Substitutability of metal-binding sites in an alcohol dehydrogenase / 生物工程学报

Covalently anchoring of a ligand/metal via polar amino acid side chain(s) is often observed in metalloenzyme, while the substitutability of metal-binding sites remains elusive. In this study, we utilized a zinc-dependent alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) as a model enzyme, analyzed the sequence conservation of the three residues Cys37, His59, and Asp150 that bind the zinc ion, and constructed the mutant library. After experimental validation, three out of 224 clones, which showed comparative conversion and ee values as the wild-type enzyme in the asymmetric reduction of the model substrate tetrahydrofuran-3-one, were screened out. The results reveal that the metal-binding sites in TbSADH are substitutable without tradeoff in activity and stereoselectivity, which lay a foundation for designing ADH-catalyzed new reactions via metal ion replacement.

Influence of in vitro capacitation time on structural and functional human sperm parameters / 亚洲男科学杂志(英文版)

A cascade of dramatic physiological events is linked to the sperm acrosome reaction and binding to the oocyte's zona pellucida during human sperm capacitation. However, structural and functional sperm changes during capacitation currently remain poorly defined. Here, we performed a multibiomarker approach based on the utilization of sperm concentration, motility, viability, morphology, acrosome reaction, tyrosine phosphorylation, DNA fragmentation, and lectin-binding sites to analyze the impact caused by swim-up selection times (uncapacitated, 1 h capacitated, and 4 h capacitated) on sperm function and structure in normozoospermic samples. We found that a 4 h swim-up capacitation increased sperm quality, because a large number of cells with normal morphology and lower DNA fragmentation rates were recovered. Furthermore, the long-term capacitation induced a higher percentage of cells with tyrosine phosphorylation of the principal piece as well as a redistribution of lectin-binding sites. Overall, the multivariate biomarkers analyzed showed a less variable distribution on spermatozoa recovered after 4 h capacitation than that with the shorter capacitation time. These findings stress the importance of capacitation time as a relevant factor in sperm quality with potential biological reproductive implications both for basic research and in assisted reproduction techniques.

Research advance on the interaction of pharmaceutical molecules with target proteins / 药学实践杂志

The function of drugs is based on the interaction between drug molecules and their targets.Qualitative analysis and quantitative detection of drug-target interactions run through the whole process from drug discovery to clinical practice.After decades of development, the study methods on the interaction between drug molecules and target proteins have been transformed from traditional biochemical experiments to a diversity of efficient and accurate technology systems supported by advanced molecular biology and biophysics theory.In this review, representative methods and techniques were introduced from aspects of target discovery and validation, affinity determination, interaction sites and structural analysis, which might provide some references for drug discovery and mechanism exploration.

Porcine transcription factor AP-2δ promotes porcine circovirus type 2 replication through enhancing the activity of the rep gene promoter / 生物工程学报

Several putative transcription factor binding sites (TFBSs) exist in the PCV2 rep gene promoter. To explore if porcine circovirus type 2 (PCV2) could regulate the viral replication by using these TFBSs, we conducted electrophoretic mobility shift assay (EMSA), DNA-pull down and liquid chromatography-tandem mass spectrometric (LC-MS/MS) assays. EMSA confirmed the binding activity of the rep gene promoter with nuclear proteins of host cells. DNA-pull down and LC-MS/MS identified the porcine transcription factor AP-2δ (poTFAP2δ) could bind the PCV2 rep gene promoter. Dual-luciferase reporter assay, quantitative real-time PCR, Western blotting and indirect immunofluorescent assay demonstrated that poTFAP2δ could not only promote the activity of the rep gene promoter, but also enhance the transcription/translation activity of the rep/cap gene and the virus titer of PCV2 during the entire life cycle of PCV2 infection. This study revealed the molecular mechanism of PCV2 using host proteins to enhance the viral replication, provided a new perspective for studying the pathogenic mechanism of PCV2 from virus and host interactions, and provided a theoretical basis for developing highly effective PCV2 vaccines.

