Covalent structure, disulfide bonding, and identification of reactive surface and active site residues of human prostatic acid phosphatase

The pairing of the half-cysteine residues of human prostatic acid phosphatase was established by proteolytic digestion and analysis of the resulting peptide mixtures by fast atom bombardment mass spectrometry (FAB-MS). An independently derived, full length cDNA clone was used as the basis for the interpretation of the FAB-MS data. The sequence of the native protein is that predicted from the present cDNA sequence, except for the carboxyl-terminal end and some possible post-translational deamidations. Isolated human prostatic acid phosphatase was found to have multiple carboxyl-terminal ends, terminating in Thr, Glu, and Asp, corresponding to residues 349-351 of the 354-residue protein that is predicted from the cDNA sequence after removal of a leader peptide. The protein contains no free sulfhydryl groups. The identical monomer chains of the dimeric native enzyme are found to contain three disulfide bonds, specifically Cys-129 to Cys-340, Cys-183 to Cys-281, and Cys-315 to Cys-319. In view of the conserved positions of cysteines in the homologous human and rat liver lysosomal acid phosphatases, an identical disulfide bonding pattern may be predicted for those proteins. The location of a potential antigenic site was established by selective labeling of proximate tyrosine residues predicted to be on the surface. A conserved RHGXRXP sequence is present in the prostatic, lysosomal, Escherichia coli, and yeast acid phosphatases and is predicted to be of mechanistic significance. In addition, residue Arg-54 is shown to be an active site residue by reaction of the enzyme with phenylglyoxal. Interestingly, this residue is present in a sequence RXRY (R,H) that is also present in lysosomal phosphatase and in recently described protein tyrosine phosphatases.     

Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase.

Site-directed mutagenesis was used to examine the catalytic importance of 2 histidine and 4 arginine residues in Escherichia coli periplasmic acid phosphatase (EcAP). The residues that were selected as targets for mutagenesis were those that were also conserved in a number of high molecular weight acid phosphatases from eukaryotic organisms, including human prostatic and lysosomal acid phosphatases. Both wild type EcAP and mutant proteins were overproduced in E. coli using an expression system based on the T7 RNA polymerase promoter, and the proteins were purified to homogeneity. Examination of the purified mutant proteins by circular dichroism and proton NMR spectroscopy revealed no significant conformational changes. The replacement of Arg16 and His17 residues that were localized in a conserved N-terminal RHGXRXP motif resulted in the complete elimination of EcAP enzymatic activity. Critical roles for Arg20, Arg92, and His303 were also established because the corresponding mutant proteins exhibited residual activities that were not higher than 0.4% of that of wild type enzyme. In contrast, the replacement of Arg63 did not cause a significant alteration of the kinetic parameters. The results are in agreement with a previously postulated distant relationship between acid phosphatases, phosphoglycerate mutases, and fructose-2,6-bisphosphatase. These and earlier results are also consistent with the conclusion that 2 histidine residues participate in the catalytic mechanism of acid phosphatases, with His17 playing the role of a nucleophilic acceptor of the phospho group, whereas His303 may act as a proton donor to the alcohol or phenol.     

Overproduction of d-xylose isomerase in Escherichia coli by cloning the d-xylose isomerase gene

The Escherichia coli d-xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) gene, xylA, has been cloned on various E. coli plasmids. However, it has been found that high levels of overproduction of the d-xylose isomerase, the protein product of the xylA gene, cannot be accomplished by cloning the intact gene on high copy-number plasmids alone. This is believed to be due to the fact that the expression of the gene through its natural promoter is highly regulated in E. coli. In order to overcome this, the xylA structural gene has been fused with other strong promoters such as tac and lac, resulting in the construction of a number of fused genes. Analysis of the E. coli transformants containing the fused genes, cloned on high copy-number plasmids, indicated that a 20-fold overproduction of the enzyme can now be obtained. It is expected that overproduction of the enzyme in E. coli can still be substantially improved through additional manipulation with recombinant DNA techniques.     

Gene expression profiling and its practice in drug development

The availability of sequenced genomes of human and many experimental animals necessitated the development of new technologies and powerful computational tools that are capable of exploiting these genomic data and ask intriguing questions about complex nature of biological processes. This gave impetus for developing whole genome approaches that can produce functional information of genes in the form of expression profiles and unscramble the relationships between variation in gene expression and the resulting physiological outcome. These profiles represent genetic fingerprints or catalogue of genes that characterize the cell or tissue being studied and provide a basis from which to begin an investigation of the underlying biology. Among the most powerful and versatile tools are high-density DNA microarrays to analyze the expression patterns of large numbers of genes across different tissues or within the same tissue under a variety of experimental conditions or even between species. The wide spread use of microarray technologies is generating large sets of data that is stimulating the development of better analytical tools so that functions can be predicted for novel genes. In this review, the authors discuss how these profiles are being used at various stages of the drug discovery process and help in the identification of new drug targets, predict the function of novel genes, and understand individual variability in response to drugs.     

