We have characterized and crystallized a human lambda I light-chain dimer, Bence-Jones protein Loc, which has variable (V) region antigenic determinants characteristic for the lambda I subgroup and constant (C) region determinants of the C lambda I gene Mcg. The crystal structure was determined to 3-A resolution; the R factor is 0.27. The angle formed by the twofold axes of the V and C domains, the "elbow bend", is 97 degrees, the smallest found so far for an antibody fragment. The antigen-binding site formed by the two V domains of the Loc light chain differs significantly from those of other immunoglobulin molecules (light-chain dimers and Fab fragments) for which X-ray crystallographic data are available. Whereas, in other antibody fragments, the V domains are related by a local twofold axis, a local twofold screw axis with a translational component of 3.5 A relates the V domains in protein Loc. In contrast to the classic antigen binding "pocket" formed by V domain interactions in the previously characterized antibody structures, the V region associations in protein Loc result in a central protrusion in the binding site, with grooves on two sides of the protrusion. The structure of protein Loc indicates that immunoglobulins are physically capable of forming a more diverse spectrum of antigen-binding sites than has been heretofore apparent. Moreover, the unusual protruding nature of the binding site may be analogous to structures required for some anti-idiotypic antibodies. Further, the complementarity-determining residues form parts of two independent grooves.(ABSTRACT TRUNCATED AT 250 WORDS)
A method to determine optimum heap leach height has resulted from design work for a uranium heap leach project on the Macusani Plateau in south-eastern Peru. An analysis of the optimum heap height estimate details its sensitivity to the constituent variables algebraically, graphically, and, in contour plot format. A comparison is drawn against a Namibian uranium project with design details in the public domain and agreement is found to be reasonable. The leach extraction model and dynamics are analysed in terms of solution to ore ratio (SOR), reaction extent and acid consumption efficiency to address model time dependence loss in the model simplification step. The rate of change in the acid efficiency is used to specify the target extraction and determine leach SOR and leach time. The validity of the optimum height estimate equation is supported by favourable comparison against the spreadsheet calculation results and data from another uranium heap leach project. The estimate is dependent on recovery discount and pad maintenance costs but is independent of theoretical recovery and pad capital costs.
Angiogenesis is essential for cancer metastasis, thus the discovery and characterization of molecules that inhibit this process is important. Thalidomide is a teratogenic drug which is known to inhibit angiogenesis and effectively inhibit cancer metastasis, yet the specific cellular targets for its effect are not well known. We discovered that CUL5 (previously identified as VACM-1), a scaffold protein in E3 ligase complexes, is involved in thalidomide-dependent inhibition of endothelial cell growth. Our results show that in human endothelial cells (HUVEC), thalidomide-dependent decrease in cell growth was associated with decreased nuclear localization of CUL5. In HUVEC transfected with anti-VACM-1 siRNA, thalidomide failed to decrease cell growth. Previously it was established that the antiproliferative effect of CUL5 is inhibited in rat endothelial cells (RAMEC) transfected with mutated CUL5 which is constitutively modified by NEDD8, a ubiquitin-like protein. In this study, the antiproliferative response to thalidomide was compromised in RAMEC expressing mutated CUL5. These results suggest that CUL5 protein is involved in the thalidomide-dependent regulation of cellular proliferation in vitro. Consequently, CUL5 may be an important part of the mechanism for thalidomide-dependent inhibition of cellular proliferation, as well as a novel biomarker for predicting a response to thalidomide for the treatment of disorders such as multiple myeloma and HIV infection.
Practical instruction of proteomics concepts was provided using high-performance liquid chromatography coupled with a mass selective detection system (HPLC-MS) for the analysis of simulated protein digests. The samples were prepared from selected dipeptides in order to facilitate the mass spectral identification. As part of the prelaboratory preparation, students calculated the parent ion patterns of the dipeptides using peptide calculator websites. Following instruction on the use of the HPLC-MS instrument, students analyzed mixtures of the dipeptides and identified the individual dipeptides in the unknowns. In addition, purchased chicken egg white lysozyme alkylated with iodoacetamide and digested with trypsin was analyzed using the same approach. Key tryptic peptides were identified from the HPLC-MS chromatogram with information generated with the FindPept tool. This experiment demonstrates that complex concepts can be taught in the undergraduate biochemistry laboratory using a problem-based approach.Keywords: Proteomics, protein sequence, mass spectrometry, HPLC-MS, lysozyme.''The great difficulty in education is to get experience out of ideas'' George Santayana Santayana, the Spanish-born philosopher and social commentator, recognized that communication of ideas in the classroom did not always result in understanding, which he called ''experience.'' The experiment described here is used in our biochemistry course to communicate complex ''ideas'' (i.e. concepts) in a practical, problembased laboratory exercise. We wanted students to understand how the researcher can use high-performance liquid chromatography-mass spectrometry (HPLC-MS) to identify peptides that are present in a mixture.Our upper-level undergraduate biochemistry laboratory is a five-hour course that meets weekly for one-half of the semester. Its purpose is to introduce students to concepts and techniques used in research labs. The course exercises include kinetic analysis of mushroom tyrosinase [1], the purification and analytical characterization of lysozyme [2], a bioinformatic exercise for lysozyme [3], and the HPLC-MS analysis of a simulated proteolytic digest using purchased dipeptides and trypsin-digested lysozyme. Hands-on use of the HPLC-MS lets students become familiar with a potent, bioanalytical technique, which can then be applied to samples that will be isolated and prepared in the research lab.Students choose to purify lysozyme from one of several avian egg whites sources. Our laboratory course uses five species of avian eggs in the exercise: Chicken (Gallus gallus), turkey (Meleagris gallopavo), duck (Anas platyrhynchos), bobwhite quail (Colinus virginianus), and ostrich (Struthio camelus). Students learn how to purify proteins by ion-exchange chromatography. They analyze their protein containing fractions for lysozyme enzyme activity against suspensions of Micrococcus lysodeikticus cell walls. Students also determine the molecular weight of their isolated lysozyme using SDS-Polyacrylamide gel electrophoresis against proteins o...
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