Recombinant Class Research Articles

Estimation of Isolation Times of the Island Species in the Drosophila simulans Complex from Multilocus DNA Sequence Data

BackgroundThe Drosophila simulans species complex continues to serve as an important model system for the study of new species formation. The complex is comprised of the cosmopolitan species, D. simulans, and two island endemics, D. mauritiana and D. sechellia. A substantial amount of effort has gone into reconstructing the natural history of the complex, in part to infer the context in which functional divergence among the species has arisen. In this regard, a key parameter to be estimated is the initial isolation time (t) of each island species. Loci in regions of low recombination have lower divergence within the complex than do other loci, yet divergence from D. melanogaster is similar for both classes. This might reflect gene flow of the low-recombination loci subsequent to initial isolation, but it might also reflect differential effects of changing population size on the two recombination classes of loci when the low-recombination loci are subject to genetic hitchhiking or pseudohitchhikingMethodology/Principal Findings New DNA sequence variation data for 17 loci corroborate the prior observation from 13 loci that DNA sequence divergence is reduced in genes of low recombination. Two models are presented to estimate t and other relevant parameters (substitution rate correction factors in lineages leading to the island species and, in the case of the 4-parameter model, the ratio of ancestral to extant effective population size) from the multilocus DNA sequence data.Conclusions/SignificanceIn general, it appears that both island species were isolated at about the same time, here estimated at ∼250,000 years ago. It also appears that the difference in divergence patterns of genes in regions of low and higher recombination can be reconciled by allowing a modestly larger effective population size for the ancestral population than for extant D. simulans.

Design Novel Selective Inhibitors of Class II Fructose‐1,6‐bisphosphate Aldolase

Fructose‐1,6‐bisphosphate (FBP) aldolase (E.C. ) catalyzes the reversible aldol condensation of dihydroxyacetonephosphate (DHAP) and glyceraldehyde‐3‐phosphate in glycolysis, gluconeogenesis and Calvin cycle. FBP aldolases are categorized into two groups based on different catalytic mechanisms: Class I aldolases form a Schiff‐base intermediate with the substrate through an active site lysine residue, whereas Class II aldolases contain a divalent metal which coordinates and stabilizes the carbanion intermediate. Noticeably, Class II aldolase does not exist in animals or plants, making this enzyme a potential drug target for pathogenic microbial organisms such as Mycobacterium tuberculosis, Magnaporthe grisea, Pseudomonas aeruginosa and Bacillus anthracis. Our research group has found that a commercially available antidote for heavy metal poisoning is a good competitive inhibitor of Class II FBP aldolase. In order to improve the binding affinity of this inhibitor for the enzyme activate site, new molecules based upon this compound were designed, synthesized and tested against recombinant Class II FBP aldolases from aforementioned microorganisms using kinetic assays developed by our group.This research is supported by NSERC (J.G.G. and G.I.D.)

Peptide-binding motif prediction by using phage display library for SasaUBA*0301, a resistance haplotype of MHC class I molecule from Atlantic Salmon ( Salmo salar)

The structure of the peptide-binding specificity of major histocompatibility complex (MHC) class I has been analyzed extensively in human and mouse. For fish, there are no crystallographic models of MHC molecules, neither are there data on the peptide-binding specificity. In this study, we describe for the first time the identification of a fish class I peptide-MHC ligand binding motif. Phage display technology using both 7 mer and 12 mer libraries enabled us to identify peptide ligands with unique specificity that interacts with the recombinant Salmon MHC class I molecule. The recombinant proteins, β 2m/SasaUBA*0301, were produced in Escherichia coli, in which the carboxyl terminus of β 2-microglobulin is joined together with a flexible (GGGGS) 3 linker to the amino terminus of the heavy chain. One hundred and seven individual phages bound to β 2m/SasaUBA*0301 were isolated after four rounds of panning from the 7 mer random-peptide library. The peptide encoding sequences were determined and peptide alignment led to the prediction of position-specific anchor residue. A prominent proline at position 2 was observed and we predict that it might be one of the anchors at the N-terminus. Meanwhile, phage display peptide library encoding random 12 mer peptides was also screened against β 2m/SasaUBA*0301. Eighty-five percentages of the corresponding peptides have an enrichment of leucine, methionine, valine, or isoleucine at the C-terminus. We predict that this particular allele of Salmon class I molecule might have a very similar binding motif at the C-terminus compared with a known mouse class I molecule H2-Kb which has L, or I, V, M at p8. Previous work showed that Atlantic Salmon carrying the allele SasaUBA*0301 are resistant to infectious Salmon aneamia virus and there is a significant association between MHC polymorphism and the disease resistance. Therefore, our study might contribute to designing a peptide vaccine against this viral disease.

