Ternary mixed monolayers for simultaneous DNA orientation control and surface passivation for label free DNA hybridization electrochemical sensing.

Target recognizing ternary mixed monolayer is constructed by simple sequential adsorption of thiol caped single stranded DNA (HS-ssDNA), 6-mercapto-1-hexanol (MCH) and 3-mercaptopropionic acid (MPA) diluents (abbreviated as HS-ssDNA/MPA/MCH) and applied for detection of DNA hybridization sequence by electrochemical method. The method is more reliable and reproducible on both conventional wire electrode and 100 nm scale gold-coated silicon chips. Detection limit of 10 pM is observed constantly on all types of electrodes. This ternary layer approach provides 60-80% discrimination effect between the hybridized and un-hybridized surfaces compared to the binary mixed monolayers HS-ssDNA/MCH and HS-ssDNA/MPA that showed 20-30% only. Characterization by impedance spectroscopy, X-ray photoelectron spectroscopy and surface Fourier Transform Infrared (FT-IR) techniques reveals 'head-to-head' anisotropic hydrogen bonding between MPA and MCH diluents that controls the HS-ssDNA orientation and enhancing the electrostatic blockade for K3[Fe(CN)6]. This is the first report characterizing the ternary layer for DNA molecular affinity sensing. Results provided unprecedented insight onto the label free electrochemical sensing and understanding of the complex sensing mechanism to develop sensors that is more reliable.

Determination of the limited trypsinolysis pathways of tumor necrosis factor-alpha and its mutant by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) is employed to directly analyze the limited trypsinolysis products of wild-type tumor necrosis factor-alpha (wtTNF-alpha) and its mutant, M3S. To determine the charge numbers of peaks of relatively small peptides in the ESI mass spectrum of a digest, a series of sodium-adduct ion peaks of each peptide are generated by adding a small quantity of NaCl to the digest before taking the spectrum. From the monitoring of the composition of proteolytic mixture as the incubation time is lengthened, it has been learned that the proteolysis of wtTNF-alpha by trypsin occurs sequentially: Arg2, Arg6, Arg32, Arg31, and Arg44, and that M3S is strongly resistant to the proteolysis. Since the cleavage sequence of wtTNF-alpha and the mutation-induced resistance of M3S are consistent with the structural features of the proteins, we can suggest a mutant more resistant to proteolysis than M3S, which has an additional point mutation, Ala35Leu or Ala35Ile.

High resolution crystal structure of a human tumor necrosis factor-alpha mutant with low systemic toxicity.

A human tumor necrosis factor-alpha (TNF-alpha) mutant (M3S) with low systemic toxicity in vivo was designed, and its structures in two different crystal packings were determined crystallographically at 1.8 and 2.15-A resolution, respectively, to explain altered biological activities of the mutant. M3S contains four changes: a hydrophilic substitution of L29S, two hydrophobic substitutions of S52I and Y56F, and a deletion of the N-terminal seven amino acids that is disordered in the structure of wild-type TNF-alpha. Compared with wild-type TNF-alpha, it exhibits 11- and 71-fold lower binding affinities for the human TNF-R55 and TNF-R75 receptors, respectively, and in vitro cytotoxic effect and in vivo systemic toxicity of M3S are 20 and 10 times lower, respectively. However, in a transplanted solid tumor mouse model, M3S suppresses tumor growth more efficiently than wild-type TNF-alpha. M3S is highly resistant to proteolysis by trypsin, and it exhibits increased thermal stability and a prolonged half-life in vivo. The L29S mutation causes substantial restructuring of the loop containing residues 29-36 into a rigid segment as a consequence of induced formation of intra- and intersubunit interactions, explaining the altered receptor binding affinity and thermal stability. A mass spectrometric analysis identified major proteolytic cleavage sites located on this loop, and thus the increased resistance of M3S to the proteolysis is consistent with the increased rigidity of the loop. The S52I and Y56F mutations do not induce a noticeable conformational change. The side chain of Phe56 projects into a hydrophobic cavity, while Ile52 is exposed to the bulk solvent. Ile52 should be involved in hydrophobic interactions with the receptors, since a mutant containing the same mutations as in M3S except for the L29S mutation exhibits an increased receptor binding affinity. The low systemic toxicity of M3S appears to be the effect of the reduced and selective binding affinities for the TNF receptors, and the superior tumor-suppression of M3S appears to be the effect of its weak but longer antitumoral activity in vivo compared with wild-type TNF-alpha. It is also expected that the 1.8-A resolution structure will serve as an accurate model for explaining the structure-function relationship of wild-type TNF-alpha and many TNF-alpha mutants reported previously and for the design of new TNF-alpha mutants.

