Protein biomarkers of external root resorption: A new protein extraction protocol. Are we going in the right direction?

OBJECTIVE: The aim of this study is to determine a protocol of gingival crevicular fluid protein extraction used for the first dimension of 2-DE gels. It also aims at conducting a review on the current candidates for protein markers of this pathology, all of which may be used to prevent the disease. METHODS: Gingival crevicular fluid was collected from two groups of 60 patients each, with and without external root resorption. Samples were extracted by means of various methods of protein extraction. SDS-PAGE gels were used to assess the quality of the method which was subsequently tested during isoelectric focusing of 2-DE gels taken from samples of patients with and without the disease. RESULTS: Milli-Q ultrapure ice cold water, without precipitation for gingival crevicular fluid protein extraction, proved the method with greatest sharpness to detect protein bands. Additionally, it allowed two-dimensional electrophoresis to be performed. CONCLUSION: The new protein extraction protocol does not interfere in isoeletric focusing of 2-DE gels. Furthermore, it provides the greatest sharpness in detecting protein bands of SDS-PAGE gels. This will allow mapping and searching of new external root resorption markers, particularly due to the difficulty in carrying out molecular tests with the current candidates for protein markers. .

OBJETIVO: o objetivo desse trabalho foi determinar o protocolo de extração proteica do fluido crevicular gengival, que pudesse ser utilizado para a realização da primeira dimensão dos géis 2-DE, bem como fazer uma revisão dos atuais candidatos a marcadores proteicos dessa patologia que podem ser utilizados na prevenção dessa doença. MÉTODOS: foi coletado o fluido crevicular gengival de dois grupos de 60 pacientes, com e sem a reabsorção radicular externa. As amostras foram extraídas por diversos métodos de extração proteica e utilizados géis SDS-PAGE para aferir a qualidade do método, que posteriormente foi testado durante a realização da focalização isoelétrica dos géis 2-DE, de amostras de pacientes com e sem a patologia. RESULTADOS: a utilização de água Milli-Q gelada ultrapura, sem nenhuma precipitação para a extração proteica do fluido crevicular gengival, foi o método com maior nitidez das bandas proteicas, além de permitir a realização da eletroforese bidimensional. CONCLUSÕES: o novo protocolo de extração proteica não interfere na focalização durante a realização dos géis 2-DE, além de maior nitidez na resolução das bandas proteicas dos géis SDS-PAGE. Isso permitirá o mapeamento e busca de novos marcadores da reabsorção radicular externa, tendo em vista a dificuldade de realização de testes moleculares com os atuais candidatos a marcadores proteicos. .

First characterisation of plasmid-mediated quinolone resistance-qnrS1 co-expressed bla CTX-M-15 and bla DHA-1 genes in clinical strain of Morganella morganii recovered from a Tunisian Intensive Care Unit.

Purpose: Aim of this study was to show the emergence of the qnr genes among fluoroquinolone-resistant, AMPC and ESBL (extended-spectrum-beta-lactamase) co-producing Morganella morganii isolate. Materials and Methods: A multi resistant Morganella morganii SM12012 isolate was recovered from pus from a patient hospitalized in the intensive care unit at the Military hospital, Tunisia. Antibiotic susceptibility was tested with the agar disk diffusion method according to Clinical and Laboratory Standards Institute guidelines. ESBLs were detected using a standard double-disk synergy test. The characterization of beta-lactamases and associated resistance genes were performed by isoelectric focusing, polymerase chain reaction and nucleotide sequencing. Results: The antimicrobial susceptibility testing showed the high resistance to penicillins, cephalosporins (MICs: 64-512 μg/ml) and fluoroquinolones (MICs: 32-512 μg/ml). But M. morganii SM12012 isolate remained susceptible to carbapenems (MICs: 4-<0.25 μg/ml). The double-disk synergy test confirmed the phenotype of extended-spectrum β-lactamases (ESBLs). Three identical β-lactamases with pI values of 6.5, 7.8 and superior to 8.6 were detected after isoelectric focusing analysis. These β-lactamases genes can be successfully transferred by the conjugative plasmid. Molecular analysis demonstrated the co-production of bla DHA-1, bla CTX-M-15 and qnrS1 genes on the same plasmid. The detection of an associated chromosomal quinolone resistance revealed the presence of a parC mutation at codon 80 (Ser80-lle80). Conclusion: This is the first report in Tunisia of nosocomial infection due to the production of CTX-M-15 and DHA-1 β-lactamases in M. morganii isolate with the association of quinolone plasmid resistance. The incidence of these strains invites continuous monitoring of such multidrug-resistant strains and the further study of their epidemiologic evolution.