Expression and characterization of recombinant single-chain salmon class I MHC fused with β 2-microglobulin with biological activity

Heterodimeric class I major histocompatibility complex (MHC) molecules consist of a putative 45-kDa heavy chain and a 12-kDa β 2-microglobulin (β 2m) light chain. The knowledge about MHC genes in Atlantic salmon accumulated during the last decade has allowed us to generate soluble and stable MHC class I molecules with biological activity. We report here the use of a bacterial expression system to produce the recombinant single-chain MHC molecules based on a specific allele Sasa-UBA*0301. This particular allele was selected because previous work has shown its association with the resistance to infectious salmon anaemia virus. The single-chain salmon MHC class I molecule has been designed and generated, in which the carboxyl terminus of β 2m is joined together with a flexible 15 or 20 amino acid peptide linker to the amino terminus of the heavy chain (Sasaβ 2mUBA*0301). Monoclonal antibodies were successfully produced against both the MHC class I heavy chain and β 2m, and showed binding to the recombinant molecule. The recombinant complex Sasaβ 2mUBA*0301 was expressed and isolated; the production was scaled up by adjusting to its optimal conditions. Subsequently, the recombinant proteins were purified by affinity chromatography using mAb against β 2m and α 3. Eluates were analyzed by Western blot and refolded by the removal of denaturant. The correct folding was confirmed by measuring its binding capacity against mAb produced to recognize the native form of MHC molecules by biosensor analysis. This production of sufficient amounts of class I MHC proteins may represent a useful tool to study the peptide-binding specificity of MHC class I molecules, in order to design a peptide vaccine against viral pathogens.

Synthesis-dependent strand annealing in meiosis.

Recent studies led to the proposal that meiotic gene conversion can result after transient engagement of the donor chromatid and subsequent DNA synthesis-dependent strand annealing (SDSA). Double Holliday junction (dHJ) intermediates were previously proposed to form both reciprocal crossover recombinants (COs) and noncrossover recombinants (NCOs); however, dHJs are now thought to give rise mainly to COs, with SDSA forming most or all NCOs. To test this model in Saccharomyces cerevisiae, we constructed a random spore system in which it is possible to identify a subset of NCO recombinants that can readily be accounted for by SDSA, but not by dHJ-mediated recombination. The diagnostic class of recombinants is one in which two markers on opposite sides of a double-strand break site are converted, without conversion of an intervening heterologous insertion located on the donor chromatid. This diagnostic class represents 26% of selected NCO recombinants. Tetrad analysis using the same markers provided additional evidence that SDSA is a major pathway for NCO gene conversion in meiosis.

Isomer-specific retinoic acid biosynthesis in HeLa cells expressing recombinant class I aldehyde dehydrogenases

Retinal dehydrogenase type 1 (RALDH1) catalyzes the oxidation of all- trans and 9- cis retinal to the respective retinoic acids (RAs), whereas another member of the aldehyde dehydrogenase family, the phenobarbital-induced aldehyde dehydrogenase (PB-ALDH), is very poorly active. We have previously generated chimeras between these two enzymes that displayed selectivity for retinal isomers in crude bacterial extracts. To examine whether the selectivity of the recombinant enzymes is retained in intact cells, we first assessed whether retinoid-isomerizing activity is present in cultured eukaryotic cells. Our results demonstrate that the only RA isomers detected in RALDH1-expressing or non-expressing cells corresponded to the same steric conformation as the supplied retinoids, indicating a lack of measurable 9- cis/all- trans retinoid-isomerizing activity. Finally, HeLa cells transfected with RALDH1 derivatives that were retinal isomer-selective in vitro produced only the corresponding RA isomers, establishing these enzymes as useful tools to assess the respective roles of the two RA isomers in vivo.