Association between the SNPs in microRNA bindingsites of ABCG5/8 and metabolism of glucose and lipid during pregnancy / 基础医学与临床

Objective To explore the relationship between SNPs in microRNA binding sites of ABCG5/8 and the glucolipid level during pregnancy.Methods 1 925 pregnant women were recruited at Peking Union Medical College hospital from 2006 to 2011.The clinical data were collected and the total genomic DNA was extracted from whole blood samples.ABCG5/8, which was reported to be related with the glucose and lipid metabolism closely, were selected as the candidate gene and the SNPs in its microRNA binding sites with minor allele frequency >5% in Han Chinese in Beijing were chosen.Then the genotyping was performed and analyzed.Results There was only one SNP matching the criteria, rs2278356, and it is significantly associated with LDL-C and TC level during pregnancy (LDL-C: b=0.104 mmol/L, 95% CI 0.023-0.185 mmol/L, P<0.05;TC: b=0.105 mmol/L, 95% CI 0.080-0.203 mmol/L, P<0.05).Conclusions The association of rs2278356 in 3′UTR of ABCG5/8 with LDL-C and TC level in pregnant Chinese Han women is found, which may provide an individualized treatment strategy for pregnant women with high cholesterol.

Prediction of Metal Ion Binding Sites in Proteins from Amino Acid Sequences by Using Simplified Amino Acid Alphabets and Random Forest Model

Metal binding proteins or metallo-proteins are important for the stability of the protein and also serve as co-factors in various functions like controlling metabolism, regulating signal transport, and metal homeostasis. In structural genomics, prediction of metal binding proteins help in the selection of suitable growth medium for overexpression’s studies and also help in obtaining the functional protein. Computational prediction using machine learning approach has been widely used in various fields of bioinformatics based on the fact all the information contains in amino acid sequence. In this study, random forest machine learning prediction systems were deployed with simplified amino acid for prediction of individual major metal ion binding sites like copper, calcium, cobalt, iron, magnesium, manganese, nickel, and zinc.

Caracterización matemática de la unión de la proteína PfNBP-1 al eritrocito mediante la aplicación de la probabilidad y entropía / Mathematical characterization of binding between PfNBP-1 protein and erythrocyite by applying probability and entropy

Objetivo: Estudiar la proteína Plasmodium falciparum Normocyte Binding Protein-1 (PfNBP-1), partiendo de un método de caracterización físico-matemática desarrollado previamente para péptidos de alta unión del merozoito de malaria al eritrocito. Materiales y métodos: Se tomaron 21 péptidos con tamaño de 20 aminoácidos no sobrelapados de los cuales dos son de alta unión, se cuantificó la frecuencia de aparición de los 20 aminoácidos esenciales en cada posición y se calculó la probabilidad, sumatoria de probabilidad y la entropía con el objetivo de diferenciar matemáticamente los péptidos de alta y baja unión. Posteriormente se calcularon los mismos valores para péptidos teóricos análogos, en los que fueron cambiados por glicinas los aminoácidos críticos confirmados experimentalmente. Resultados: Los péptidos de PfNBP-1 comprobados experimentalmente de alta unión, presentaron valores de probabilidad, sumatoria de probabilidad y entropía ubicados dentro del macroestado de unión y sus péptidos teóricos análogos presentaron resultados que se diferenciaban cada vez más del macroestado de unión a medida que se reemplazaban aminoácidos críticos por glicinas. En cuanto a las secuencias de no unión de PfNBP-1, se encontró que los valores calculados son diferentes a los asociados al macroestado de unión, comprobando que en el 100 % de casos estudiados es posible diferenciar los péptidos de no unión y alta unión matemáticamente. Conclusiones: La probabilidad y la entropía permiten caracterizar adecuadamente los péptidos de alta unión de PfNBP-1, y evidenciar el orden matemático subyacente al proceso de unión de proteínas de malaria.

Objective: To study the Plasmodium falciparum Normocyte Binding Protein-1 (PfNBP-1) based on a of physicalmathematical characterization method previously developed for high binding peptides of malaria merozoite to erythrocyte. Materials and methods: 21 non overlapped peptides with size of 20 amino acids, including two of high binding were taken; the frequency of occurrence of the 20 essential amino acids in each position was quantified and probability, summation of probability and entropy were calculated in order to mathematically differentiate high and low binding peptides. Later the same values were calculated for theoretical analogs peptides, where the critical amino acids confirmed experimentally were changed by glycine. Results: The experimentally validated high binding peptides of PfNBP-1 showed values of probability, summation of probability and entropy located within the binding macrostate peptides and their theoretical analogues peptides presented results that differed increasingly of the binding macrostate as critical amino acids were replaced by glycine. For the PfNBP-1 sequences of non-binding, it was found that the calculated values are different from those associated with the macrostate of binding and it was verified that in 100% of studied cases it is possible to mathematically differentiate binding and non-binding peptides. Conclusions: The probability and entropy allow to adequately characterize the high-binding peptides of PfNBP-1 and show the mathematical order underlying the process of protein binding of malaria to the erythrocyte.

