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At least 307 records · Page 17Linked to original sources
PubMed1993

Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency (or bare lymphocyte syndrome).

Hereditary major histocompatibility complex (MHC) class II deficiency (or bare lymphocyte syndrome) is a form of severe primary immunodeficiency with a total lack of MHC class II expression. It is due to a defect in the regulation of MHC class II genes. A novel gene was isolated by complementation cloning, using an MHC class II-negative mutant cell line. This gene (CIITA) functions as a transactivator of MHC class II gene expression and restores expression of all MHC class II isotypes in mutant cells. In addition, CIITA fully corrects the MHC class II regulatory defect of cells from patients with bare lymphocyte syndrome. In this disease we have identified a splicing mutation that results in a 24 amino acid deletion in CIITA, resulting in loss of function of the transactivator. Hence, the CIITA gene is essential for MHC class II gene expression and has been shown to be responsible for hereditary MHC class II deficiency.

Amino Acid Sequence↗
PubMed1996

Stat1 alpha expression is involved in IFN-gamma induction of the class II transactivator and class II MHC genes.

Class II MHC Ags are critical in the regulation of immune responses by presenting Ag to T lymphocytes, resulting in their activation and differentiation. Class II expression is rare in the normal central nervous system, but elevated expression on glial cells has been observed in several neurologic diseases. We have previously demonstrated that IFN-gamma-induced class II expression in glial cells involves activation of both tyrosine kinase and protein kinase C. IFN-gamma induces tyrosine phosphorylation of the tyrosine kinases Jak1 and Jak2 and of Stat1 alpha. In addition, IFN-gamma enhances expression of Stat1 alpha mRNA and protein. We utilized antisense oligonucleotides against Stat1 alpha to determine directly whether IFN-gamma-induced activation and/or enhancement of Stat1 alpha is involved in class II expression. Antisense oligonucleotides complementary to Stat1 alpha mRNA were introduced in CH235-MG astroglioma cells by transient transfection; such treatment inhibited both constitutive and IFN-gamma-enhanced expression of Stat1 alpha. IFN-gamma-induced class II MHC expression was also inhibited in cells exposed to Stat1 alpha antisense oligonucleotides. The fact that the class II promoter does not contain IFN-gamma-activated sequences for binding Stat1 alpha suggests that Stat1 alpha must activate another protein that is directly involved in class II expression. A likely candidate is the class II MHC transactivator (CIITA). IFN-gamma induction of CIITA mRNA was also inhibited in cells treated with antisense oligonucleotides against Stat1 alpha. These findings demonstrate that Stat1 alpha is involved in IFN-gamma induction of CIITA expression, resulting in class II MHC expression.

Astrocytes↗
PubMed1997

Identification of residues in the class II-associated Ii peptide (CLIP) region of invariant chain that affect efficiency of MHC class II-mediated antigen presentation in an allele-dependent manner.

Invariant chain (Ii) associates with class II MHC molecules and is crucial for Ag presentation by class II molecules. A general explanation for how invariant chain (Ii) associates with polymorphic MHC class II molecules has been suggested by the crystallographic structure of CLIP (class II-associated Ii peptide) complexed with an HLA class II molecule, HLA-DR3. We show here that methionine residues at positions 93 and 99 in Ii are important in MHC class II-mediated Ag presentation, but function in an allele-dependent manner. Introduction of a Met-->Ala mutation at position 99 in Ii (M99AIi) impaired presentation of peptides derived from exogenous proteins by I-Ad and I-Au class II molecules. Mutating Met-->Ala in Ii at position 93 (M93AIi) abrogated presentation by I-Au molecules, but not by I-Ad. Impaired Ag presentation was associated with conformationally altered expression of I-A molecules on the surface of cells expressing mutated Ii. Cell surface CLIP staining and immunoprecipitation studies showed that both I-Ad and I-Au molecules were associated with an increased abundance of Ii peptides, CLIP, in cells expressing mutated Ii. These results show that methionine 93 and methionine 99 play an important physiologic role in Ii association with class II molecules by regulating release of CLIP from class II in the endocytic compartments to allow binding of cognate peptides.

Alanine↗
PubMed1990

Reconstitution of H-2 class I expression by gene transfection decreases susceptibility to natural killer cells of an EL4 class I loss variant.