Expansion of CD8+ Cytotoxic T Cells in vitro and in vivo Using MHC Class I Tetramers

Background: The expansion of cytotoxic CD8+ T lymphocytes (CTLs) which recognize peptide epitopes of tumour or viral origin has been a major aim of immunotherapy research for the past decade. Alongside the established dendritic cell-based methods, more recent approaches using recombinant MHC class I peptide complexes have been developed. Methods: In this study we have explored the potential of a simplified system using soluble streptavidin-linked MHC class I tetramers to expand antigen-specific CTLs in vitro and in vivo. Results: In vitro tetramer-mediated expansion of CD8+ CTLs recognizing HLA-2/Melan-A and HLA-A2/Gag complexes was demonstrated with PBMCs from healthy donors or HIV+ donors, respectively. With 3 weekly rounds of tetramer stimulation, cell numbers expanded 100-fold from 0.05 to 5.0%. The lytic function of HLA-A2/Melan-A-expanded cells was demonstrated in ⁵¹Cr release assays by specific killing of T2 cells pulsed with Melan-A, but not other peptides. Similarly, murine CD8+ T cells specific for the HY epitope H2-D^b/Uty could be expanded in vitro over a wide range of tetramer concentrations (0.008–1.0 µg/ml), with a single exposure producing substantial T cell expansion from 0.11 to 36%. Intraperitoneal administration of H2-D^b/Uty tetramers to primed C57BL/6 mice produced over 5-fold expansion of D^b/Uty-specific CTL in vivo. Conclusion: The results in this paper demonstrate that simple, multimeric MHC complexes may be of value in expanding CTLs in vitro for adoptive immunotherapy and also potentially in vivo. Further studies will be necessary to clarify the optimum protocols and schedules of administration for T cell expansion using recombinant MHC multimers.

HLA Class I Epitopes: Recognition of Binding Sites by mAbs or Eluted Alloantibody Confirmed With Single Recombinant Antigens

Development of beads coated with single recombinant HLA antigens has permitted the confirmation and further definition of HLA class I epitopes. In this study, monoclonal antibodies (mAbs) or alloantibodies eluted from recombinant cell lines were tested for reactivity with Luminex beads individually coated with 79 recombinant HLA class I single antigen (rHLA SA). Published amino acid sequences were used to map epitopes common to sets of antigens reactive with each antibody. While several epitopes have already been demonstrated, this study confirmed them by adsorption of allosera with transfectants or SA beads having a single HLA antigen and specific binding of the eluted antibody on SA beads. The allosera and mAbs used in this study recognized a total of at least 58 HLA class I epitopes, as demonstrated by their different adsorption/reactivity patterns. Of these, 25 epitopes were characterized by a single unique common amino acid, 30 shared 2 signature amino acids in close proximity, and 3 epitopes involved 3 specific amino acids in a non-linear sequence. Since these epitopes may be targets for antibody-mediated allograft rejection, epitope analysis should complement HLA and CREG assignment for defining complex antibodies and identifying suitable donors for highly sensitized transplant patients.

319 POSTER Biochemical characterization of PXD101, a small molecule HDAC inhibitor, and a library of additional compounds on recombinant class I and II HDAC isoforms

319 POSTER Biochemical characterization of PXD101, a small molecule HDAC inhibitor, and a library of additional compounds on recombinant class I and II HDAC isoforms

HLA class I mono-specific APCs and target cells: A method to standardise in vitro CD8+ T cell expansion and functional assays

The introduction of in vitro T cell expansion and assay methods that are robust and easy to use would be welcome in cancer vaccine and infectious disease research. By coating HLA class I −ve B cells with recombinant HLA class I peptide complexes, we are able to produce antigen presenting cells and target cells expressing a single defined antigen in the context of costimulatory and adhesion molecules. HLA class I mono-specific cells promoted the in vitro expansion of CMV epitope specific CD8+ T cells from 0.03% to 30.6% in 2 weeks, which was comparable to using peptide-loaded dendritic cells. The HLA class I mono-specific cells were also shown to promote in vitro antigen specific T cell function in assays based on measuring cytokine production and cytolytic activity. HLA class I mono-specific cells are simple to prepare, can be used with any recombinant HLA class I allele/peptide combination and should provide a useful system for in vitro T cell expansion and functional analysis.

