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PubMed2002

Poly(C) synthesis by class I and class II CCA-adding enzymes.

The CCA-adding enzymes [ATP(CTP):tRNA nucleotidyl transferases], which catalyze synthesis of the conserved CCA sequence to the tRNA 3' end, are divided into two classes. Recent studies show that the class II Escherichia coli CCA-adding enzyme synthesizes poly(C) when incubated with CTP alone, but switches to synthesize CCA when incubated with both CTP and ATP. Because the poly(C) activity can shed important light on the mechanism of the untemplated synthesis of CCA, it is important to determine if this activity is also present in the class I CCA enzymes, which differ from the class II enzymes by significant sequence divergence. We show here that two members of the class I family, the archaeal Sulfolobus shibatae and Methanococcus jannaschii CCA-adding enzymes, are also capable of poly(C) synthesis. These two class I enzymes catalyze poly(C) synthesis and display a response of kinetic parameters to the presence of ATP similar to that of the class II E. coli enzyme. Thus, despite extensive sequence diversification, members of both classes employ common strategies of nucleotide addition, suggesting conservation of a mechanism in the development of specificity for CCA. For the E. coli enzyme, discrimination of poly(C) from CCA synthesis in the intact tRNA and in the acceptor-TPsiC domain is achieved by the same kinetic strategy, and a mutation that preferentially affects addition of A76 but not poly(C) has been identified. Additionally, we show that enzymes of both classes exhibit a processing activity that removes nucleotides in the 3' to 5' direction to as far as position 74.

Adenosine Triphosphate↗

PubMed2001

Immunomodulation of glioma cells after gene therapy: induction of major histocompatibility complex class I but not class II antigen in vitro.

OBJECTIVE: Acquired immunity has been demonstrated in Fischer rats bearing syngeneic 9L tumors after herpes simplex virus (HSV) thymidine kinase (TK) gene transfection and ganciclovir treatment. The nature of this immunity in rats and its relevance to the HSV TK/ganciclovir protocol for human subjects remain to be determined. In this study, levels of major histocompatibility complex (MHC) Class I and II antigen expression were measured before and after HSV TK transfection, in an effort to document immunomodulatory changes caused by gene therapy. METHODS: Tumor cells from the 9L gliosarcoma cell line, three primary human glioma cultures, and the human glioma cell line U87 MG were transduced with HSV TK vector-containing supernatant from fibroblast-producing cells (titer of 5 x 10(6) colony-forming units/ml) and selected in G418 medium for neomycin resistance. Clones were pooled or individually selected for cell-killing assays with ganciclovir, to confirm TK expression (10(3) cells/well in a 96-well dish). Northern analyses using MHC Class I and Class II complementary deoxyribonucleic acid probes were performed on blots containing total ribonucleic acid from wild-type tumor cells and HSV TK transfectants. A beta-actin complementary deoxyribonucleic acid probe served as an internal control. Cell surface expression was confirmed with flow cytometry. The induction of MHC Class I was tested for cycloheximide and genistein sensitivity. RESULTS: All cell cultures exhibited increases in MHC Class I but not MHC Class II expression, as determined by Northern analysis densitometry and flow cytometry. Cycloheximide treatment did not diminish the up-regulation of MHC Class I after retroviral transfection, implicating a signal transduction pathway that does not require ongoing protein synthesis. Genistein pretreatment of cell cultures did diminish the up-regulation of MHC Class I, implicating a tyrosine kinase in the signaling cascade. CONCLUSION: Induction of MHC Class I in rat and human glioma cells after HSV TK retroviral gene therapy is a primary effect that is dependent on tyrosine kinase activity. Specific immune responses generated after transfection may represent an important general side effect of gene therapy protocols. Elucidation of the mechanism of immunomodulation after gene therapy will likely yield safer and more effective clinical protocols.

Animals↗

PubMed2002

Streptomyces spp. contain class Ia and class II ribonucleotide reductases: expression analysis of the genes in vegetative growth.