Several reports have suggested that an inverse correlation exists between major histocompatibility complex class I expression and the susceptibility to natural killer (NK)-mediated lysis. For example, the increased class I expression induced by interferon-gamma was always accompanied by an increased resistance to NK lysis. Likewise, class I loss variants were often more NK susceptible than their normal counterparts. To investigate whether the inverse correlation between class I expression and NK susceptibility was fortuitous or whether the class I molecules were directly responsible for this effect we resorted to gene transfection studies. From the murine thymoma line EL4 and H-2Db- and Kb-negative variant S3 was selected. This variant was highly susceptible to NK lysis. S3 was found to have a defect in beta 2-microglobulin gene expression. Therefore, restoration of Db and Kb expression could be achieved by transfection with the beta 2-microglobulin gene. This resulted in a strong decrease in susceptibility to NK lysis to the level of the H-2+ parental EL4. Transfection with class II genes had no effect. Blocking of the class I molecules on the H-2+ cells with anti-H-2b F(ab')2 fragments increased the susceptibility to NK cells to the level of the H-2- variant S3. These data demonstrate that the class I molecules on the targets are directly responsible for regulation of NK susceptibility but the mechanism is not clear. Possibly the class I molecules interfere with the unknown NK target structures.

Animals↗
PubMed1988

Comparison of the effects of class 1 and class 2 heparin-binding growth factors on protein synthesis and actin mRNA expression in BALB/c-3T3 cells.

Biological effects of class 1 or class 2 heparin-binding growth factors (HBGFs) were compared in BALB/c-3T3 cells. Changes in protein synthesis, as monitored by two-dimensional gel electrophoresis, reveal that while both HBGFs induce the same changes in the synthesis of intracellular proteins, class 2 HBGF selectively increases the synthesis of a 43-kD extracellular protein. Heparin, which potentiates the mitogenic activity of class 1 but not class 2 HBGF, does not potentiate the changes in protein synthesis elicited by HBGF-1. Since each HBGF increases actin synthesis, regulation of actin mRNA expression was examined. Actin mRNA levels increase rapidly and transiently in response to either HBGF, and similar superinduction responses are observed in the presence of HBGF and cycloheximide. Although the maximum increase in actin mRNA stimulated by either HBGF is similar, the levels of mRNA induced by class 2 HBGF remain elevated up to 48 hours compared to the level induced by class 1 HBGF. These results imply that in the same cell type class 1 and class 2 HBGFs may modulate some biological effects differently.

Actins↗
PubMed1997

Fluorimetric detection of aldehyde dehydrogenase activity in human blood, saliva, and organ biopsies and kinetic differentiation between class I and class III isozymes.

Two highly fluorogenic aldehydes, 7-methoxy-1-naphthaldehyde (MONAL-71) and 6-methoxy-2-naphthaldehyde (MONAL-62), were examined as indicators of the aldehyde dehydrogenase (ALDH) activity in human tissue homogenates and accessible body fluids. Both compounds were previously found to be excellent substrates for the ALDH from erythrocytes and for the purified class I (cytosolic) ALDH from human liver. By contrast, only MONAL-62, but not the isomeric MONAL-71, was oxidized by class III ALDH present in human saliva. The apparent Km for the former compound reacting with salvia ALDH is 0.24 microM, with the reaction rate (Vmax) close to that of benzaldehyde oxidation. There is also a fully competitive inhibition of the fluorogenic oxidation of the MONAL-62 by benzaldehyde. Both NAD+ and NADP+ can be used as oxidants in this reaction, with comparable rates, a fact previously reported for the human class III aldehyde dehydrogenase. In human liver homogenate (cytosolic + microsomal fraction), the ALDH activity is easily detectable using either MONAL-71 or MONAL-62, with specific activities of approximately 2.5 and 3.2 units per gram of protein, respectively. The low apparent Km values, 0.85 and < 0.03 microM, respectively, together with the inhibition profile by propionic aldehyde (ID50 in the micromolar range) indicate that both compounds are oxidized primarily by the class I ALDH, further confirmed by low activity (0.4 U/g) with NADP+ as oxidant. By contrast, in human stomach, containing mostly class III ALDH, the activity measured with MONAL-71, 0.4 U/g, is much lower than that with MONAL-62 (5.1 U/g with NAD+ and 3.1 U/g with NADP+), the latter being virtually insensitive to 1 mM propionic aldehyde. Hence, in a stomach homogenate, class I and class III ALDH activities can be measured selectively with the two fluorogenic substrates described. In all experiments, the activity of aldehyde oxidase was at least 10-fold lower than that of the ALDH. Addition of 5 mM 4-methylpyrazole, a known inhibitor of the alcohol dehydrogenase, did not change the resultant ALDH activities by more than 10%, indicating lack of interference by the former enzyme. A preliminary screening of two liver tumour samples showed diminished class I ALDH activities (0.7 and 0.03 U/g), but no evidence for class III ALDH induction. The above observations are discussed in relation to the mechanism of detoxication of cyclophosphamide.