(256) Investigation into the Poor Seedling Establishment Trait Associated with Compact Phenotype in PI308916 and Possible Relationship to Apical Hook Formation

Cucumber PI308916 has a compact growth habit (short internodes and main stem length) due to a single recessive gene cp. We also observed that this PI has lower incidence of Phytophthora fruit rot, likely due to its upright fruit bearing habit. Previous researchers reported that compact cucumber lines derived from PI308916 exhibited the potential for higher yields due to increased planting density. Despite these potentially beneficial traits, efforts to develop PI308916-derived lines were terminated due to poor seedling establishment. The objective of this research is to determine the relationship between the compact phenotype and poor seedling establishment. Short internodes can be caused by deficiency in gibberellins or brassinosteroids that can also impact germination or apical hook formation, a trait important for seedling emergence from the soil. Germination rate and apical hook angle was recorded for `Wautoma' (control inbred line that forms normal apical hook), PI308916, and their F1, F2, and BC progeny. Germinating `Wautoma' seeds showed consistent, large apical hook angles (mean 147), while hook angles of PI308916 were broadly distributed from 0 to 180 (mean 96). F1 progeny for the reciprocal crosses had a similar angle (mean 134 and 133) to `Wautoma'. Segregation ratios for apical hook angle in the F2 and BC populations were consistent with a single recessive gene. Evaluation of the relationship between the apical hook and compact phenotype, showed an absence of the recombinant class of long internodes and small apical hook angle in the F2 population, suggesting that the two traits may be conferred by a single gene or two tightly linked genes.

Cloning, purification and crystallization of Bacillus anthracis class C acid phosphatase.

Cloning, expression, purification and crystallization studies of a recombinant class C acid phosphatase from the Category A pathogen Bacillus anthracis are reported. Large diffraction-quality crystals were grown in the presence of HEPES and Jeffamine ED-2001 at pH 7.0. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 53.4, b = 90.1, c = 104.2 angstroms. The asymmetric unit is predicted to contain two protein molecules with a solvent content of 38%. Two native data sets were collected from the same crystal before and after flash-annealing. The first data set had a mosaicity of 1.6 degrees and a high-resolution limit of 1.8 angstroms. After flash-annealing, the apparent mosaicity decreased to 0.9 degrees and the high-resolution limit of usable data increased to 1.6 angstroms. This crystal form is currently being used to determine the structure of B. anthracis class C acid phosphatase with experimental phasing techniques.

Functional Assessment of Human Alcohol Dehydrogenase Family in Ethanol Metabolism: Significance of First‐Pass Metabolism

Alcohol dehydrogenase (ADH) is the principal enzyme responsible for ethanol metabolism in mammals. Human ADH constitutes a unique complex enzyme family with no equivalent counterpart in experimental rodents. This study was undertaken to quantitatively assess relative contributions of human ADH isozymes and allozymes to hepatic versus gastric metabolism of ethanol in the context of the entire family. Kinetic parameters for ethanol oxidation for recombinant human class I ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, and ADH1C2; class II ADH2; class III ADH3; and class IV ADH4 were determined in 0.1 M sodium phosphate at pH 7.5 over a wide range of substrate concentrations in the presence of 0.5 mM NAD+. The composite numerical formulations for organ steady-state ethanol clearance were established by summing up the kinetic equations of constituent isozymes/allozymes with the assessed contents in livers and gastric mucosae with different genotypes. In ADH1B*1 individuals, ADH1B1 and ADH1C allozymes were found to be the major contributors to hepatic-alcohol clearance; ADH2 made a significant contribution only at high ethanol levels (> 20 mM). ADH1B2 was the major hepatic contributor in ADH1B*2 individuals. ADH1C allozymes were the major contributor at low ethanol (< 2 mM), whereas ADH1B3 the major form at higher levels (> 10 mM) in ADH1B*3 individuals. For gastric mucosal-alcohol clearance, the relative contributions of ADH1C allozymes and ADH4 were converse as ethanol concentration increased. It was assessed that livers with ADH1B*1 may eliminate approximately 95% or more of single-passed ethanol as inflow sinusoidal alcohol reaches approximately 1 mM and that stomachs with different ADH1C genotypes may remove 20% to 30% of single-passed alcohol at the similar level in mucosal cells. This work provides just a model, but a strong one, for quantitative assessments of ethanol metabolism in the human liver and stomach. The results indicate that the hepatic-alcohol clearance of ADH1B*2 individuals is higher than that of the ADH1B*1 and those of the ADH1B*3 versus the ADH1B*1 vary depending on sinusoidal ethanol levels. The maximal capacity for potential alcohol first-pass metabolism in the liver is greater than in the stomach.