Targeting transforming growth factor-βreceptor Ⅱ aptamer binding sites prediction and validation studies / 重庆医学

Objective To predict the binding sites of transforming growth factor-βreceptor Ⅱ (TβRⅡ ) ectodomain and the aptamer S58 specifically targeted TβRⅡ ,and to confirm the structure stability of the aptamer S 58 in vitro .Methods We created three-dimensional structure by utilizing ssDNA aptamer sequences ,the crystal structure of the TβRⅡ was searched by protein data bank database .According to the results of the molecular docking experiments on aptamer S 58 and TβRⅡ ectodomain ,we sheared the aptamer sequences ,then verified its affinity respectively by biosensor technology and Western blot .Results Binding sites of aptamers S58 and TRβⅡ ectodomain included site Ⅰ(T4 ,T5 ,G6 ,C7) ,site Ⅱ(G13 ,A14 ,T15 ,C16 ,G17 ,C18 ) ,site Ⅲ (T31 ,G32 , T33 ,C34) and site Ⅳ(G40 ,A41 ,T42 ,T43 ,T44 ,G45 ,G46) .We validated the high affinity between aptamer S58 and TRβⅡ ectodo-main .The expression of α-smooth muscle actin(α-SMA) protein in the human tenon′s capsule fibroblasts was descended obviously after the experiment of the aptamer S58 in comparing with the control of DMEM (P< 0 .05) .But the new ssDNA by shear the aptamer ssDNA S58 according to the results were poor than aptamers S58 .Conclusion The aptamer S58 targeted TβRⅡ was high-ly specific with a certain stability ,any changing of structure will reduce the affinity of TβRⅡ .Computer-aided molecular docking technology has become an important means of an exploratory intermolecular interaction ,and can provides a good theoretical basis on medical research .

Anti-tumor activity and mechanism of new microtubuIe-targeting compound WX-127-07 / 中国药理学与毒理学杂志

OBJECTIVE To evaluate the anti-tumor activities of WX-127-07,a new microtubule-tar-geting agent invitroand probe its molecular mechanism. METHODS The well-known microtubule-targe-ting anti-tumor drugs taxol,vincristine and anti-gout drug colchicine were used as positive controls. The anti-proliferation activity was examined in five different cell lines after treatment with WX-127-07(0.3 -300 nmol·L-1 )for 72 h by SRB assay. The cell cycle arrest profile was assayed by flow cytometry. The multiparameters of cytotoxicity,cell morphology,apoptosis and different signaling pathways related to tumorigenesis and inflammation were analyzed using the high content analysis platform. Tubulin tryptic digestion and competition inhibition assay for colchicine or vinblastine site were used to confirm the bind-ing site in microtubules at a molecular level. RESULTS All the tested compounds obviously inhibited the growth of A549,HepG2,HeLa,HLF and HUVEC cells. The lC50 values of WX-127-07 were 4.47±0.05, 5.18±0.08,4.90±0.19,4.10±0.16 and(5.04±0.08)nmol·L-1 respectively,lower than those of colchicine〔the lC50 values were 21. 17 ± 1. 22,14. 19 ± 0. 53,43. 80 ± 1. 64,145. 89 ± 10. 97 and( 27. 67 ± 1.79)nmol·L-1 ,respectively〕and those of vincristine〔the lC50 values were 16.51±0.36,16.76±0.33, 27.80±2.75,43.80±1.48 and(9.15±0.78)nmol·L-1 ,respectively〕,but were similar to or lower than those of taxol〔the lC50 values were 10. 68 ± 0. 61,12. 86 ± 0. 25,4. 81 ± 0. 61,102. 07 ± 15. 17 and( 3. 04 ± 0.12)nmol·L-1 ,respectively〕. High content multi-parameter analysis revealed that WX-127-07 induced a concentration-dependent microtubular depolymerization(P=0.0075)with the same pattern as colchicine and vincristine,but at a lower concentration. Both WX-127-07 and positive drugs could induce cell cycle arrest in A549 cells,increase nuclear membrane permeability and early signs of apoptosis in HepG2 cells,but neither cancer related pathways nor inflammation related pathways were affected. Microtubular competition inhibition assay showed that WX-127-07 inhibited the binding of colchicine with tubulin(P =0.0259). Tryptic digestion of tubulin-WX-127-07 premixture showed a similar electrophoretic band to that of tubulin-colchicine premixture. CONCLUSION WX-127-07 is a novel microtubule-depolymerizing agent with anti-proliferation activity and acting on the colchicine binding site.

