Victor Sivozhelezov

Immunosuppressive drug‐free operational immune tolerance in human kidney transplants recipients. Part II. Non‐statistical gene microarray analysis

Kidney transplant is the reference treatment for patients with end‐stage renal disease, but patients may develop long‐term rejection of the graft. However, some patients do not reject the transplant, but instead are operationally tolerant state despite withdrawal of immunosuppressive treatment. In this second article we outline a microarray‐based identification of key leader genes associated respectively to rejection and to operational tolerance of the kidney transplant in humans by utilizing a non/statistical bioinformatic approach based on the identification of “key genes,” either as those mostly changing their expression, or having the strongest interconnections. A uniquely informative picture emerges on the genes controlling the human transplant from the detailed comparison of these findings with the traditional statistical SAM (Tusher et al. 2001 Proc Natl Acad Sci USA 98:5116–5121) analysis of the microarrays and with the clinical study carried out in the accompanying part I article. J. Cell. Biochem. 103: 1693–1706, 2008.

Gene expression in the cell cycle of human T lymphocytes: I. Predicted gene and protein networks

The key genes involved in the cell cycle of human T lymphocytes were identified by iterative searches of gene‐related databases, as derived also from DNA microarray experimentation, revealing and predicting interactions between those genes, assigning scores to each of the genes according to numbers of interaction for each gene weighted by significance of each interaction, and finally applying several types of clustering algorithms to genes basing on the assigned scores. All clustering algorithms applied, both hierarchical and K‐means, invariably selected the same six “leader” genes involved in controlling the cell cycle of human T lymphocytes. Relations of the six genes to experimental data describing switching between stages of cell cycle of human T lymphocytes are discussed. J. Cell. Biochem. 97: 1137–1150, 2006.

Comparison of lysozyme structures derived from thin-film-based and classical crystals.

The present report is dedicated to a systematic comparison of crystal structures produced by the nanobiofilm template method and by the classical hanging-drop vapour-diffusion method. Crystals grown by the innovative nanostructured template method appear indeed radiation-resistant even in the presence of a third-generation highly focused beam at the European Synchrotron Radiation Facility. The implications of this finding for protein crystallography are discussed here in terms of water redistribution and of the detailed atomic resolution comparative studies of the two crystal structures with or without nanobiofilm template, as emerging also from circular-dichroism and thermal denaturation studies.

SMILE silencing and PMA activation gene networks in hela cells: Comparison with kidney transplantation gene networks†

Recent findings indicated that the SMILE gene may be involved in kidney graft operational tolerance in human. This gene was found to be up‐regulated in blood from patients with a well functioning kidney transplant in the absence of immunosuppression compared to other transplanted recipients with clinically different status. A microarray study of SMILE knock‐down and phorbol 12‐myristate 13‐acetate (PMA) activation in HeLa cells was herein compared to our earlier analysis based on microarray data of kidney allograft tolerance and rejection in humans and in a rat model of allograft transplantation to determine possible new genes and gene networks involved in kidney transplantation. The nearest neighbors at the intersection of the SMILE knock‐down network with the human tolerance/rejection networks are shown to be NPHS1 and ARRB2, the former (Nephrin) being involved in kidney podocyte function, and the decrease of the latter (Arrestin β2) being recently shown to be involved in monocyte activation during acute kidney allograft rejection in rat. Moreover, another one of the neighbors at the intersection of SMILE network and tolerance/rejection networks is XBP‐1, that we report previously to be increased, at a transcript level, after ER stress in SMILE silenced cells. Finally, in this study, we also show that topological properties (both local and global) of joint SMILE knock‐down network—tolerance/rejection networks and joint PMA activation network—tolerance/rejection networks in rat and human are essentially different, likely due to the inherent nature of the gene SMILE and the mitogen PMA, that do not act the same way on genes and do not interfere the same way on networks. We also show that interestingly SMILE networks contain more feed‐forward loop (FFL) motifs and thus SMILE calls for a more fine‐tuned genetic regulation. J. Cell. Biochem. 113: 1820–1832, 2012.

AKT1 leader gene and downstream targets are involved in a rat model of kidney allograft tolerance

Tolerance is the so‐called “Holy Grail” of transplantation but achieving this state is proving a major challenge, particularly in the clinical settings. This tolerance state can be induced in rodent models using a variety of maneuvers. This phenomenon is classically characterized by donor specificity (recipients accept a secondary donor‐specific allograft but reject third‐party allograft) as well as by the absence of chronic rejection lesion. We previously showed that administration and anti‐donor anti‐class II serum on the day of transplantation induce tolerance to a kidney allograft in the LEW‐1W to LEW‐1A strain combination. In this study, we used DNA microarrays to compare gene patterns involved in anti‐donor anti‐class II tolerated or untreated syngeneic kidney transplants in this strain combination. Statistical and non‐statistical analyses were combined with ab initio analysis, using the recently developed leader gene approach, to shed new light on this phenomenon. Theoretical and experimental results suggest that tolerance and rejection outcome may be in large part determined by low expression variations of some genes, which can form a core gene network around specific genes such as Rac1, NFKB1, RelA, AKT1, IKBKB, BCL2, BCLX, and CHUK. Through this model, we showed that AKT1 gene, WNT pathway and NO synthesis are strictly connected to each other and may play an important role in kidney tolerance and rejection processes, with AKT1 gene being the center of this complex network of interactions. J. Cell. Biochem. 111: 709–719, 2010.