Orally Administered Recombinant Metallo-β-Lactamase Preserves Colonization Resistance of Piperacillin-Tazobactam-Treated Mice

Antibiotics that are excreted into the intestinal tract may disrupt colonization resistance (i.e., the ability of the indigenous microflora to provide resistance against colonization by potentially pathogenic microorganisms) (1, 5). Because antibiotic activity within the intestinal lumenis is not needed for the treatment of most infections, we hypothesized that the enzymatic inactivation of the portion of antibiotic that is excreted into the intestinal tract would result in the preservation of colonization resistance during parenteral antibiotic therapy. We previously demonstrated that an orally administered recombinant class A β-lactamase preserved the colonization resistance of piperacillin-treated mice against nosocomial pathogens (4). Here, we show that a recombinant class B metallo-β-lactamase that is resistant to tazobactam inactivation preserves colonization resistance during treatment with the more widely used broad-spectrum antibiotic piperacillin-tazobactam. Targeted recombinant β-lactamase 2 (TRBL-2) containing amino acid residues 31 to 257 of the metalloenzyme of a clinical Bacillus cereus isolate (98ME 1552 from Helsinki University Hospital) was overproduced in Bacillus subtilis using a bacillar secretion vector (4). The hydrolysis rate of TRBL-2 for piperacillin with or without tazobactam was 275 μg/minute/μg of enzyme. Individually housed female CF-1 mice weighing 25 to 30 g (Harlan Sprague-Dawley, Indianapolis, Indiana) were used to examine the efficacy of TRBL-2 in the preservation of colonization resistance against vancomycin-resistant enterococci (VRE). At 24 h and again at 12 h prior to the orogastric inoculation of 104 CFU of VRE strain C68 (4), mice received either subcutaneous (0.2 ml) and orogastric (0.5 ml) phosphate-buffered saline (PBS), subcutaneous piperacillin-tazobactam (4 mg) and orogastric PBS, subcutaneous piperacillin-tazobactam (4 mg) and orogastric TRBL-2 (60 mg/kg of body weight), or subcutaneous piperacillin-tazobactam (4 mg) and orogastric TRBL-2 (60 mg/kg) that had been inactivated by boiling for 10 min. Stool VRE density was monitored as previously described (2). Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified bacterial rRNA genes was performed on stool samples collected 3 days after the inoculation of VRE in order to monitor changes in the indigenous microflora (3, 4). One-way analysis of variance was performed to compare VRE densities among treatment groups, with P values adjusted for multiple comparisons using the Scheffe correction. DGGE similarity indices were compared using Student's t test. Computations were performed using Stata software (version 5.0; Stata, College Station, Texas). Mice treated with piperacillin-tazobactam developed high-density VRE stool colonization; saline controls and mice treated with piperacillin-tazobactam in conjunction with TRBL-2 did not (P < 0.001) (Fig. ​(Fig.1).1). The protective effect of TRBL-2 was eliminated by heat inactivation. DGGE analysis showed that piperacillin-tazobactam caused a significant disruption of the indigenous microflora but that piperacillin-tazobactam in conjunction with TRBL-2 caused a relatively minor alteration of the microflora (mean similarity indices of mice treated with piperacillin-tazobactam and mice treated with piperacillin-tazobactam and TRBL-2 in comparison to those of saline controls were 33% and 86%, respectively; P < 0.001) (Fig. ​(Fig.22). FIG. 1. Efficacy of oral β-lactamase in preventing piperacillin-tazobactam-induced overgrowth of VRE. The densities (log10 CFU/g) of stool VRE are shown after the orogastric inoculation of 104 CFU of VRE on day 0. Prior to inoculation, none of the mice ... FIG. 2. DGGE analysis of stool microflora of individual mice. Lane 1, controls containing rRNA genes amplified from strains of Bacteroides thetaiotaomicron, Bacteroides uniformis, and Escherichia coli (top to bottom); lanes 2 to 4, saline control mice; lanes ... These findings provide further evidence that oral β-lactamase treatment may be an effective means to preserve colonization resistance during therapy with broad-spectrum, parenteral β-lactam antibiotics. Additional studies are needed to determine the efficacy of this strategy as a means to limit the dissemination of nosocomial pathogens in humans.