Genes encoding two ribonucleotide reductases (RNRs) were identified in members of the genus Streptomyces. One gene, nrdJ, encoded an oligomeric protein comprising four identical subunits each with a molecular mass of approximately 108 kDa. The activity of this protein depended on the presence of 5'-deoxyadenosylcobalamine (coenzyme B12), establishing it as a class II RNR. The Streptomyces clavuligerus nrdJ gene was cloned, using internal peptide sequences from the purified protein, and was found to encode a polypeptide of 961 aa. Molecular phylogenetic analysis showed that the S. clavuligerus class II RNR shares significant similarity with most other bacterial and archaeal class II RNRs. Two other genes, nrdA and nrdB, were initially identified in the Streptomyces coelicolor genome database in unannotated ORFs as encoding a class Ia RNR. Southern analysis demonstrated that the nrdAB genes were present in different Streptomyces spp. The S. coelicolor nrdAB genes were cloned and expressed in Escherichia coli, and the recombinant proteins were shown to represent a class I RNR. It was shown, using quantitative real-time PCR, that the S. clavuligerus class Ia and class II RNR genes were differentially transcribed during vegetative growth. The copy number of the class II nrdJ transcripts was approximately constant throughout the exponential phase of vegetative growth (3-5x10(5) copies per 400 ng total RNA after reverse transcription). In contrast, the copy number of the class Ia nrdAB transcripts was some 10- to 20-fold less than that of nrdJ in the early-exponential growth phase (2.8x10(4) copies), and decreased markedly at the mid-exponential (4x10(3) copies) and late-exponential phases (1.1x10(3) copies) of growth. A possible role for the involvement of two RNRs during vegetative growth is discussed.

Actinomycetales↗

PubMed1994

Plastid Class I and Cytosol Class II Aldolase of Euglena gracilis (Purification and Characterization).

The plastidic class I and cytosolic class II aldolases of Euglena gracilis have been purified to apparent homogeneity. In autotrophically grown cells, up to 81% of the total activity is due to class I activity, whereas in heterotrophically grown cells, it is only 7%. The class I aldolase has been purified to a specific activity of 20 units/mg protein by anion-exchange chromatography, affinity chromatography, and gel filtration. The native enzyme (molecular mass 160 kD) consisted of four identical subunits of 40 kD. The class II aldolase was purified to a specific activity of 21 units/mg by (NH4)2SO4 fractionation, anion-exchange chromatography, chromatography on hydroxylapatite, and gel filtration. The native enzyme (molecular mass 80 kD) consisted of two identical subunits of 38 kD. The Km (fructose-1,6-bisphosphate) values were 12 [mu]M for the class I enzyme and 175 [mu]M for the class II enzyme. The class II aldolase was inhibited by 1 mM ethylenediaminetetraacetate (EDTA), 0.8 mM cysteine, 0.5 mM Zn2+, or 0.5 mM Cu2+. Na+, K+, Rb+, and NH4+ (but not Li+ or Cs+) enhanced the activity up to 7-fold. After inactivation by EDTA, the activity could be partially restored by Mn2+, Cu2+, or Co2+. A subclassification of class II aldolases is proposed based on (a) activation/inhibition by Cys and (b) activation or not by divalent ions.

Journal Article↗

PubMed2006

Three-class ROC analysis--the equal error utility assumption and the optimality of three-class ROC surface using the ideal observer.

Previously, we have developed a decision model for three-class receiver operating characteristic (ROC) analysis based on decision theory. The proposed decision model maximizes the expected decision utility under the assumption that incorrect decisions have equal utilities under the same hypothesis (equal error utility assumption). This assumption reduced the dimensionality of the "general" three-class ROC analysis and provided a practical figure-of-merit to evaluate the three-class task performance. However, it also limits the generality of the resulting model because the equal error utility assumption will not apply for all clinical three-class decision tasks. The goal of this study was to investigate the optimality of the proposed three-class decision model with respect to several other decision criteria. In particular, besides the maximum expected utility (MEU) criterion used in the previous study, we investigated the maximum-correctness (MC) (or minimum-error), maximum likelihood (ML), and Nyman-Pearson (N-P) criteria. We found that by making assumptions for both MEU and N-P criteria, all decision criteria lead to the previously-proposed three-class decision model. As a result, this model maximizes the expected utility under the equal error utility assumption, maximizes the probability of making correct decisions, satisfies the N-P criterion in the sense that it maximizes the sensitivity of one class given the sensitivities of the other two classes, and the resulting ROC surface contains the maximum likelihood decision operating point. While the proposed three-class ROC analysis model is not optimal in the general sense due to the use of the equal error utility assumption, the range of criteria for which it is optimal increases its applicability for evaluating and comparing a range of diagnostic systems.

