Construction of cTnC-linker-TnI (P) Genes, Expression of Fusion Protein and Preparation of Lyophilized Protein / 生物医学工程学杂志

In order to construct and express human cardiac troponin C-linker-troponin I(P) [ cTnC-linker-TnI(P)] fusion protein, detect its activity and prepare lyophilized protein, we searched the CDs of human cTnC and cTnI from GenBank, synthesized cTnC and cTnI(30-110aa) into cloning vector by a short DNA sequence coding for 15 neutral amino acid residues. pCold I-cTnC-linker-TnI(P) was constructed and transformed into E. coli BL21(DE3). Then, cTnC-linker-TnI(P) fusion protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Soluable expression of cTnC-linker-TnI(P) in prokaryotic system was successfully obtained. The fusion protein was purified by Ni²⁺ Sepharose 6 Fast Flow affinity chromatography with over 95% purity and prepared into lyophilized protein. The activity of purified cTnC-linker-TnI(P) and its lyophilized protein were detected by Wondfo Finecare™ cTnI Test. Lyophilized protein of cTnC-linker-TnI(P) was stable for 10 or more days at 37 °C and 4 or more months at 25 °C and 4 °C. The expression system established in this research is feasible and efficient. Lyophilized protein is stable enough to be provided as biological raw materials for further research.

Paraspinal Muscle Sparing Versus Percutaneous Screw Fixation: A Comparative Enzyme Study of Tissue Injury during the Treatment of L4-L5 Spondylolisthesis

OBJECTIVE: Screw fixation via the paraspinal muscle sparing approach and by percutaneous screw fixation are known to diminish the risk of complications, such as, iatrogenic muscle injury as compared with the conventional midline approach. The purpose of this study was to evaluate tissue injury markers after these less traumatic screw fixation techniques for the treatment of L4-L5 spondylolisthesis. METHODS: Twenty-two patients scheduled for posterior lumbar interbody fusion (PLIF) at the L4-L5 segment for spondylolisthesis were prospectively studied. Patients were divided into two groups by screw fixation technique (Group I: paraspinal muscle sparing approach and Group II: percutaneous screw fixation). Levels of serum enzymes representing muscle injury (CK-MM and Troponin C type 2 fast), pro-inflammatory cytokine (IL-8), and anti-inflammatory cytokine (IL-1ra) were analyzed using ELISA techniques on the day of the surgery and 1, 3, and 7 days after the surgery. RESULTS: Serum CK-MM, Troponic C type 2 fast (TNNC2), and IL-1ra levels were significantly elevated in Group I on postoperative day 1 and 3, and returned to preoperative levels on postoperative day 7. No significant intergroup difference was found between IL-8 levels despite higher concentrations in Group I on postoperative day 1 and 3. CONCLUSION: This study shows that percutaneous screw fixation procedure is the preferable minimally invasive technique in terms of minimizing muscle injury associated with L4-L5 spondylolisthesis.

Cardiac injury in traumatic subarachnoid hemorrhagea: prospective study in 35 patients