Mutation Screening of Modifying Genes (Atox1 and Commd1) in Association with Wilson Disease.

Wilson disease (WD) results from accumulation of copper and is caused due to mutations in ATP7B, a copper transporting ATPase. Although WD is an established monogenic disorder, heterogeneity in phenotype is observed even among patients harboring mutations in ATP7B that would affect the mutant protein similarly. Such observations led to the speculation that there might be modifying loci that modulate the phenotype resulting from the aberration in the ATP7B gene. The expected genes coding for proteins that interact either directly with ATP7B or influence it indirectly might fit the role of modifier locus. ATOX1 and COMMD1 are the candidate genes that might play the role of a modifier locus having copper homeostasis pathway with such potential. To understand the role of modifying genes, we screened ATOX1 and COMMD1, a gene implicated in canine copper toxicosis, in 45 WD patients along with 50 healthy controls. This study did not yield satisfactory results concluding more patients to be analyzed. Keywords: Wilson Disease, ATOX1, COMMD1.

Plasticity of regulation of mannitol phosphotransferase system operon by CRP-cAMP complex in Vibrio cholerae / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>The complex of the cyclic AMP receptor protein (CRP) and cAMP is an important transcriptional regulator of numerous genes in prokaryotes. The transport of mannitol through the phosphotransferase systems (PTS) is regulated by the CRP-cAMP complex. The aim of the study is to investigate how the CRP-cAMP complex acting on the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype.</p><p><b>METHODS</b>The crp mutant strain was generated by homologous recombination to assess the need of CRP to activate the mannitol PTS operon of V. cholerae El Tor. Electrophoretic mobility shift assays (EMSA) and the reporter plasmid pBBRlux were used to confirm the role that the CRP-cAMP complex playing on the mannitol PTS operon mtl.</p><p><b>RESULTS</b>In this study, we confirmed that CRP is strictly needed for the activation of the mtl operon. We further experimentally identified five CRP binding sites within the promoter region upstream of the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype and found that these sites display different affinities for CRP and provide different contributions to the activation of the operon.</p><p><b>CONCLUSION</b>The five binding sites collectively confer the strong activation of mannitol transfer by CRP in V. cholerae, indicating an elaborate and subtle CRP activation mechanism.</p>

Analysis of human papillomavirus 16 subtypes E2 gene and long control region sequence in Chengdu / 中华传染病杂志

Objective To explore the relevance between sequence variation of human papillomavirus (HPV)16 subtypes E2 gene or long control region (LCR) and cervical lesions.Methods Fifty specimens from HPV16 infected people in Chengdu were collected,including cervical exfoliated cells from 38 HPV carriers,papilloma tissues from 8 cases of genital warts,2 with cervical intraepithelial neoplasia (CIN) Ⅱ and 2 with CIN Ⅲ in this study.Polymerase chain reaction was used to amplify E2 gene and LCR,then an evolutionary tree was constructed.Results In all the 50 specimens,there were 12 mutation sites in E2 gene,among which,C→A existed in one specimen of genital warts,and ≥2 mutation sites existed in all the other 48 specimens.There were 28 mutation sites of LCR sequence of all the specimens.Ten specimens were chosen to construct evolutionary tree and were sequenced.The data showed that 8 specimens were Asian variants,E2 gene mutation existed in all the specimens while the LCR 7867 G→A only existed in the four CIN.Conclusion LCR 7867G→A is a correlative mutation site of cervical lesions in Chengdu.