Algorithms↗

PubMed1999

Molecular classification of cancer: class discovery and class prediction by gene expression monitoring.

Although cancer classification has improved over the past 30 years, there has been no general approach for identifying new cancer classes (class discovery) or for assigning tumors to known classes (class prediction). Here, a generic approach to cancer classification based on gene expression monitoring by DNA microarrays is described and applied to human acute leukemias as a test case. A class discovery procedure automatically discovered the distinction between acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) without previous knowledge of these classes. An automatically derived class predictor was able to determine the class of new leukemia cases. The results demonstrate the feasibility of cancer classification based solely on gene expression monitoring and suggest a general strategy for discovering and predicting cancer classes for other types of cancer, independent of previous biological knowledge.

Acute Disease↗

PubMed1963

Adsorption and fluorescence of fat-soluble fluorescent dyes on class I and class III Saccharomyces cerevisiae.

Lycette, R. M. (Illinois Institute of Technology, Chicago) and L. R. Hedrick. Adsorption and fluorescence of fat-soluble fluorescent dyes on class I and class III Saccharomyces cerevisiae. J. Bacteriol. 85:1-6. 1963.-Class III cells of the brewery yeast Saccharomyces cerevisiae flocculate, but class I cells do not, when cultured in an organic YM medium. Class III cells adsorb the fat-soluble stilbyl-triazole fluorescent dye (STC) more readily and produce more fluorescence than do class I cells. This suggests that cells of the class III yeasts have more lipids or more phospholipids on their surface than do cells of class I yeasts. Fluorescence of STC in the absence of cells is pH-independent, but adsorption of the compound on the surface of cells, with the associated fluorescence, is pH-dependent. As the pH level was decreased within the range of pH 9 to 0, and as the temperature was elevated within the range of 0 to 40 C, there was an increase in adsorption and a corresponding increase in fluorescence. Maximal adsorption was attained in less than 15 min when the cells and dye were shaken at 26 C. Cells of class III adsorbed 2.5 mug of STC per 3.25 mg (dry wt) of cells. The kinetics of adsorption follow the Langmuir monomolecular model.

Adsorption↗

PubMed1987

Effects of a temperature-sensitive mutation in the immediate-early gene of pseudorabies virus on class II and class III gene transcription.

The pseudorabies virus immediate-early protein activates transcription of both class II and class III genes. At present it is not known whether the activation of class II genes occurs through an activation of cellular factors or by a direct interaction of the immediate-early protein with factors or DNA. It is also not known whether the activation of class II and class III genes occurs by a similar or different mechanism. We utilized tsG, a temperature-sensitive mutation in the immediate-early gene of pseudorabies virus, to study the activation of viral genes transcribed by RNA polymerases II and III. Previous studies have shown that tsG inhibits wild-type adenovirus early gene transcription in coinfections at the nonpermissive temperature (L. T. Feldman and S. E. Ahlers, J. Virol. 57:13-17, 1986). Using this system of mixed infections as an assay, we obtained several results which allowed us to draw certain conclusions about the mode of action of the pseudorabies virus immediate-early protein (IEP). First, the tsG mutation inhibits the formation of new transcription complexes on class II genes, but does not affect transcription from preestablished transcription complexes formed in the presence or absence of the adenovirus E1A protein. Second, tsG does not inhibit transcription from class III genes, suggesting that the activation by IEP of class II and class III genes occurs by different mechanisms. Third, activation of transcription by the adenovirus E1A protein is not dominant to inhibition by tsG, suggesting that the temperature-sensitive IEP is involved in some physical interaction which actively inhibits transcription of viral genes.

Adenovirus Early Proteins↗

PubMed1989

Mechanisms of termination of reentrant atrial arrhythmias by class I and class III antiarrhythmic agents.