Various electrocardiographic abnormalities have been noted since 1954 in patients with head trauma complicated by subarachnoid hemorrhage [SAH]. However, very few studies have interested to these ECG modifications in the case of post traumatic SAH [t-SAH]. To assess the incidence of ECG abnormalities during the first five days after admission and the predictive value of these cardiac complications on the mortality in t-SAH. This prospective study included 35 patients out of 125 with traumatic SAH diagnosed in the emergency unit in Rabta's hospital [2001-2009]. Patients with cardio vascular history, thoracic trauma, non neurological coma and vascular-related neurological coma were excluded. An electrocardiogram monitoring was performed. A brain CT scan was performed in admission, 48 h after and case of neurological aggravation. Serum cardiac troponin 1C levels were determined on hospital admission and then on the third and fifth days of hospitalization. The statistical analysis was based on the non-parametric variance test of Kruskal-Wallis to compare the means; on the chi 2 and Fisher tests to compare percentage, with a significant result at 0.05 percentile and on the Odds ratio non-parametric factors for death. Association between 2 quantitative variables have been analyzed by Pearson coefficient of correlation. Mean age of the 35 patients was 39 +/- 17 years. Sex ratio was 4 in favor of men. The prevalence of electrocardiographic changes was of 57% [20 patients] Serum Troponin I level showed a peak on the 3rd day then it decreased. The majority of electrical abnormalities occurred during the third after admission and are associated to a markedly increased Troponin I plasma level and to the highest rate of mortality. Statistical analysis showed a significant correlation between T wave changes and the increase of serum Tn 1C level [p= 0; 0002]. The relative risk of mortality was higher than 7.2 times in cases with increase serum Tnlc level. We demonstrated that ECG changes were common in patients with t SAH and the major predictive factors of mortality were the increase of serum TnIC and T wave changes Subarachnoid hemorrhage [SAH] is a complication of head trauma inducing frequently cerebral vasospasm and even cerebral infarct. Various electrocardiographic abnormalities have been noted in patients with head trauma complicated by subarachnoid hemorrhage [1-5]. They are considered to be secondary to the massive catecholamine discharge in systemic circulation [6, -9]. Serum cardiac troponin I [Tnl] was considered a highly sensitive and specific marker of myocardial cell lesion and might be regularly performed in these patients to detect early myocardial ischemia. We carried out a prospective study in 35 patients with traumatic SAH [tSAH] in order to assess the incidence of coronary complications during the first five days after admission and to demonstrate the interest of troponin le blood assay in the diagnosis of coronary abnormalities

Lactic acid- the innocent culprit of muscle fatigue

What causes muscle fatigue? Is lactic acid considered to be a major culprit in the underlying mechanisms of muscle fatigue? These are very important questions and the answers are difficult and sophisticated. For decades, lactic acid was the major culprit of muscle fatigue. This review reveals that muscle fatigue would occur more badly if lactic acid was not produced in anaerobic cellular conditions to regenerate NAD[+] required for continuation of glycolysis, the major pathway for generation of energy needed for muscle performance

The research progress of one member of the EF-hand superfamily--troponin C / 生物工程学报

The EF-hand superfamily is a large group of proteins which contain EF-hand motif formed by helix-loop-helix. These proteins always have the ability of binding metal ions or forming dimmers. Troponin C, known as having ability of binding Ca2+, is one member of the EF-hand superfamily. Troponin C interacts with troponin I and troponin T, forming a troponin complex which takes part in regulating muscle contraction. It is interesting that troponin C was also found in non-muscular tissue, and its function was proved to be different from that of troponin C found in muscular tissue. To date, a lot of researches about troponin C have been carried out widely. However, most of them focused on vertebrate, seldom were done on invertebrate. Our group carried out a research on troponin C from silkworm, a model organism of insects, aiming to clarify the structure and function of silkworm troponin C. Here, we mainly discuss the characters of the EF-hand superfamily and the classification, structure and function of troponin C . We also introduced our work about silkworm troponin C briefly, hoping of making a little contribution to the research of invertebrate troponin C.

Diversity and plasticity of vertebrate skeletal muscle: insights from hybrid fibres

It is now generally accepted that hybrid skeletal muscle fibres are not experimental artefacts, but complex molecular systems that expand the functional repertoire of the muscle to which they belong. The purpose of this review is to highlight the cognitive value of hybrid fibres by discussing several insights into skeletal muscle biology produced by studies using hybrid fibres and/or muscles containing hybrid fibres. There is strong evidence that hybrid fibres can be used as indicators of muscle remodeling and specialization. Also, there is increasing evidence that hybrid fibres are suitable for investigating issues related to (i) the coexpression of different myosin heavy chain (MHC) isoforms and their assembly in the sarcomeric structure, (ii) the operation of the muscle cell as a multinuclear system, (iii) the tightness of the relationship between MHC isoform expression and expression of other polymorphic muscle proteins, (iv) the tightness of the relationship between MHC isoform expression and various contractile parameters, and (v) the extent of the neural input into defining the molecular and functional phenotype of skeletal muscle cells. It is predicted that, when used together with imaginatively designed methods, the hybrid fibres will further our (still limited) understanding of the regulation of muscle gene expression in multinuclear cells and of the interactions of gene products within and across different intracellular signalling pathways.