Computational Analysis of miRNA and Target mRNA Interactions: Combined Effects of The Quantity and Quality of Their Binding Sites / 生物化学与生物物理进展

MicroRNAs (miRNAs) act by binding to complementary sites on target messenger RNA (mRNA) to induce mRNA degradation and/or translational repression. To investigate the influence of miRNAs at transcript levels, two human miRNAs (miR-1 and miR-124) were transfected into HeLa cells and microarrays used to examine changes in the mRNA profile showed that many genes were downregulated and that the fold decreases in levels of these target mRNAs differed remarkably. Features depicting interactions between miRNAs and their respective target mRNAs, such as the number of putative binding sites, the strength of complementary matches and the degree of stabilization of the binding duplex, were extracted and analyzed. It was found that, for a given target mRNA, both the quality and quantity of miRNA binding sites significantly affected its degree of destabilization. To delineate these types of interactions, a simple statistical model was proposed, which considers the combined effects of both the quality and quantity of miRNA binding sites on the degradation levels of target mRNAs. The analysis provides insights into how any animal miRNA might interact with its target mRNA. It will help us in designing more accurate methods for predicting miRNA targets and should improve understanding of the origins of miRNAs.

Transcriptome analysis and promoter sequence studies on early adipogenesis in 3T3-L1 cells

To identify regulatory molecules which play key roles in the development of obesity, we investigated the transcriptional profiles in 3T3-L1 cells at early stage of differentiation and analyzed the promoter sequences of differentially regulated genes. One hundred and sixty-one (161) genes were found to have significant changes in expression at the 2nd day following treatment with differentiation cocktail. Among them, 86 transcripts were up-regulated and 75 transcripts were down-regulated. The 161 transcripts were classified into 10 categories according to their functional roles; cytoskeleton, cell adhesion, immune, defense response, metabolism, protein modification, protein metabolism, regulation of transcription, signal transduction and transporter. To identify transcription factors likely involved in regulating these differentially expressed genes, we analyzed the promoter sequences of up- or -down regulated genes for the presence of transcription factor binding sites (TFBSs). Based on coincidence of regulatory sites, we have identified candidate transcription factors (TFs), which include those previously known to be involved in adipogenesis (CREB, OCT-1 and c-Myc). Among them, c-Myc was also identified by our microarray data. Our approach to take advantage of the resource of the human genome sequences and the results from our microarray experiments should be validated by further studies of promoter occupancy and TF perturbation.

Computational Analysis of miRNA and Target mRNA Interactions:Combined Effects of The Quantity and Quality of Their Binding Sites / 生物化学与生物物理进展

MicroRNAs(miRNAs) act by binding to complementary sites on target messenger RNA(mRNA) to induce mRNA degradation and/or translational repression.To investigate the influence of miRNAs at transcript levels,two human miRNAs(miR-1 and miR-124) were transfected into HeLa cells and microarrays used to examine changes in the mRNA profile showed that many genes were downregulated and that the fold decreases in levels of these target mRNAs differed remarkably.Features depicting interactions between miRNAs and their respective target mRNAs,such as the number of putative binding sites,the strength of complementary matches and the degree of stabilization of the binding duplex,were extracted and analyzed.It was found that,for a given target mRNA,both the quality and quantity of miRNA binding sites significantly affected its degree of destabilization.To delineate these types of interactions,a simple statistical model was proposed,which considers the combined effects of both the quality and quantity of miRNA binding sites on the degradation levels of target mRNAs.The analysis provides insights into how any animal miRNA might interact with its target mRNA.It will help us in designing more accurate methods for predicting miRNA targets and should improve understanding of the origins of miRNAs.