Aldehyde Dehydrogenase↗
PubMed1996

Analysis of domain structural class using an automated class assignment protocol.

The extent to which the contemporary dataset of protein structures can be segregated into four structural "classes" as originally defined by Levitt & Chothia in 1976 is examined and a simple method presented for the assignment of protein domains into these classes. Assignments are based on known three-dimensional structures, and for successful assignment it was found that helix/sheet content, contacts between secondary structures and their sequential order had to be used. The procedure attempts to maximise the automatic separation into classes for a dataset of 197 manually classified, non-homologous domains. It was found that approximately 90% of the structures were classified automatically; the remainder were borderline and were left for manual inspection. The method was then applied to a test set of 43 protein domains with similar results. The data support the concept of distinct classes of protein structure, although a few intermediate structures are found, demonstrating that it is possible to define relatively simple parameters complying with commonly accepted nomenclature that automatically define 90% of protein domains with essentially 100% accuracy. However, re-examination of the data also suggested that the previously separate alpha/beta and alpha + beta classes show considerable overlap and are more naturally represented as a single alpha beta class. This large alpha beta class can then be most easily subdivided by consideration of whether the sheets are mainly parallel, antiparallel or mixed. The correlation between structural class and function is discussed, together with the conservation of class within a sequence superfamily. This represents the first step in an automated phenetic description of protein structure complementing the usual phylogenetic approach to protein structure classification.

Algorithms↗
PubMed1987

An epitope common to HLA class I and class II antigens, Ig light chains, and beta 2-microglobulin.

The homology of class I major histocompatibility complex (MHC) antigens, class II MHC antigens, and immunoglobulin molecules has suggested their divergence from a common ancestral gene. We report here a monoclonal antibody (mAb), PAC.M1, which reacts with HLA class I heavy chains, HLA class II alpha and beta chains, and the light chain of human immunoglobulin by Western blot analysis. PAC.M1 reacted with 44 kd, 33 kd, and 29 kd species when tested on membrane glycoproteins from TRal, a B-lymphoblastoid cell line (B-LCL). Two-dimensional electrophoresis and Western blotting of TRal glycoproteins showed that these species had the appropriate electrophoretic mobilities for class I heavy chain and class II alpha and beta subunits. The presence of the epitope was verified on class II alpha and beta subunits by Western blotting of purified alpha beta-invariant chain complexes, and on class I heavy chains by Western blotting of purified class I antigens. The PAC.M1 mAb also reacted with immunoglobulin light chains when Western blotting was performed with normal human serum and purified IgG and IgM as antigens. While reactivity of the mAb with beta-2 microglobulin (beta 2m) was difficult to detect by Western blotting, binding of PAC.M1 to purified beta 2m was detectable in a solid-phase binding assay. Thus, PAC.M1 reacts with a determinant shared by a number of members of the immunoglobulin superfamily.

Antibodies, Monoclonal↗
PubMed1996

Analysis of forces and moments in arch guided molar protraction using Class I and Class II elastics. An in-vitro study.

The use of class I and II elastics in arch guided tooth movement of the lower molars belongs to the proven clinical methods to achieve space closure even though risks are present. The vertical force component of class II elastics tends to interact with the sagittal force and thus the vertical force may change the desired sagittal force and movement direction. The objective of the study presented here was to investigate friction behavior and the movement dynamics of the arch guided protraction of the lower first molar being acted on by differing class I and class II elastic band geometries. The influence of class I and class II elastics at different force levels (1 N and 2 N) were studied. The pattern of the force line varied in the area of angulation from 0 degree to 40 degrees relative to the arch plane. The orthodontic measurement and simulation system (OMSS) was employed to determine force loss due to friction and to analyze side effects. In the arch guided mesialization of the lower first molar, the vertical component of class II elastics induces a minor force loss in comparison with class I elastics. This holds, however, only for the lower 1 N force level. When employing class II bands at a greater force level and with increased angulation, relatively greater force loss and increased side effects, such as extrusion and mesial tipping of the first molar, occur.

Biomechanical Phenomena↗
PubMed1987

Role of HLA class I and class II antigens in activation and differentiation of B cells.

