-10 V) was used for comfortable paraesthesia. The study consisted of two parts: a run-in period (1 week) to standardise the stimulation when mobilisation was performed. A treatment period (18 months) to determine the patient's working capacity after continuous stimulation (Table 2). After a successful run-in period a Medtronic receiver was implanted, connected to the electrode and stimulated by external pulse generator. Different variables were used to assess the effect: pulse rate, blood pressure, the product of pulse rate and systolic blood pressure, estimated anginal pain, and ST changes in the electrocardiogram (ECG) before, during and after mobilisation. RESULTS. The stimulation was carried out for 30 min 10-12 times a day during the run-in period and five to six times a day during the treatment period. Altogether there was slight lowering of heart rate and systolic blood pressure. Consequently the product of heart rate and systolic blood pressure was diminished. In one case (NYHA II) the distinct disorder of repolarisation reverted to the normal condition as shown on ECG. In the other case (NYHA III) the ECG remained unchanged because of a severe aneurysm of the cardiac wall. Both patients experienced nearly complete pain relief after a few days for 6 and 12 months respectively. However, an increasing effort tolerance could be demonstrated in both patients by reducing the extent of the heart failure (NYHA II/III to NYHA I/II) (Table 2). DISCUSSION. Our two hospitalised patients had clinically intractable angina pectoris and severe manifestations of heart disease corresponding to at least NYHA functional class II-III. Both were unsuitable for operation and showed no improvement on individually titrated maximal oral antianginal drug treatment. During SCS treatment significant improvement was obvious: chest pain, ST-segment depression, and the extent of heart failure could be reduced. Both patients reached a better NYHA functional class, exhibited increased working capacity and reported reductions in anginal attacks and pain. Th
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Angina de Pecho/fisiopatología , Manejo del Dolor , Médula Espinal/fisiología , Estimulación Eléctrica Transcutánea del Nervio , Anciano , Femenino , Humanos , Persona de Mediana EdadRESUMEN
Polyacrylamide capillary gels were prepared with constant (5% C) cross-linker concentration and with total acrylamide concentration ranging from 2.5 to 6% T. At each acrylamide concentration, peak spacing was constant for DNA sequencing fragments ranging from 25 to 250 nucleotides in length. Peak spacing increased linearly with the total acrylamide concentration. The intercept of the retention time vs. fragment length plot was independent of % T. Ferguson plots were constructed for short DNA fragments; the polyacrylamide pore size falls in the 2.5 to 3.5 nm range for the gels studied. Theoretical plate count is independent of total acrylamide concentration; longitudinal diffusion, and not thermal gradients, limit the plate count. A phenomenological model is presented that predicts retention time, plate count, and resolution for sequencing fragments ranging in size from 25 to 250 bases and gels that range from 2.5 to 6% total acrylamide.
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Resinas Acrílicas/química , Secuencia de Bases , ADN/análisis , Electroforesis en Gel de Poliacrilamida/instrumentación , Difusión , Modelos TeóricosRESUMEN
Complicated electropherograms are produced in the separation of fluorescently labeled peptides. Incomplete labeling of epsilon-amino groups on lysine residues results in the production of 2n-1 reaction products, where n is the number of alpha and epsilon amino groups in the peptide. A single label is attached to the peptide by first taking the peptide through one cycle of the Edman degradation reaction. All epsilon-amino groups are converted to the phenyl thiocarbamyl and the cleavage step exposes one alpha-amino group at the N-terminus of the peptide; the fluorescent label is attached to the N-terminus.
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Péptidos/análisis , Secuencia de Aminoácidos , Electroforesis , Colorantes Fluorescentes , Indicadores y Reactivos , Datos de Secuencia Molecular , Espectrometría de FluorescenciaRESUMEN
We report a modification to the peak-height encoded DNA sequencing technique of Tabor and Richardson. As in the original protocol, the sequencing reaction uses modified T7 polymerase with manganese rather than magnesium to produce very uniform incorporation of each dideoxynucleoside. To improve sequencing accuracy, two fluorescently labeled primers are employed in separate sequencing reactions. As an example, one sequencing reaction uses a FAM-labeled primer with dideoxyadenosine triphosphate and dideoxycytosine triphosphate; the concentrations of ddATP and ddCTP are adjusted to produce a 2:1 variation in the relative intensity of fragments. The second sequencing reaction uses a TAMRA labeled primer with dideoxythymidine triphosphate and dideoxyguanidine triphosphate; the concentrations of ddTTP and ddGTP are adjusted to produce a 2:1 variation in relative intensity of fragments. The pooled reaction products are separated by capillary gel electrophoresis and identified by one of three different detector systems. Use of a 2:1 peak height ratio typically produces a sequencing accuracy of 97.5% for the first 350 bases; a 3:1 peak height ratio improves accuracy to 99.5% for the first 400 bases. For these experiments, capillary electrophoresis is performed at an electric field of 200 V/cm; two to three hours are required to separate sequencing fragments up to 400 nucleotides in length.
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ADN/genética , Análisis de Secuencia de ADN/métodos , Bacteriófago T7/enzimología , ADN/química , ADN Polimerasa Dirigida por ADN , Desoxirribonucleótidos/análisis , Electroforesis en Gel de Poliacrilamida/instrumentación , Electroforesis en Gel de Poliacrilamida/métodos , Indicadores y Reactivos , Rayos Láser , Análisis de Secuencia de ADN/instrumentación , Espectrometría de FluorescenciaRESUMEN
Compressions are occasionally found during the separation of DNA sequencing fragments, particularly in G/C-rich regions and in gels operated at room temperature. Addition of at least 10% formamide to urea/polyacrylamide sequencing gels improves the denaturing capacity of the gel, minimizing compressions. Addition of 20% or more formamide decreases the separation rate, theoretical plate count, and resolution for normally migrating fragments. An optimum concentration of 10% formamide improves resolution of compressed regions without degrading the other characteristics of the gel. Operation of gels at room temperature simplifies the engineering associated with automated sequencers based on capillary gel electrophoresis.
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ADN/química , Electroforesis en Gel de Poliacrilamida/métodos , Geles/química , Secuencia de Bases , Formamidas , Desnaturalización de Ácido Nucleico , TemperaturaRESUMEN
Capillary gel electrophoresis is demonstrated for the four-spectral-channel sequencing technique of Smith, the two-spectral-channel sequencing technique of Prober, and the one-spectral-channel sequencing technique of Richardson and Tabor. Sequencing rates up to 1000 bases/h are obtained at electric field strengths of 465 V/cm. At lower electric field strengths, capillary electrophoresis produces useful data for fragments greater than 550 nucleotides in length with 2 times better resolution than slab gel electrophoresis. An on-column detector produces detection limits of 200 zmol (1 zmol = 10(-21) mol = 600 molecules) for the four-spectral-channel technique. A postcolumn detector, based on the sheath flow cuvette, produces detection limits of 20 and 2 zmol for the two- and one-spectral-channel techniques, respectively.