Targeting T lymphocytes for immune monitoring and intervention in autoimmune diabetes.

Recognition of a peptide-MHC complex by the T cell receptor (TCR) is a key interaction that initiates T lymphocyte activation or silencing during an immune response. Fluorochrome-labeled recombinant MHC class II-peptide reagents function as soluble mimetics of this interaction, bind to their specific TCR, and allow for detection of antigen-specific CD4+ T cells. These reagents are now under scrutiny for "immune staging" of patients at risk of type 1 diabetes, in an effort to diagnose islet autoimmunity early enough to block immune-mediated beta cell destruction. Several issues are currently being addressed to improve the performance of these T cell assays: enrichment steps for better sensitivity, multiplexing of several islet epitopes, simultaneous monitoring of CD4+ and CD8+ responses, detection of low avidity T cells, combination of quantitative (number of positive cells) and qualitative (cytokine secretion, naive/memory phenotype) readouts. CD4+ T cells are key effectors of autoimmunity, and these MHC class II peptide reagents, through their signaling properties, might also provide therapeutics to block the autoimmune process at its onset, analogous to the use of OKT3gammao1(AlaAla) anti-CD3 antibody but in an antigen-specific fashion. The aim of such therapeutics is to potentiate different physiological control mechanisms to restore immune tolerance. Mechanisms initiated by this pathway may be capable of triggering elimination of pathogenic T cells through antigen-specific apoptosis and anergy, combined with the induction of regulatory T cells with broad suppressive function.

Expansion of Functional Endogenous Antigen-Specific CD4+CD25+ Regulatory T Cells from Nonobese Diabetic Mice

CD4+CD25+Foxp3+ regulatory T cells (T(reg)) are critical for controlling autoimmunity. Evidence suggests that T(reg) development, peripheral maintenance, and suppressive function are dependent on Ag specificity. However, there is little direct evidence that the T(reg) responsible for controlling autoimmunity in NOD mice or other natural settings are Ag specific. In fact, some investigators have argued that polyclonal Ag-nonspecific T(reg) are efficient regulators of immunity. Thus, the goal of this study was to identify, expand, and characterize islet Ag-specific T(reg) in NOD mice. Ag-specific T(reg) from NOD mice were efficiently expanded in vitro using IL-2 and beads coated with recombinant islet peptide mimic-MHC class II and anti-CD28 mAb. The expanded Ag-specific T(reg) expressed prototypic surface markers and cytokines. Although activated in an Ag-specific fashion, the expanded T(reg) were capable of bystander suppression both in vitro and in vivo. Importantly, the islet peptide mimic-specific T(reg) were more efficient than polyclonal T(reg) in suppressing autoimmune diabetes. These results provide a direct demonstration of the presence of autoantigen-specific T(reg) in the natural setting that can be applied as therapeutics for organ-specific autoimmunity.