Mecanismos celulares y moleculares de la neurotoxicidad por plomo

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Abstract: Lead, a heavy metal, has been used by humans for many technological aims, a fact that has determined its actual widespread distribution. Although various actions have been taken to diminish the use and distribution of lead in the environment, it remains a significant health problem. The evolution of technological processes applied in the industry has followed economic interest. Its only in recent times that criteria related to health and ecology have been considered while designing new industries. Particularly susceptible groups are children and workers involved in mining, metallurgy, paint manufacturing and battery recycling. The communities living in areas where those industries are settled have also a higher lead exposure risk. Its high biological toxicity has determined lead to become one of the most significant environmental contaminants with pathogenic potential for humans. The toxic mechanism of lead is essentially due to its capability to substitute other polyvalent cations (particularly divalent cations such as calcium and zinc) into the molecular machinery of living organisms. Thanks to its ionic structure, lead establishes very favorable interactions, usually with higher affinity, with chemical groups that normally coordinate divalent cations in proteins. The coordination of cations in proteins is usually achieved by negatively charged acidic residues. These residues establish ionic interactions with the positively charged ion, resulting in a change in the structure and electric charge of the protein. These interactions determine that lead may affect different biologically significant processes, including metal transporting proteins, ionic channels, cell adhesion molecules, diverse enzymes which have metallic cofactors, signaling molecules such as calmodulin and protein kinase C and DNA binding proteins, among other molecular targets. Lead interactions with the coordinating amino acid residues in proteins may induce an abnormal conformational configuration of proteins, as compared to the conformational structure acquired when interacting with commonly active cations, thus significantly altering its functional properties in the very complex molecular machinery. Among the biologically active sites usually occupied by lead, those related to calcium seem to have the most significant pathological importance for lead toxicity due to their widespread distribution and highly significant functional relevance for the normal cell function. Two of the principal calcium binding motifs in proteins, the EF-hand motif and the C2 motif, have an intrinsic high affinity for lead. In the case of EF-hand motifs, calmodulin is one of the most remarkable targets for lead due its importance in regulating cellular processes, being activated by lead at lower concentrations than required for calcium and displaying an abnormal activity. The C2 motif is expressed mainly in calcium dependent membrane associated proteins such as protein kinase C (PKC) or synaptotagmin. The principal characteristic in these motifs is an electrical change in the protein after the calcium binding, allowing its interaction with biological membranes. In synaptotagmin, according with the electrical characteristics of lead, the interaction of the complex lead-synaptotagmin with biological membranes is similar to the interaction calcium-synaptotagmin with membranes, which is eminently electrical. Hence, the conformation of this complex is probably different to the conformation with calcium, fact evidenced by the failure of lead-synaptotagmin to interact with other proteins of the exocitic machinery. In relation to lead neurotoxicity, membrane ionic channels seem to be among the most relevant molecular targets of lead. In particular, calcium and potassium channel function may be significantly impaired by lead, affecting the activation of calcium activated potassium channels, the inactivation process of calcium channels, and the ionic conductance of calcium channels. As occurs with other heavy metals, lead is capable of blocking the calcium channel, probably at the selectivity filter. The high affinity lead binding to the acidic residues of the filter provokes a slow flux of the metal trough the channel pore, blocking the calcium conductance. The regulation of ionic channels will be significantly altered also. Calmodulin is a common calcium sensing protein for many ionic channels and its alteration by lead could affect the channel operation. Abnormal functioning of regulatory and signaling proteins such as calmodulin, protein kinase C and synaptotagmins, which normally require calcium for its activity, may also display an abnormal functioning, thus determining a widespread metabolic influence of lead poisoning. Lead distributes evenly into the cell thus reaching intracellular organelles, including the endoplasmic reticulum, mitochondria and the cell nucleus. This results in significant alterations of intracellular calcium metabolism and regulation due in part to the malfunctioning of calcium channels and ionic pumps in plasma membrane, endoplasmic reticulum and mitochondria. Inadequate energy generation due to mitochondrial damage and malfunctioning in cation dependent enzymes, alterations in protein folding due to the direct binding of lead to calcium activated reticular chaperones, or indirectly, altering the intrareticular calcium levels, and the disruption of the structure of DNA binding motifs such as zinc fingers, among others, promotes alterations in gene expression and DNA reparation. Lead poisoning is one of the most important chronic environmental illnesses affecting children in modern life. Developing central nervous system is particularly susceptible to lead toxicity. At critical times in development, lead may have a disorganizing influence with long-lasting effects which may continue into teenage and beyond. Mechanisms originating this disorganizing influence in the central nervous system are a consequence of the interaction of lead with various targets as previously described; alterations of cell molecular machinery, at the systems level induce excitotoxic phenomena, interferes with neurotransmission at neurotransmitter synthesis, release and receptor activation levels, alters intracellular signaling and produce cell membrane peroxidative damage. Compared to adult lead poisoning, pediatric lead is most common and its effects may occur at reduced blood levels with subclinical symptoms; thus a high index of suspicion is necessary for physicians when dealing with pediatric patients. Long-term effects of lead may produce cognitive and motor impairment, with behavioral alterations. The particular vulnerability of the immature nervous system to the lead poisoning is probably due to the fact that in this stage of development the establishment of appropriate neural networks is highly dependent on the synaptic activity, which in turn could be altered by lead. Lead poisoning has been considered as a potential co-factor in complex neuropsychological alterations such as schizophrenia. In this sense it is worth to note the possibility that the physical and psychic symptoms of Vincent Van Gogh may have been due to chronic lead poisoning. The following are among the clinical symptoms described by Van Gogh in his autographed letters: initial debilitation, stomatitis with loss of teeth, recurring abdominal pains, anemia (with a "plumbic" skin tone), neuropathy of the radial and saturnine encephalopathy, including epileptic crises, progressive character changes and periods of delirium, all of which meet present criteria for diagnosis of Organic Mental Disorder due to cerebral lesion or somatic illness, and Organic Character Disorder (DSM-IV-R). Apha-thujone, found in absinthe and in many popular herbal medicines, may also have contributed to Van Gogh symptoms since he was a well-known absynthe drinker. Many countries, including Mexico, have implemented politics aimed to eliminate lead from the majority of their industrial processes. This has been carried out with considerable effort, and in some cases, with open confrontations between the scientific community and industrial sector. Although there have been actually significant advances to eliminate lead from many products (gasoline, painting manufacturing, etc.), lead is not degradable, thence once it is released in the environment it remains there for long periods of time. This implies that we should have to deal with lead poisoning in the years to come and to be aware of this diagnostic possibility in any suspicious case. This review is centered in the description of the molecular mechanisms of lead toxicity and its repercussion in the cellular excitability and central nervous system function.