Biological controls upon the physical taphonomy of exceptionally preserved salamanders from the Miocene of Rubielos de Mora,northeast Spain

McNamara, M.E., Orr, P.J., Manzocchi, T., Alcalá, L., Anadón, P. & Peñalver, E. 2011: Biological controls upon the physical taphonomy of exceptionally preserved salamanders from the Miocene of Rubielos de Mora, northeast Spain. Lethaia, Vol. 45, pp. 210–226. The middle Miocene Rubielos de Mora Konservat‐Lagerstätte of northeast Spain is hosted within profundal, finely laminated, lacustrine mudstones. The diverse biota includes abundant salamanders. Most individuals died during separate episodes and sank rapidly postmortem. Specimens are typically preserved in dorso‐ventral aspect, the most hydrodynamically stable orientation. The near‐cylindrical morphology of the body, however, allowed some carcasses to settle in or subsequently re‐orientate into, lateral orientations. Loss of skeletal elements (i.e. reduced completeness) reflects their location within the body and followed a distal to proximal trend. Two stages are identified: initial loss of a small number of phalanges, followed by loss of more proximal limb bones plus additional phalanges. Disarticulation is more complex: it occurred via several mechanisms (notably, abdominal rupture and re‐orientation of part of the body and limbs during decay) and shows no consistent pattern among specimens. The physical taphonomy of the salamanders is controlled predominantly by intrinsic biological factors, i.e. the geometry of the body and of individual skeletal elements, the orientation, inherent strength and location of specific joints and the extent to which soft tissues, particularly the skin, persist during decay. These biological factors probably control patterns of physical taphonomy of other fossil tetrapods with a similar skeletal configuration. □Articulation, completeness, Konservat‐Lagerstätten, orientation, quantitative taphonomy, salamanders.     

Systems analysis of primary Sjögren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model

Bone tissue has an exceptional quality to regenerate to native tissue in response to injury. However, the fracture repair process requires mechanical stability or a viable biological microenvironment or both to ensure successful healing to native tissue. An improved understanding of the molecular and cellular events that occur during bone repair and remodeling has led to the development of biologic agents that can augment the biological microenvironment and enhance bone repair. Orthobiologics, including stem cells, osteoinductive growth factors, osteoconductive matrices, and anabolic agents, are available clinically for accelerating fracture repair and treatment of compromised bone repair situations like delayed unions and nonunions. Preclinical and clinical studies using biologic agents like recombinant bone morphogenetic proteins have demonstrated an efficacy similar or better than that of autologous bone graft in acute fracture healing. A lack of standardized outcome measures for comparison of biologic agents in clinical fracture repair trials, frequent off-label use, and a limited understanding of the biological activity of these agents at the bone repair site have limited their efficacy in clinical applications.     

Infrared Thermography and Ultrasonography to Indirectly Monitor the Influence of Liner Type and Overmilking on Teat Tissue Recovery

Eight Danish Holstein cows were milked with a 1-mm thick specially designed soft liner on their right rear teat and a standard liner mounted under extra high tension on their left rear teat. Four of the animals were overmilked for 5 min. Rear teats were subjected to ultrasound examination on the first day and to infrared thermography on the second day. Teats were submersed in ethanol 20 min post-milking on the second day. Ultrasonography measurements showed that teat canal length increased by 30–41% during milking. Twenty minutes after milking, teats milked with modified standard liners still had elongated teat canals while teats milked with the soft liner were normalized. Overmilking tended to increase teat wall thickness. Approximately 80% of variability in teat canal length, from before teat preparation to after milking, could be explained by changes during teat preparation. Thermography indicated a general drop in teat temperature during teat preparation. Teat temperature increased during milking and continued to increase until the ethanol challenge induced a significant drop. Temperatures approached pre-challenge rather than pre-milking temperatures within 10 minutes after challenge. Teat temperatures were dependent on type of liner. Mid-teat temperatures post-challenge relative to pre-teat preparation were dependent on overmilking. Thermography and ultrasound were considered useful methods to indirectly and non invasively evaluate teat tissue integrity.     

重组腺相关病毒2型转导人外周血单核细胞来源树突状细胞的研究

To find out the infection efficiency of recombinant adeno-as sociated virus 2-mediated exogenou s genes in human peripheral blood monocyte-derived dendritic cells(D Cs),the process of transfection wa s investigated by FITC-labeled rAA V2,and observed under confocal mic roscope.Newly separated dendritic cells were tranfected by rAAV2-luc and rAAV2-GFP at different MOI,and transfection efficiency were detec ted by luminometer for rAAV2-luc a nd flow cytometry for rAAV2-GFP.Th e results were elucidated in four different assay systems:(1)60min w as needed for rAAV2 to bind on den dritic cells,and got into cells in the following 10min;(2)the express ion of luc could be detected at th e MOI as low as 1×10~5v.g/cell,and the expression plateau was reached by the MOI of 10~6～10~7v.g/cell,furt her increase of MOI had no functio n on expression level;(3)transgene expression was detected after 48h, and maintained a higher expression level from 96h to 240h after infec tion;(4)7days postinfection of rAA V2-GFP,5%～18% dendritic cells were GFP positive. These data suggest th at rAAV2 vector can efficiently in fect monocyte-derived dendritic ce lls and mediate exogenous gene exp ression,and that the application o f rAAV2 as vector may be useful fo r gene transfer to dendritic cells in ex vivo immunotherapy.