Africanization in the United States

The expansion of Africanized honeybees from South America to the southwestern United States in <50 years is considered one of the most spectacular biological invasions yet documented. In the American tropics, it has been shown that during their expansion Africanized honeybees have low levels of introgressed alleles from resident European populations. In the United States, it has been speculated, but not shown, that Africanized honeybees would hybridize extensively with European honeybees. Here we report a continuous 11-year study investigating temporal changes in the genetic structure of a feral population from the southern United States undergoing Africanization. Our microsatellite data showed that (1) the process of Africanization involved both maternal and paternal bidirectional gene flow between European and Africanized honeybees and (2) the panmitic European population was replaced by panmitic mixtures of A. m. scutellata and European genes within 5 years after Africanization. The post-Africanization gene pool (1998-2001) was composed of a diverse array of recombinant classes with a substantial European genetic contribution (mean 25-37%). Therefore, the resulting feral honeybee population of south Texas was best viewed as a hybrid swarm.

Preparation of enzymatically active recombinant class III protein deacetylases

Class III histone deacetylases, or sirtuins, are homologous to the Saccharomyces cerevisiae transcriptional regulator SIR2. The class III enzymes are characterized by their dependence on nicotinamide adenine dinucleotide (NAD +). This cofactor serves as an acetyl-group acceptor in the deacetylation reaction generating O-acetyl-ADP-ribose. Enzymatic activity of sirtuin can be measured in vitro using recombinant proteins purified from mammalian cells after overexpression or after purification from Escherichia coli. This review discusses protocols for the purification of enzymatically active human sirtuin 1, 2, and 3 and their activities on histone and nonhistone substrates.

Investigation of Peptide Involvement in T Cell Allorecognition Using Recombinant HLA Class I Multimers

Alloreactive T cells are involved in injurious graft rejection and graft-vs-host disease. However, they can also evoke beneficial responses to tumor Ags restricted by foreign MHC molecules. Manipulation of these alloreactivities requires information on the basis of T cell allorecognition. The vigorous T cell response to foreign MHC molecules may arise from peptide-independent recognition of polymorphic residues of foreign MHC molecules or peptide-specific recognition of novel peptides presented by foreign MHC molecules. We investigated CD8+ T cell allorecognition using recombinant HLA class I/peptide complexes. Peptide-specific allorecognition was examined using tetramers of HLA-A*0201 representing five peptides derived from ubiquitously expressed self-proteins that are known to bind endogenously to HLA-A*0201. Distinct subsets of CD8+ T cells specific for each HLA-A*0201/peptide combination were detected within four in vitro-stimulated T cell populations specific for foreign HLA-A*0201. Peptide-independent allorecognition was investigated using artificial Ag-presenting constructs (aAPCs) coated with CD54, CD80, and functional densities of a single HLA-A*0201/peptide combination for four different peptides. None of the four T cell populations specific for foreign HLA-A*0201 were stimulated by the aAPCs, whereas they did produce IFN-gamma upon stimulation with cells naturally expressing HLA-A*0201. Thus, aAPCs did not stimulate putative peptide-independent allorestricted T cells. The results show that these alloreactive populations comprise subsets of T cells, each specific for a self-peptide presented by foreign class I molecules, with no evidence of peptide-independent components.

Unusual segregation products in sperm from a pericentric inversion 17 heterozygote

Chromosome segregation and interchromosomal effect were studied in spermatozoa from a carrier of a pericentric chromosome 17 inversion, 46,XY,inv(17)(p13.1q25.3). Sperm chromosome segregation, lymphocytes of the inversion carrier, and cells from his offspring were analysed by multicolour fluorescence in situ hybridization. The frequency of balanced sperm was 73%. An unusual segregation of recombinants was observed, viz. deletion of the p arm (14.6%) or duplication of the p arm with the presence of one q arm (8.4%), instead of the expected recombinants, viz. duplication of one arm with deletion of the other and vice versa. These unusual recombinants were explained by the position of the 17q breakpoint, which was between the q arm telomere-associated repeats and the unique q subtelomere region. The offspring of the donor were found to have a 17p deletion including the Miller-Dieker critical region, similar to the most frequent recombinant sperm class. The disomy frequency was significantly increased for chromosome 17 compared with other autosomes, suggesting that pairing and recombination of the inversion may predispose to non-disjunction. There was no significant difference between the frequencies of aneuploidy for chromosomes 13, 21, X and Y in the chromosome inversion heterozygote compared with controls. Thus, this unique pericentric inversion of chromosome 17 produces unusual recombinant products; no evidence was apparent of an interchromosomal effect in any of the tested chromosomes.