We studied atrial flutter due to circus movement in chronically instrumented conscious dogs to identify the mechanism by which class I and class III antiarrhythmic drugs terminate reentrant excitation. We used a crossover experimental design administering five class I agents and one class III agent, by intravenous bolus followed by intravenous infusion. The class I agents other than lidocaine were almost uniformly effective in terminating the arrhythmia (disopyramide in six of seven dogs, propafenone in six of six, flecainide in seven of seven, and SC-40230 in seven of seven). Termination was preceded by a marked increase in cycle length (ranging from +78% with propafenone to +55% with disopyramide), but with the exception of disopyramide, class I agents did not significantly shorten the excitable gap. With disopyramide the gap decreased from 49 +/- 3% to 28 +/- 3% of the cycle length. With no class I agent did the wavelength of effective refractoriness increase to approach the cycle length of the arrhythmia. Lidocaine, used as a negative control, terminated the reentry in one dog with modest prolongation of the cycle length. Terminations with class I agents correlated with depression of conduction rather than prolongation of refractoriness. In contrast with class I agents, D-sotalol prolonged the cycle length minimally (+10%) and terminated the arrhythmia in six of seven dogs. It decreased the excitable gap from 42 +/- 4% to 26 +/- 6% of the cycle, but it still did not cause the wavelength of effective refractoriness to equal the cycle length. Terminations by D-sotalol seemed to result from either failure of the lateral boundaries of the circus path or reflection within the path.

Animals↗

PubMed2004

MFV-class: a multi-faceted visualization tool of object classes.

Classes are key software components in an object-oriented software system. In many industrial OO software systems, there are some classes that have complicated structure and relationships. So in the processes of software maintenance, testing, software reengineering, software reuse and software restructure, it is a challenge for software engineers to understand these classes thoroughly. This paper proposes a class comprehension model based on constructivist learning theory, and implements a software visualization tool (MFV-Class) to help in the comprehension of a class. The tool provides multiple views of class to uncover manifold facets of class contents. It enables visualizing three object-oriented metrics of classes to help users focus on the understanding process. A case study was conducted to evaluate our approach and the toolkit.

Computer Graphics↗

PubMed2001

Regulation of MHC class II antigen presentation by sorting of recycling HLA-DM/DO and class II within the multivesicular body.

MHC class II molecules bind antigenic peptides in the late endosomal/lysosomal MHC class II compartments (MIIC) before cell surface presentation. The class II modulatory molecules HLA-DM and HLA-DO mainly localize to the MIICs. Here we show that DM/DO complexes continuously recycle between the plasma membrane and the lysosomal MIICs. Like DMbeta and the class II-associated invariant chain, the DObeta cytoplasmic tail contains potential lysosomal targeting signals. The DObeta signals, however, are not essential for internalization of the DM/DO complex from the plasma membrane or targeting to the MIICs. Instead, the DObeta tail determines the distribution of both DM/DO and class II within the multivesicular MIIC by preferentially localizing them to the limiting membrane and, in lesser amounts, to the internal membranes. This distribution augments the efficiency of class II antigenic peptide loading by affecting the efficacy of lateral interaction between DM/DO and class II molecules. Sorting of DM/DO and class II molecules to specific localizations within the MIIC represents a novel way of regulating MHC class II Ag presentation.

Amino Acid Motifs↗

PubMed1987

Expression of major histocompatibility complex class I and class II antigens in human Schwann cell cultures and effects of infection with Mycobacterium leprae.

Recent experiments on rats have raised the possibility that Schwann cells can present antigens to T lymphocytes. We have investigated whether this mechanism might be relevant in leprosy by determining under what conditions human Schwann cells express class I and class II antigens, and whether infection with Mycobacterium leprae affects this expression. The distribution of these antigens was examined on human Schwann cells in dissociated cell cultures derived from human fetal peripheral nerves. We find that both Schwann cells and fibroblastic cells in these cultures normally express class I antigens but not class II antigens. When Schwann cells are infected with live Mycobacterium leprae for 48 h, 73% of Schwann cells phagocytose the bacteria. Mycobacterium leprae prevents 3H-thymidine incorporation into cultured human Schwann cells, but does not affect class I expression in these cells. Treatment of normal and Mycobacterium leprae infected cultures with gamma-interferon for 72 h induces class II expression on most Schwann cells but not on the majority of fibroblastic cells. The fact that human Schwann cells infected with Mycobacterium leprae can be induced by gamma-interferon to express class II antigens suggests that they may be able to present Mycobacterium leprae antigens to T lymphocytes and thus initiate immune responses against the bacteria. We suggest that a failure of this response, such as that seen within nerve trunks in lepromatous leprosy, is caused by deficient class II expression on Schwann cells. This deficiency in class II expression, in turn, may be caused by the reduced gamma-interferon production characteristic of lepromatous leprosy.