Levosimendan: uma nova alternativa no menejo da insuficiência cardíaca em terapia intensiva / Levosimedan: a new alternative for managing cardiac insufficiency in intensive care

Introdução: O usuo de levosimendan em pacientes criticamente enfermos e, principalmente, nos que se apresentam com pressão arterial média inferior a 60mmHG, ainda não teve a sua eficácia e a sua segurança estabelecidas.Objetivos: avaliar a resposta do levosimendan em cardiopatas graves já em uso de dobutamina e noradrenalina.Métodos: foram avaliados de forma propspectiva 37 pacientes internados em ambiente de terapia intensiva geral e cardiológica, sendo 51,3 por cento (n=19) do sexo masculino e 48,7 por cento (n=18) do sexo feminino. A média de idade doi de 65,3 anos, variando entre 49 e 84 anos, todos em classe funcional IV, segundo a classificação da NYHA, e dependentes da infusão venosa contínua de dobutamina com doses Superiores a 5ug/kg/min, sendo que 15 deles (40,5 por cento) estavam dependentes também de noradrenalina (dose acima de 0,05ug/kg/min)...

Poder pronóstico precoz de la mioglobina en los síndromes coronarios agudos sin elevación del segmento ST / Prognostic value of myoglobin in non-ST elevation acute coronary syndromes

Antecedentes: La mioglobina es un marcador precoz de necrosis miocárdica. Sin embargo, su valor pronóstico aún no se ha aclarado en forma prospectiva en las primeras horas de evolución de pacientes con síndromes coronarios agudos (SCA) sin supradesnivel del segmento ST. Objetivos: Determinar la relación entre los valores de mioglobina al ingreso y la ocurrencia de muerte o infarto a los 30 días en pacientes con SCA sin supradesnivel del ST. Material y métodos: Se realizó un estudio de cohortes prospectivo y multicéntrico que incluyó 366 pacientes para evaluar el valor pronóstico de la mioglobina en el momento de la admisión. Se determinó también la concentración de troponina T, la proteína C reactiva (PCR) y la CK-MB masa a las 12 horas del comienzo de los síntomas. Las determinaciones fueron realizadas a doble ciego por un laboratorio central e independiente. El punto final primario fue muerte y/o reinfarto no fatal a los 30 días. Se realizó un análisis multivariado para determinar la asociación entre los niveles de mioglobina y el punto final clínico. Resultados: La mediana de tiempo síntomas-admisión fue de 4 horas. La tasa de eventos a los 30 días fue del 10,1 por ciento. De acuerdo con la regresión logística, se observó una asociación entre los niveles elevados de mioglobina al ingreso y los eventos clínicos (OR 3,5 [IC 95 por ciento 1,6-7,6]), independiente de marcadores clínicos y bioquímicos convencionales. Conclusiones: La elevación precoz de la mioglobina agrega información pronóstica independiente a marcadores clínicos y bioquímicos convencionales y además se asocia con mala evolución clínica a los 30 días en pacientes con SCA sin supradesnivel del ST.

Usefulness of Immunohistochemistry in Various Types of Sudden Cardiac Death / 대한법의학회지

Postmortem diagnosis of early myocardial infarction is still a puzzling problem in forensic pathology practice, especially in sudden cardiac death. This study was undertaken to evaluate the distribution patterns of several immunohistochemical markers in various types of sudden cardiac death; 27 cases of obstructive coronary atherosclerosis (SCD-CAD) including 13 cases of critical coronary atherosclerosis, 6 cases of atherosclerotic heart disease and 8 cases of acute myocardial infarction, 26 cases of sudden cardiac death with obvious other causes (SCD-miscellaneous) including hypertensive heart disease, arrhythmogenic right ventricular cardiomyopathy, etc. and 14 cases of sudden cardiac death without any myocardial changes and/or minimal coronary atherosclerosis(SCDunknown). 10 cases of unnatural death were included in control group. Imnunohistochemical reactivity and the severity of reactivity loss were evaluated. The immunoreactivity against myoglobin and troponin C were excellent, and their reactivity loss were statistically significant in SCD-CAD compared to control group, SCD-unknown and SCD-miscellaneous (p<0.05). There were increasing pattern of loss of immunoreactivity in SCD-unknown and SCD-miscellaneous compared to control group. The pattern of expression and the severity of reactivity loss against vascular endothelial growth factor(VEGF) and basic fibroblast growth factor (bFGF) were with no differences in experimental group and control group. Immunohistochemistry may be partially useful in determination of early myocyte necrosis in sudden cardiac death, and myoglobin and troponin C may be better. But, its practical application in forensic pathology may be still limited.