CD99 (MIC2) regulates the LFA-1/ICAM-1-mediated adhesion of lymphocytes, and its gene encodes both positive and negative regulators of cellular adhesion.

Despite the fact that integrin-mediated lymphocyte adhesion is a crucial event for an appropriate immune response, little is known about the mechanisms that control the adhesion and deadhesion processes generated by the engagement of CD99 between various types of immune cells. Here we report that the CD99 gene encodes two distinct proteins with opposite functions in the LFA-1/intercellular adhesion molecule 1 (ICAM-1)-mediated cell adhesion process. The two forms of the CD99 protein are produced by alternative splicing of the CD99 gene transcript. The major form induced homotypic adhesion of the human B lymphoblastoid cell line IM-9, whereas the minor, truncated form inhibited the adhesion process. Activation of the major form of CD99 with anti-CD99 monoclonal antibodies induced rapid aggregation of IM-9 cells, which was blocked by the addition of mAbs to LFA-1 or intracellular adhesion molecule 1. Overexpression of the minor truncated form of CD99 markedly down-regulated the expression of LFA-1. The two forms of CD99 are differentially expressed in most human cells tested and are highly conserved in monkey. Taken together, these observations suggest that the two forms of CD99 function in vivo in both positive and negative regulation of LFA-1-mediated adhesion of lymphocytes during an immune response.

DN4: a novel cell surface molecule on cortical epithelial cells of the human thymus.

A novel cell surface molecule, DN4, defined by an mAb raised against human thymic epithelial cells, showed a specificity for epithelial cells of the thymic cortex. This antigen was not expressed at detectable levels on any other types of tissues in the human body except for the thymus and bone marrow. Immunohistochemical analysis revealed that the reactivity of anti-DN4 mAb was restricted to the thymic cortex, and the antigen-expressing cells were arranged in a reticular network with long processes between thymocytes. The cellular nature of DN4-positive cells was identified as cortical epithelial cells, as DN4 was expressed in a subpopulation of freshly prepared thymic stromal cells which contain a large amount of keratin and expression of DN4 was strictly confined to the cortical area within the thymus on immunohistochemical analysis of frozen tissues. Immunofluorescence and flow cytometric analysis revealed that a subpopulation of bone marrow cells was also positive for DN4 (20%). The large blasts of normal bone marrow cells were clearly labeled with anti-DN4 mAb, in contrast to small-sized bone marrow cells. This finding suggests that DN4 seems to be transiently expressed in certain blastic stages during the differentiation of bone marrow cells. Immunoprecipitation of 125I-labeled cell lysates from THP-1 and U937 cell lines with anti-DN4 mAb yielded a single chain glycoprotein with an approximate size of 80-85 kd. There was a reduction in apparent molecular weight of approximately 40 kd in the immunoprecipitation of cell lysates after endoglycosidase F treatment. Thus, DN4 seemed to have a considerable amount of carbohydrate group. DN4 appears to be a novel cortical epithelial cell antigen of the human thymus, and although the role of this molecule has not been well established experimentally, the possibility can be suggested that the DN4 molecule might be involved in the positive selection of thymocytes which occurs predominantly in the thymic cortical area.