Cells, Cultured↗

PubMed1994

Characterization of class II and class III ADP-ribosylation factor genes and proteins in Drosophila melanogaster.

ADP-ribosylation factors (ARFs) are ubiquitous approximately 20-kDa guanine nucleotide-binding proteins that enhance the ADP-ribosyltransferase activity of cholera toxin and are involved in intracellular vesicular transport. Based on size, phylogenetic analysis, amino acid identity, and gene structure, mammalian ARFs fall into three classes (class I, ARF1, -2, and -3; class II, ARF4 and -5; class III, ARF6). A class I ARF had been identified in Drosophila melanogaster. To search for ARFs of other classes in Drosophila, polymerase chain reaction-based techniques were used, resulting in cloning of Drosophila ARF (dARF) II and dARF III with deduced amino acid sequences similar to those of class II and class III mammalian ARFs, respectively. The three Drosophila ARF genes map to different chromosomes and the coding regions have different splicing sites. dARF II mRNA, like ARF I mRNA, is fairly uniformly distributed throughout adult flies, whereas dARF III mRNA is significantly more abundant in heads than in legs or bodies. Recombinant dARF II and dARF III have biochemical and immunological properties similar to those of human ARF5 (hARF5) and hARF6, respectively. These observations are consistent with the conclusion that the three classes of ARFs are present in non-mammalian as well as mammalian species.

ADP-Ribosylation Factor 1↗

PubMed1999

Distinct regulation of HLA class II and class I cell surface expression in the THP-1 macrophage cell line after bacterial phagocytosis.

Expression of HLA and CD1b molecules was investigated in the THP-1 macrophage cell line within 2 weeks following phagocytosis of mycobacteria or Escherichia coli. During the first 2-3 days, cell surface expression of HLA class II and CD1b was drastically down-modulated, whereas HLA class I expression was up-modulated. In the following days both HLA class II and CD1b expression first returned to normal, then increased and finally returned to normal with kinetics similar to that observed for the steadily increased HLA class I. The initial down-modulation of HLA class II and CD1b cell surface antigens was absolutely dependent on phagocytosis of bacteria. Further studies indicated that initial HLA class II cell surface down-modulation (1) was not due to reduced transcription or biosynthesis of mature HLA class II heterodimers, (2) was only partially, if at all, rescued by treatment with IFN-gamma, although both mRNA and corresponding intracellular proteins increased up to sixfold with respect to untreated cells, and (3) resulted in failure of THP-1 cells to process and present mycobacterial antigens to HLA-DR-restricted antigen-specific T cell lines. The existence of a transient block of transport of mature HLA class II heterodimers to the cell surface in the first days after phagocytosis of bacteria may have negative and positive consequences: it decreases APC function early but it may increase it later by favoring optimal loading of bacterial antigens in cellular compartments at high concentration of antigen-presenting molecules.

Antigen Presentation↗

PubMed1992

Electroporation of exogenous antigen into the cytosol for antigen processing and class I major histocompatibility complex (MHC) presentation: weak base amines and hypothermia (18 degrees C) inhibit the class I MHC processing pathway.