Utilidad del ecocardiograma y de marcadores séricos en la estratificación de riesgo en pacientes cursando con embolia pulmonar submasiva / Utility of echocardiogram and serum markers in the risk stratification in patients attending with submassive pulmonary embolism

Los pacientes que cursan tromboembolismo, dependiendo de su severidad, pueden recibir variados tratamientos no exentos de efectos adversos. Existe, por lo tanto, la necesidad de estratificar riesgo -magnitud- mortalidad con criterios que le permitan al clínico tomar las mejores decisiones. En este artículo se pretende establecer un sistema de aproximación al tromboembolismo pulmonar de acuerdo a la información hasta ahora disponible.

A ligação de cálcio à troponina C analisada por diálise de fluxo e por espectroscopia de fluorescência / Calcium binding to troponin C analized by flow dialysis and by fluorescence spectroscopy

A troponina C é o componente do complexo heterotrimérico troponina ao qual o Ca²+ se associa. Pearlstone et al. (Biochemistry 31, 6545, (1992)) utilizaram o cDNA da troponina C de músculo esquelético de galinha (sTnC) para construir um mutante onde a fenilalanina da posição 29 foi substituída por triptofano (mutante F29W). Esse mutante permitiu que a ligação de Ca²+ aos sítios regulatórios amino terminais fosse acompanhada através de técnicas fluorescentes. Entretanto, algumas propriedades da sTnC foram alteradas pela mutação (Li et al. (1995) Biochemistry 34, 8330). No presente estudo, ensaios de ligação direta de Ca²+ bem como titulações de Ca²+ seguidas por fluorescência foram usadas para melhor se entenderem...

Troponina: un marcador util en isquemia cardíaca / Troponin: a marked use in heart ischemia

En la búsqueda de realizar diagnósticos precisos y en el menor tiempo posible, se han desarrollado nuevos marcadores bioquímicos de elevada especifícidad y sensibilidad para el diagnóstico de lesión isquémica cardíaca, sobre todo cuando estamos en presencia de un paciente con dolor torácico en el cual ni las enzimas ni el electrocardiograma son concluyentes. Estos nuevos marcadores son proteínas constituyentes del músculo cardíaco conocidas como complejo de troponina (I, C y T) presentando una configuración genética que les permite alta especificidad, distinguiéndolas de las proteínas del músculo esquelético. También las caracteriza una adecuada cinética que las hace ser sensibles aún cuando existen lesiones muy pequeñas, o cuando ha pasado un período considerable de haberse iniciado el evento isquémico. Presentamos una revisión de los últimos trabajos publicados sobre estos marcadores

Kinetics of a recombinant protein production by E. coli BL21

Troponina C, TnC, uma proteína do músculo esquelético de galinha, foi produzida em células de Escherichia coli BL21 (DE3) pLysS contendo o cDNA no plasmídio pET sob controle do promotor lac UV5. Neste sistema, a expressäo do gene da TnC depende da presença de uma molécula indutora, o que permitiu a realizaçäo de ensaios com uma fase de crescimento independente da fase de produçäo. O balanço gasoso aplicado à fase de produçäo da TnC em cultivos em biorreator, resultou em um fator de conversäo de oxigênio a células, Yx/o, entre6.5 e 142 mg/mmol, e porcentagem de TnC na célula com relaçäo ao teor de proteína celular, TnC (porcentagem), entre 11.7 e 26.1 por cento, sendo que para os maiores valores de TnC (porcentagem) correspondem aos menores valores de Yx/o. Além disso, para o maior o valor de y no início da fase de induçäo, obteve-se maior valor de TnC (porcentagem). Os dados apresentados indicam que na produçäo de TnC sob controle do gene lac UV5 e promotor de T7 em E.coli, a energia do metabolismo aeróbico é direcionada principalmente à síntese da proteína recombinante com conseqüente reduçäo da atividade de crescimento.