The monoclonal antibodies (MoAb) CR10-214, CR11-115, and Q1/28 to distinct monomorphic determinants of HLA class I antigens, the MoAb CL413 and PTF29.12 recognizing monomorphic determinants of HLA-DR antigens, the anti-HLA-DQw1 MoAb KS11, the anti-HLA-DPw1 MoAb B7/21, and the anti-HLA-DR,DP MoAb CR11-462 were tested for their ability to modulate human B-lymphocyte proliferation and maturation to IgM-forming cells. Purified tonsillar B cells were stimulated with Staphylococcus aureus bacteria of the Cowan first strain (SAC) or anti-human mu-chain xenoantibodies, as well as in growth factor- or T-cell-dependent activation cultures. The B-cell proliferative responses induced by SAC or by mitogenic concentrations of anti-mu-chain xenoantibodies were inhibited by some of the anti-HLA class I and anti-HLA class II monoclonal antibodies tested. The same antibodies were effective inhibitors of the proliferation of B cells stimulated with interferon-gamma (IFN-gamma) or interleukin-2 (IL-2) and with submitogenic concentrations of anti-mu-chain xenoantibodies. The proliferation induced by IL-2 of SAC-preactivated B cells was inhibited by some of the anti-HLA class II monoclonal antibodies, but not by the anti-HLA class I monoclonal antibodies tested. This inhibition appeared to reflect at least in part a direct effect on later events of the B-cell activation cascade, since some anti-HLA class II monoclonal antibodies still exerted considerable inhibitory activity when added together with IL-2 to SAC-preactivated B cells after the third day of culture. Anti HLA-DR, DQ, and DP monoclonal antibodies consistently inhibited the IgM production induced in B cells by T cells alone, T cells plus pokeweed mitogen (PWM), SAC plus IL-2, or IL-2 alone. In contrast, two of the three anti-HLA class I monoclonal antibodies tested inhibited the IgM production in cultures stimulated with SAC plus IL-2 and one the IgM production induced by IL-2 alone, but none of them had inhibitory effects on T-cell dependent IgM production. The results reported herein indicate that HLA class II molecules directly participate in different phases of the B-cell activation cascade. In addition, our data also suggest that HLA class I molecules can be involved in the events leading to B-cell proliferation and differentiation into immunoglobulin-secreting cells.

Antibodies, Monoclonal↗
PubMed1997

Mapping of tubule and intertubule surface areas available for bonding in Class V and Class II preparations.

OBJECTIVES AND METHODS: The aim of this in vitro study was to evaluate the morphology of dentine in Class V and the Class II preparation walls in terms of tubule orientation, density and increase in surface area after conditioning. Six circular V-shaped preparations were cut at the cementum-enamel junction (CEJ) of anterior teeth and six Class II cavities with the cervical margin 1 mm below the CEJ were prepared in posterior teeth. The preparations were conditioned with 10% maleic acid. The samples were directly studied by SEM. The observations were organized according to location in the preparation, tubule density was counted and the increase in area available for bonding after acid etching was calculated. RESULTS: Dentine morphology of Class V and Class II preparation walls was mostly regular according to predictable patterns. Between the tubules, along the walls of the cavity, the etched dentine surface exhibited a porous network of collagen fibers. More than 50% of Class V and Class II cavity surface presented oblique or parallel tubule orientation with respect to the cut surface. Because of the presence of a structureless cementum layer. the morphology of the cervical area turned out to be less predictable. The tubule density varied considerably in different regions of the preparations. The intertubular dentine surface area increased after conditioning in Class V preparations from 20.9% to 50.3% on the walls where tubules were cut parallel to their long axis, and from 5.8% to 47.8% where tubules were cut perpendicularly. These same values for Class II preparations ranged from 0.6% to 46.4% on the walls where tubules were cut parallel to their long axis, and 29.8% of the tubules were cut perpendicularly. CONCLUSIONS: Based on this morphological investigation, the increase in intertubular dentine surface area might very well be responsible for the enhanced bond strength after acid etching of dentine, but not all areas exhibited equal responses to etching. In particular, the bonding substrate at the gingival margins may contribute little in terms of micromechanical retention.

Acid Etching, Dental↗
PubMed1992

The major piscine liver alcohol dehydrogenase has class-mixed properties in relation to mammalian alcohol dehydrogenases of classes I and III.