Abnormal haemoglobins: detection & characterization.

Haemoglobin (Hb) abnormalities though quite frequent, are generally detected in populations during surveys and programmes run for prevention of Hb disorders. Several methods are now available for detection of Hb abnormalities. In this review, the following are discussed: (i) the methods used for characterization of haemoglobin disorders; (ii) the problems linked to diagnosis of thalassaemic trait; (iii) the strategy for detection of common Hb variants; and (iv) the difficulties in identification of rare variants. The differences between developing and industrialized countries for the strategies employed in the diagnosis of abnormal haemoglobins are considered. We mention the limits and pitfalls for each approach and the necessity to characterize the abnormalities using at least two different methods. The recommended strategy is to use a combination of cation-exchange high performance chromatography (CE-HPLC), capillary electrophoresis (CE) and when possible isoelectric focusing (IEF). Difficult cases may demand further investigations requiring specialized protein and/or molecular biology techniques.

accurate prediction of rare hemoglobin variants using a combination of high performance liquid chromatography, retention time and isoelectric focusing electrophoresis position

To investigate the predictive accuracy of using a combination of the high pressure liquid chromatography [HPLC] retention time and the relative isoelectric focusing [IEF] position to diagnose rare hemoglobin variants. A selected group of 40 patients with a rare beta-chain variant were assigned a presumed diagnosis following HPLC and IEF screening and then the variant identified in each case by DNA analysis. The study was conducted at the National Hemoglobinopathy Reference Laboratory, Oxford, United Kingdom, from August 2008 to October 2008. Thirteen out of 14 different variants were predicted accurately in 39 [97.5%] cases, compared to only one each for HPLC and IEF when used individually. A novel amplification refractory mutation system-polymerase chain reaction test was developed for Hb J-Baltimore and used successfully to provide a simple, rapid, and inexpensive diagnosis. The use of both HPLC retention time and isoelectric focusing position provides an accurate presumed diagnosis of a rare hemoglobin variant in the majority of cases. Amplification refractory mutation system-polymerase chain reaction test can provide a simple, rapid and inexpensive molecular diagnostic method for rare beta-chain variants

Study of some biochemical and genetic aspects of schizophrenic disorders

Molecular genetics and Biochemistry have been devoted to establish the genetic contribution to aetiology of schizophrenia. The biochemical changes in brain neurotransmitters may contribute to the patho genesis of schizophrenia. The human platelets contain monoamine oxidase [MAO] which is similar in many physiochemical properties to that of the brain, the similarity was also established between brain catechol-O-methytransferase [COMT] and acetyicholinesterase [AChE] and that of RBCs. So, this study was directed towards monitoring the platelet MAO and RBCs, COMT and AchE as possible indices for the CNS cellular events. The present study was carried out on 144 subjects classified into normal control group free of any psychiatric manifestation and schizophrenic patients group. Assessment of the changes in neurotransmitters metabolism, was tested e.g. that of catecholamine and acetylcholine by determination of the activity of the enzymes involved in its catabolism e.g. MAO, COMT and AChE either by fluorimetric method or colorimetric method. Our results indicated a highly significant reduction in platelets MAO activity among schizophrenic patients than control group [P<0.001]. Concerning the COMT activity, there was no statistical significant difference between control and patients group. Assessment of AChE activity indicated a significant reduction in patients group [P<0.02]. So, the changes in cholinergic activity in relation of that catecholamine may play a role in the explanations of schizophrenic dysfunction. The genetic contribution was conducted by phenotyping of group specific component [Gc] and phosphoglucomutase I [PGMI] as genetic makers of schizophrenia using isoelectro focusing techniques. In the present study analyzing the distribution of different Gc genotypes among control and schizophrenic groups demonstrated the increase of Gc 2-1 genotype frequency among schizophrenics [P<0.001] with a relative risk factor of RR=2.56. There was significant difference in distribution of PGM1 1+1+ between normal control group and schizophrenic group [P<0.001]. No correlation could be detected between MAO, COMT, AChE enzyme activity and Gc genotypes or PGM1 phenotypes