Langmuir-Blodgett based lipase nanofilms of unique structure-function relationship.

Proteins represent versatile building blocks for realization of nanostructured materials of unique structure-function relationship to be applied in nanobiotechnology. Following a recent work [Bruzzese, D., Pastorino, L., Pechkova, E., Sivozhelezov, Nicolini, C., Increase of catalytic activity of lipase towards olive oil by Langmuir-Film Immobilization of Lipase, Enzyme and Microbial Technology, submitted for publication.], the Langmuir-Blodgett technique was utilized to develop nanostructured crystal materials based on enzymes interfacially activated with olive oil as substrate. Particularly, thin films of lipase from both Mucor miehei and Candida rugosa were fabricated and characterized by UV-vis spectroscopy, Atomic force microscopy and biochemical assays. As the first step the M. miehei protein films were studied at the air-water interface and then transferred onto a solid support for further characterization of the enzymatic activity also versus surface pressure, proving that Langmuir-Blodgett film provides a better catalytic effect in lipase than a mere oil-water boundary. Moreover, improvement of lipase catalytic performance was achieved for the M. miehei versus the C. rugosa, despite its almost random distribution of hydrophobic patches and the low purity of its preparation.

Interpretation of ionic strength dependencies of the electron transfer in the Cytochrome c-Cytochrome b 5 system for the wild-type and lysine-mutated yeast Cytochrome c

Ionic strength dependencies of electron transfer between Cytochrome b5 and variants of yeast Cytochrome c were analyzed by curve fitting to the simple model of the electrostatic interaction between the two proteins assuming the process to be non-diffusion-controlled. Mutagenesis of Lys79, but not Lys72, leads to an increase of effective radius of the interacting charged species, suggesting that the mutation effects of the two residues on the electrostatic field distribution near the contact site are different, even within the crude electrostatic model used. Extrapolation of the ionic strength dependencies to infinite ionic strength resulted in similar values, around (2–3)×10-6 for all Cyt c variants considered thus showing the lysine residue mutations to primarily affect protein association rather than the electron transfer directly. Based on the ionic strength dependencies of binding constants of the two proteins into an electrostatically stabilized complex, the monomolecular electron transfer rate constant was estimated to be 1.1×104–1.6×105 s-1. The electrostatic part of the binding energy of the complex at I=0.19 was estimated to be-2.4 kcal/mol, strongly at variance with the values-13.0 and-6.4 kcal/mol reported for the two types of complexes identified using Brownian dynamics techniques. Possible reasons for this discrepancy are discussed.

Controlling the electron transfer rate between oxymyoglobin and ferricytochrome C by alterations in the myoglobin contact site. The role of histidines and electrostatic and steric interactions in the mechanism of the reaction

The kinetics of the redox reaction of sperm whale and pig oxymyoglobins (MbO2) with ferricytochrome C (CytC) from pig heart has been studied in the pH range 5–8. Also, the effects of histidine (His) modification and of the complexing of both myoglobins with Zn2+, on the electron transfer rate, has been investigated. It has been shown that pig MbO2 reduces Cyt C much more effectively than sperm whale MbO2. The pH dependence of the reaction rate is shown to result from the influence of two histidines, His 12(A10) and His 119(GH1), in the case of sperm whale myoglobin and only of His GH1 in the case of pig MbO2. The protonation of His A10 at pH<7.5 decreases the rate of the reaction with Cyt C whereas the ionization of His GH1, on the contrary, increases the electron transfer rate 10–30 times (atI=0.03). The His residues of Cyt C are shown to have no effect on the reaction. Complexing of His GH1 with a zinc ion strongly inhibits the reaction of both sperm whale and pig MbO2 with Cyt C. The reaction of the zinc-MbO2 complexes, as distinct from the intact oxymyoglobins, becomes independent of pH and ionic strength. Unlike His A10, His GH1 plays a very important role in the formation of the electron transfer complexes, and is probably directly involved in the charge transfer step. Based on the data obtained, the reactive site of the Mb surface has been identified in the A-GH region. The spatial arrangement of the charged groups in the reactive sites of the two myoglobins has been obtained. The solvent accessibilities of all amino acid residues situated there have been calculated, according to Lee and Richards. In order to explain the different reactivities of sperm whale and pig myoglobins, their electrostatic properties and the steric features of the contact sites have been compared.

Molecular Mechanisms of Cognitive Impairment in Patients with HIV Infection: Application of Bioinformatics and Data Mining

AIDS patients often suffer from cognitive impairment, including distractibility, delirium, and dementia. In fact, global brain atrophy was recognized from MRI images in HIV-associated neurocognitive disorders. A number of studies have shown that a complex network of inflammatory molecules including cytokines, chemokines, growth factors, and excitatory compounds is associated with brain inflammation and damage in HIV-infected patients.