The major alcohol dehydrogenase of cod liver has been purified, enzymatically characterized, and structurally analyzed in order to establish original functions and relationships among the deviating classes of the enzyme in mammalian tissues. Interestingly, the cod enzyme exhibits mixed properties--many positional identities with a class III protein, but functionally a class I enzyme--blurring the distinction among the classes of alcohol dehydrogenase. The two domain interfaces, affected by movements upon coenzyme binding, both exhibit substitutions in a manner thus far unique to the cod enzyme. In contrast, coenzyme-binding residues are highly conserved. At the active site, inner and outer parts of the substrate pocket show different extents of amino acid replacement. In total, no less than 7-10 residues of 11 in the substrate binding pocket differ from those of all the mammalian classes, explaining the substrate specificities. However, the inner part of the substrate pocket is very similar to that of the class I enzymes, which is compatible with the observed characteristics of the cod enzyme: ethanol is an excellent substrate (Km = 1.2 mM) and 4-methylpyrazole is a strong inhibitor (Ki = 0.1 microM). These values are about as low as those typical for the ethanol-active class I mammalian enzyme and do not at all resemble those for class III, for which ethanol is hardly a substrate and pyrazole is hardly an inhibitor. Further out in the substrate pocket, several residues differ from the mammalian classes, affecting large substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗
PubMed1991

Amphibian alcohol dehydrogenase, the major frog liver enzyme. Relationships to other forms and assessment of an early gene duplication separating vertebrate class I and class III alcohol dehydrogenases.

Submammalian alcohol dehydrogenase structures can be used to evaluate the origins and functions of the different types of the mammalian enzyme. Two avian forms were recently reported, and we now define the major amphibian alcohol dehydrogenase. The enzyme from the liver of the Green frog Rana perezi was purified, carboxymethylated, and submitted to amino acid sequence determination by peptide analysis of six different digests. The protein has a 375-residue subunit and is a class I alcohol dehydrogenase, bridging the gap toward the original separation of the classes that are observable in the human alcohol dehydrogenase system. In relation to the human class I enzyme, the amphibian protein has residue identities exactly halfway (68%) between those for the corresponding avian enzyme (74%) and the human class III enzyme (62%), suggesting an origin of the alcohol dehydrogenase classes very early in or close to the evolution of the vertebrate line. This conclusion suggests that these enzyme classes are more universal among animals than previously realized and constitutes the first real assessment of the origin of the duplications leading to the alcohol dehydrogenase classes. Functionally, the amphibian enzyme exhibits properties typical for class I but has an unusually low Km for ethanol (0.09 mM) and Ki for pyrazole (0.15 microM) at pH 10.0. This correlates with a strictly hydrophobic substrate pocket and one amino acid difference toward the human class I enzyme at the inner part of the pocket. Coenzyme binding is highly similar, while subunit-interacting residues, as in other alcohol dehydrogenases, exhibit several differences.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗
PubMed1989

Inducible (class 3) aldehyde dehydrogenase from rat hepatocellular carcinoma and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated liver: distant relationship to the class 1 and 2 enzymes from mammalian liver cytosol/mitochondria.

Peptides from rat liver aldehyde dehydrogenase (AIDH) induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment match the AIDH structure from HTC rat hepatoma cells (HTC-AIDH) at all positions examined, indicating induction of the same gene product by two independent routes. This 452 amino acid residue, class 3 AIDH structure differs substantially from the 500-residue AIDH structures isolated from normal liver cytosol (class 1) and mitochondria (class 2). Despite a 29.8% identity in 429 overlapping amino acids vs the human class 1 enzyme (27.7% vs class 2), neither the N- nor C-termini coincide, and gaps are introduced to optimize the alignment. Two residues placed in the active site of human liver AIDH by chemical modification, Cys-302 and Glu-268, are conserved in class 3 AIDH as Cys-243 and Glu-209. Cys-243/302 is the only cysteine residue conserved in all known AIDH structures. Gly-245 and Gly-250 of class 1/2 AIDHs, fitting the patterns of glycine residues in coenzyme binding fold of other dehydrogenases, are also conserved. Otherwise, Cys-49, Cys-162, and Glu-487, to which functional importance has also been ascribed, are not retained in the class 3 structure. Overall, a high conservation of Gly, Pro, and Trp and similar patterns of predicted secondary structure indicate general conservation of tertiary structure, as noted with other distantly related proteins. Three exon boundaries from the human liver mitochondria AIDH gene directly correspond to the N-terminus of the rat class 3 protein and to two of the gaps in the alignment.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehyde Dehydrogenase↗