Caracterización bioquímica de prolactina de diferentes especies / Biochemical characterization of prolactin of different species

Se utilizaron diferentes técnicas para determinar las características bioquímicas de las preparaciones de prolactina (Prl) de origen hipofisario con preparados que se utilizan en centros de investigaciones para el desarrollo de sistemas in vivo e in vitro, bioanálisis, radioinmunoanálisis y análisis inmunoenzimáticos. Los preparados se analizaron por electroforesis bajo condiciones reductoras (SDS-PAGE), electrotransferencia e inmunodetección, enfoque isoeléctrico, cromatografía líquida de alta presión (HPLC), y capacidad de unión a receptores microsomales (RRA). Se encontró que en las Prl (s) porcina (pPrl), ovina (oPrl) y humana (hPrl) existen proporciones significativas de la forma glucosilada (30-40 porciento), no así en la Prl bovina (bPrl) y en la de rata (rPrl). Se comprobó que es posible detectar impurezas o mezclas de las diferentes formas moleculares de la Prl presentes en estos preparados hipofisarios utilizando HPLC. El análisis por enfoque isoeléctrico de las Prl (s) hipofisarias de diferentes especies reveló que en la bPrl existen 4 isoformas de carga eléctrica, la oPrl y la hPrl están compuestas por 3 isoformas mientras que la rPrl y la pPrl presentan 1 y 2 isoformas, respectivamente. Aún se desconoce cuál podría ser el significado fisiológico de las isoformas de carga eléctrica en la Prl hipofisaria de diferentes especies. Según los resultados obtenidos a partir de las curvas de desplazamiento y de las gráficas de Scatchard, se encontró que existen diferencias entre las constantes de disociación (Kd) y la capacidad de unión de las prolactinas a los receptores microsomales. Finalmente es necesario conocer con mayor exactitud las propiedades bioquímicas, inmunológicas y biológicas de los preparados de las prolactinas que son utilizadas en diferentes estudios tanto in vivo como in vitro, porque en esas residen las funciones biológicas de la hormona. Se confirmó y se amplió la heterogeneidad estructural de la Prl en diferentes especies de mamíferos, que dan lugar a la existencia de diferentes variantes moleculares de la hormona y a la diversidad de funciones que ejerce. Se comprobó la necesidad de desarrollar nuevos métodos para el estudio del significado biológico de la heterogeneidad estructural de la Prl y de obtener las diferentes variantes moleculares de la Prl con un alto grado de pureza, que permitan establecer nuevos métodos cuantitativos, útiles para ser aplicados en estudios clínicos y bioquímicos(AU)