Procedimientos para la detección e identificación de anticuerpos eritrocitarios / Procedures for the detection and identification of red cell antibodies

En Inmunohematología se ha desarrollado una amplia gama de procederes de detección e identificación de anticuerpos eritrocitarios in vitro, por lo cual se realiza una revisión de técnicas y métodos empleados con este objetivo, como son el método que utiliza eritrocitos pretratados con enzimas proteolíticas y las técnicas de Polibreno, que utiliza solución de baja fuerza iónica (LISS), la de antiglobulina indirecta, la de aglutinación en gel, la inhibición de la aglutinación, la hemólisis y la adherencia de eritrocitos en fase sólida. Se abordan los problemas que afectan a la reacción de aglutinación entre el antígeno y el anticuerpo; para una mejor comprensión la reacción de aglutinación se subdivide en su primera y segunda etapa. En la primera etapa los factores que se analizan son concentración de antígeno y anticuerpo, pH, temperatura, fuerza iónica y tiempo de incubación; en la segunda etapa la característica del anticuerpo, localización y número de sitios antigénicos, fuerzas que mantienen la distancia entre los eritrocitos, uso de la albúmina bovina, uso de enzimas, efecto de dosis y efecto de moléculas con carga positiva.

Awide range of procedures for the detection and identification of red cell antibodies in vitro has been developed in Immunohematology. Therefore, it is made a review of the techniques and methods used with this aim, such as the method using erythrocytes pretreated with proteolytic enzimes and the techniques of Polibreno that utilize low ionic force solution (LIFS), the indirect antiglobulin test, the gel agglutination test, the agglutination inhibition test, hemolysis and the solid phase erythrocyte adherence test. The problems affecting the agglutination reaction between the antigen and the antibody are also dealt with. The agglutination reaction is subdivided into its first and second phase for a better understanding. In the first phase, the antigen and antibody concentration, pH, temperature, ionic force and incubation time are analyzed. The characteristic of the antibody, localization and number of antigenic sites, forces maintaining the distance among the erythrocytes, use of bovine albumin, use of enzimes, dose effect and effect of molecules with positive charge are studied in the second phase.