While endogenous antigens are presented by class I major histocompatibility complex (MHC) molecules, exogenous antigens generally require a means for penetration into the cytosol for processing prior to class I MHC presentation. We have optimized conditions for electroporation as a means to experimentally introduce exogenous antigens into the cytosol, providing a system with a number of advantages for dissecting the class I MHC processing pathway. Presentation was assessed by the response of class I or class II MHC-restricted T hybridoma cells. Essentially instantaneous antigen delivery by electroporation facilitated kinetic analysis of the class I pathway and investigation of the effects of various inhibitors or hypothermic conditions on class I MHC antigen processing. This pathway was inhibited by weak base amines (e.g. chloroquine and NH4Cl), cycloheximide, and hypothermia (18 degrees C, which inhibits certain intracellular vesicular processing pathways). The electroporation technique provides a simple, consistent approach for rapid cytosolic antigen delivery for analysis of class I MHC processing.

Ammonium Chloride↗

PubMed2006

MHC class I and class II molecules are expressed in both human and mouse prostate tumor microenvironment.

BACKGROUND: There has been a determined search for therapies specifically aimed at eradicating tumor cells while leaving normal host cells unaffected. This goal can potentially be accomplished by engaging tumor antigen-specific T-cell repertoire to attack the tumor. A pre-requisite for a successful T-cell-mediated attack against tumors or pathogens is that the target tissues express major histocompatibility complex (MHC) molecules. Using newer anti-MHC class I and MHC class II antibody reagents, we re-examined the expression of MHC in both human and mouse prostate tumors and their microenvironments. METHODS: Using immunocytochemistry, we examined the expression of MHC class I, class II, and CD3 molecules on cryopreserved human and mouse prostate tumor samples. RESULTS: MHC class I molecules are expressed by the entire spectrum of different stages of both human and mouse prostate tumor cells. Additionally, cells of the hematopoietic lineage, dispersed in the tumor microenvironment, showed significant expression of MHC class II molecules. Human prostate tumors also show a significant infiltrate of CD3+ T cells. CONCLUSIONS: Expression of MHC class I and class II molecules within the prostate tumor microenvironment are consequential for T-cell-mediated immunotherapeutic approaches against prostate cancer.

Aged↗

PubMed2005

Major histocompatibility genes in the Lake Tana African large barb species flock: evidence for complete partitioning of class II B, but not class I, genes among different species.

The 16 African 'large' barb fish species of Lake Tana inhabit different ecological niches, exploit different food webs and have different temporal and spatial spawning patterns within the lake. This unique fish species flock is thought to be the result of adaptive radiation within the past 5 million years. Previous analyses of major histocompatibility class II B exon 2 sequences in four Lake Tana African large barb species revealed that these sequences are indeed under selection. No sharing of class II B alleles was observed among the four Lake Tana African large barb species. In this study we analysed the class II B exon 2 sequences of seven additional Lake Tana African large barb species and African large barbs from the Blue Nile and its tributaries. In addition, the presence and variability of major histocompatibility complex class I UA exon 3 sequences in six Lake Tana and Blue Nile African large barb species was analysed. Phylogenetic lineages are maintained by purifying or neutral selection on non-peptide binding regions. Class II B intron 1 and exon 2 sequences were not shared among the different Lake Tana African large barb species or with the riverine barb species. In contrast, identical class I UA exon 3 sequences were found both in the lacustrine and riverine barb species. Our analyses demonstrate complete partitioning of class II B alleles among Lake Tana African large barb species. In contrast, class I alleles remain for the large part shared among species. These different modes of evolution probably reflect the unlinked nature of major histocompatibility genes in teleost fishes.

Alleles↗

PubMed2000

Nonlinkage of major histocompatibility complex class I and class II loci in bony fishes.

In tetrapods, the functional (classical) class I and class II B loci of the major histocompatibility complex (Mhc) are tightly linked in a single chromosomal region. In an earlier study, we demonstrated that in the zebrafish, Danio rerio, order Cypriniformes, the two classes are present on different chromosomes. Here, we show that the situation is similar in the stickleback, Gasterosteus aculeatus, order Gasterosteiformes, the common guppy, Poecilia reticulata, order Cyprinodontiformes, and the cichlid fish Oreochromis niloticus, order Perciformes. These data, together with unpublished results from other laboratories suggest that in all Euteleostei, the classical class I and class II B loci are in separate linkage groups, and that in at least some of these taxa, the class II loci are in two different groups. Since Euteleostei are at least as numerous as tetrapods, in approximately one-half of jawed vertebrates, the class I and class II regions are not linked.

Animals↗