Different techniques were used to determine the biochemical characteristics of the Prolactin (Prl) preparations of hypophyseal origin with preparations that are used in research centers for the development of in vitro and in vivo systems, bioanalyses, radioimmunoanalyses and immunoenzimatic analyses. The preparations were analyzed by electrophoresis under reducing conditions (SDS-PAGE), electrotrasnference and immunodetection, isoelectric focussing, high pressure liquid chromatography (HPLC) and binding capacity to microsomal receptors (RRA). It was observed that in the porcine Prl (pPrl), ovine Prl (oPrl) and human Prl (hPrl) there are significant proportions of the glycosilated form (30-40%) that are not found in the bovine Prl (bPrl) and in the rat Prl (rPrl). It was proved that it is possible to detect impurities or mixtures of the different molecular forms of Prl that appear in these hypophyseal preparations by using HPLC. The analysis by isoelectric focussing of the hypophyseal prolactins of different species revealed that in the bPrl there are 4 isoforms of electric charge, that the oPrl and the hPrl are composed of 3 isoforms, whereas the rPrl and the pPrl have 1 and 2 isoforms, respectively. The physiological meaning of the isoforms of electric charge in the hypophyseal Prl of different species is still unknown. According to the results obtained from the displacement curves and from the Scatchard graphs, it was found that there are differences among the dissociation constants (Kd) and the binding capacity of prolactins to microsomal receptors. Finally, it is necessary to know with more precision the biochemical, immunological and biological properties of the preparations of prolactins that are used in different studies, both in vivo and in vitro, since that's where the biological functions of the hormone are. The structural heterogeneity of the Prl was confirmed and widened in different species of mammals giving rise to the existence of different molecular variants of the hormone and to the diversity of functions it performs. The need to develop new methods for studying the biological meaning of the structural heterogeneity of Prl and to obtain different molecular variants of Prl with a degree of purity that allow to establish new quantitative methods useful to be applied in clinical and biochemical studies was proved. The adequate understanding of the structural modifications of prolactins and of their biological consequences will make possible to establish the synthesis and secretion mechanisms of this hormone, including its multifunctional properties(AU)

A study on antigenic and allergenic changes during storage in three different biological extracts.

The stability of three allergens common in tropical countries was evaluated under different storage conditions. Prosopis juliflora (PJ), Rhizopus nigricans (RN), and wheat dust (WD), were taken as representatives of various groups of allergens viz, pollen, fungi and dust. The extracts were stored in buffer containing phenol (0.4%) or glycerol (50%) at temperatures ranging from 4-55 degrees C for 15 to 60 days. Protein content of PJ extract was reduced remarkably when it was stored at 40 degrees C for 45 days. Thin layer isoelectric focusing and rocket immunoelectrophoresis of PJ showed that certain antigenic proteins degrade rapidly even at 25 degrees C as early as day 15. However, two to three proteins of PJ remain stable at a higher temperature (40 degrees C) for two months. Relative radioallergosorbent test (RAST) inhibition showed substantial loss of allergenic activity in all the three extracts, when stored at higher temperatures (25-55 degrees C) even for short durations, i.e., 15 days. Extracts (PJ and RN) containing 50% glycerol were found to be stable, retaining more than 50% activity, even when stored at 55 degrees C for 40 days, while extracts without glycerol lost more than 75% of their allergenic activity. However, addition of glycerol did not change the stability of wheat dust allergenic extract. The present findings indicate that allergenic extracts behave differently when stored. Hence, the stability of each extract should be determined individually.

Pesquisaje de hemoglobinas anormales en el recién nacido por la técnica de enfoque isoeléctrico / Screening for abnormal hemoglobins in the newborn using isoelectric focusing technique

Se analizaron 4246 muestras de sangre de recién nacidos de Ciudad de la Habana, en un período de ocho meses (enero-agosto de 1983) por la técnica de enfoque isoeléctrico de cadenas de hemoglobina. En el estudio se detectaron 128 FAS, 8 FAC, 1 FBS subíndice otal, 1 FSC y 2 FAG Philadelphia. Se analiza las ventajas de esta técnica en el estudio de la Hbs al nacimiento, así como la importancia de la realización conjunta de un programa de prevención de la AHF y la fenilcetonuria al nacimiento