The study of bidirectional regulation effect on inducible nitric oxide synthase gene promoter by hepatitis B virus preS1 promoter DNA-binding protein 1 / 中华传染病杂志

Objective To investigate the regulating effect of hepatitis B virus(HBV)SPⅠbinding protein 1(SBP1)on inducible nitric-oxide synthase(iNOS)gene promoter activity.Methods Polymerase chain reaction(PCR)technique was employed to amplify the coding sequence of iNOS promoter DNA by using HepG2 genomic DNA as template,and 3 deletion mutants were amplified. The products were cloned into pGEM-T vector,respectively.The iNOS gene and 3 deletion mutants were cut from T-iNOS by KpnⅠand XhoⅠ,and then were cloned into pCAT3-Basic.The construc- ted vectors were named as p1-iNOSp,p2-iNOSp,p3-iNOSp and p4-iNOSp,respectively.Each of the vectors containing different iNOSp DNA fragments was transfected into the HepG2 cell line and cotransfected into HepG2 cells with pcDNA3.1(-)-SBP1 by FuGENE 6 transfection reagents.The HepG2 cells transfected with pCAT3-Basic were used as negative control.The activity of chloram- phenicol acetyltransferase(CAT)in transfected HepG2 cells was detected by an enzyme-linked immu- nosorbent assay(ELISA)kit after 48 hours,which would reflect the regulating effect of SBP1 on iNOS gene promoter activity.Results The expression vector pcDNA3.1(-)-SBP1 and report vector pCAT3-iNOS were constructed and confirmed by restriction enzyme digestion and sequencing.The expression of pcDNA3.1(-)-SBP1 in HepG2 cells up-regulated the activity of p1-iNOSp and down- regulated the activity of p3-iNOSp.The inhibiting rate was 31.3%.Conclusions It is suggested that SBP1 can regulate iNOS gene promoter bidirectionally by influencing the binding sites of nuclear factor (NF)-IL6,A activator domain binding site and NF-?B in iNOS gene promoter.

Expression and characterization of the flavoprotein domain of gp91phox

Truncated forms of gp91(phox) were expressed in E. coli in which the N-terminal hydrophobic transmembrane region was replaced with a portion of the highly soluble bacterial protein thioredoxin (TRX). TRX-gp91(phox) (306-569), which contains the putative FAD and NADPH binding sites, showed NADPH-dependent NBT (nitroblue tetrazolium) reductase activity, whereas TRX-gp91(phox) (304-423) and TRX-gp91(phox) (424-569) were inactive. Activity saturated at about a 1:1 molar ratio of FAD to TRX-gp91(phox) (306- 569), and showed the same Km for NADPH as that for superoxide generating activity by the intact enzyme. Activity was not inhibited by superoxide dismutase, indicating that it was not mediated by superoxide, but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium (DPI). In the presence of Rac1, the cytosolic regulatory protein p67(phox) stimulated the NBT reductase activity, but p47(phox) had no effect. Truncated p67(phox) containing the activation domain (residues 199- 210) stimulated activity approximately 2-fold, whereas forms mutated or lacking this region failed to stimulate the activity. Our data indicate that: 1) TRX-gp91(phox) (306-569) contains the binding sites for both pyridine and flavin nucleotides; 2) this flavoprotein domain shows NBT reductase activity; and 3) the flavin-binding domain of gp91(phox) is the target of regulation by the activation domain of p67(phox).

Imidazoline binding sites and receptors related with cardiovascular functions / 中国药理学通报

I 2 Imidazoline binding sites have been shown to exist on cardiac myocytes of human beings and rat. Both of I 1 and I 2 imidazoline binding sites have been identified on vascular smooth muscle cells of various species. Vascular I 2 imidazoline binding sites may participate in vascular smooth muscle proliferation. The sympathetic nerves supplying the cardiovascular system are endowed with presynaptic inhibitory imidazoline receptors. Being different from most of the imidazolines, moxonidine does not activate presynaptic imidazoline receptors, but SR141716A, which is considered as a selective antagonist at cannabinoid receptors, antagonizes presynaptic imidazoline receptors. It has been shown that imidazolines exhibit antiarrhythmic action. Agamatine, which is endogenous ligand at imidazoline receptors, not only decreases the rate of pacemaker firing in sinoatrial node of animal, prolongs action potential duration on cardiac myocytes of human beings and animal, but also inhibits afterdepolarizations induced by isoproterenol. On the other hand, imidazolines and guanidines inhibit the cardiovascular K ATP channel in a noncompetitive manner, those effects might interfere with the cardioprotective effects of K ATP channel.