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Results for “Functional characterization”

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PubMed2005

Functional characterization of the mouse [corrected] solute carrier, SLC41A2.

We have recently demonstrated that the human solute carrier, SLC41A1, is a Mg 2+ transporter that is regulated by extracellular magnesium. A BLAST search found a closely related protein encoded by SLC41A2 that may have related functional properties. In order to determine the function of SLC41A2, the corresponding cRNA was expressed in Xenopus laevis oocytes and Mg2+ currents were determined under voltage-clamp conditions. Further, real-time RT-PCR was performed to determine if SLC41A2 expression is regulated by magnesium. When expressed in oocytes, SLC41A2 mediates voltage-dependent and saturable Mg2+ uptake with a Michaelis constant of 0.34+/-0.05 mM. Expressed SLC41A2 transports a range of other divalent cations: Ba2+, Ni2+, Co2+, Fe2+, or Mn2+, but not Ca2+, Zn2+, or Cu2+. Mg2+ transport was inhibited by large concentrations of Ca2+. Real-time reverse transcription polymerase chain reaction of RNA isolated from renal distal tubule epithelial (MDCT) cells cultured in low-magnesium media relative to normal media and in kidney cortex of mice maintained on low-magnesium diets compared to those animals consuming normal diets showed that SLC41A2 transcript, unlike SLC41A1 mRNA, is not responsive to magnesium. These studies suggest that SLC41A2 is a Mg2+ transporter that might be involved in magnesium homeostasis in epithelial cells.

Animals↗

PubMed2005

Functional characterization of monocyte-derived dendritic cells generated under serumfree culture conditions.

The culture of human monocyte-derived dendritic cells (DCs) is typically performed in media containing human or fetal calf serum, supplements with the potential to influence the cells phenotype and their functional properties. Published clinical trails based on serumfree cultured DCs reported the use of the commercially available medium AIMV. In this study, we directly compared DCs generated in AIMV medium ("AIMV/sf-DCs") with DCs generated in RPMI supplemented with 2% human serum ("RPMI/HS-DCs") in functional assays of potential relevance for vaccine application. Using TNF-alpha/PGE(2)/IL-1beta/IL-6 as maturation stimulus, AIMV/sf-DCs revealed to be comparable with RPMI/HS-DCs with regard to phenotypic expression of maturation markers, survival in vitro, migratory capacity and stimulation of lymphocyte proliferation except for CD1a which was expressed on a fraction of DCs only when cultured in serumfree AIMV medium. However, IL-12p70 production in response to Toll-like receptor (TLR) stimulating agents plus IFN-gamma was consistently lower in AIMV medium although also under serumfree culture conditions, nanogram quantities of IL-12 were produced. Together, DCs with functional characteristics important for in vivo application can be generated under defined serumfree conditions; however, medium and/or serum conditions appear to have strong influence on the production of relevant T cell differentiating cytokines.

Antigens, CD1↗

PubMed1999

Functional characterization and expression of thalamic GABA(B) receptors in a rodent model of Parkinson's disease.

Increased GABAergic neurotransmission of the basal ganglia output nuclei projecting to the motor thalamus is thought to contribute to the pathophysiology of Parkinson's disease. We investigated the functional role of thalamic GABA(B) receptors in a rodent model of Parkinson's disease. First, we examined the effects of blockade of GABA(B) receptors in the ventromedial thalamic nucleus of rats with a unilateral 6-OHDA lesion of the substantia nigra on locomotor activity. In addition we studied the expression of GABA(B) receptor mRNA in the basal ganglia and thalamus using in situ hybridisation. Unilateral microinjections of the GABA(B) receptor antagonist 2-hydroxysaclofen into the ventromedial thalamic nucleus ipsilateral to the nigrostriatal lesion induced contralateral rotations in a dose-dependent manner. However, microinjection of antagonists with higher affinity for the GABA(B) receptor SCH 50911, CGP 56433 and CGP 55845 did not result in rotational behaviour, but did induce convulsions at higher doses. GABA(B) receptor mRNA expression was found throughout the basal ganglia and thalamus, including the ventromedial thalamic nucleus. No statistically significant differences in GABA(B) mRNA expression were observed in the ventromedial thalamic nucleus following a unilateral 6-OHDA lesion of the substantia nigra. These results make it improbable that thalamocortical GABA(B) receptors play an important role in the pathophysiology of parkinsonism. Therefore, GABA(B) receptors do not appear to be a promising target for novel antiparkinsonian drugs.

Adrenergic Agents↗

PubMed2000

Structure of the murine Pit1 phosphate transporter/retrovirus receptor gene and functional characterization of its promoter region.

The Pit1 phosphate transporter (formerly also called Glvr-1) probably plays an important role in regulated phosphate handling in bone-forming cells. In this study, we describe the structure of the mouse Pit1 gene, as well as some functional characteristics of its promoter region in murine bone cells. Screening of a genomic library led to the isolation of two overlapping lambda clones containing 7kb of 5' flanking region, as well as the 10 exons of the mouse Pit1 gene corresponding to the published cDNA. The translation start site is located within exon I and the stop codon within exon X. The overall structure of the mouse gene is very similar to that of its human homolog, except for the presence of an additional 5' untranslated exon in human. The structure of the 5' untranslated region of the mouse gene was thus further investigated using rapid amplification of cDNA ends in murine ATDC5, MC3T3-E1 and Swiss 3T3 cells. The results indicate that, compared to the published cDNA, the mouse Pit1 gene contains in fact one additional 5' exon, which we named exon IA. Reporter gene assays demonstrate the presence of a functional TATA box containing promoter upstream of exon IA. This description of the murine Pit1 gene and of its promoter region paves the way to more detailed analyses concerning the regulation of Pit1 transcription in mouse cells. Furthermore, a comparison of mouse and human promoters will hopefully allow a better understanding of general mechanisms regulating Pit1 expression in different species.

3T3 Cells↗

PubMed1997

Functional characterization of the 5'-flanking region of the gene encoding human 2-oxoglutarate dehydrogenase.

The 5'-flanking region of the gene (OGDH) encoding human 2-oxoglutarate dehydrogenase (OGDH) does not contain TATA or CAAT boxes, but contains an inverted GC box. To identify a functional OGDH promoter, systematic transient expression analysis of the 5'-flanking region (wild type, -3276/+212) of the OGDH was performed using serially nested deletions and linker-scanning mutations. The OGDH (wild type)-luciferase (LUC) reporter vector (pGV-E) construct and its deletion or linker-mutant constructs were transfected into BHK-21 cells and LUC activity was assessed. The OGDH-LUC construct expressed reproducibly >12-fold more LUC activity than did a control pGV-E vector. The promoter activity was up-regulated by treatment with 2-oxoglutarate and 2-oxoglutarate/glutamate. Deletions of sequences between nt -563 and -62 (M6 and M7) resulted in a 2-fold increase in LUC activity. Further deletion of the sequence between nt -61 and +212 (M8-M10) abolished LUC activity. High resolution mutagenesis within the -113 to -14 region indicated that the -53 to -44 and -33 to -24 sequences were required for positive regulation and the -93 to -84 sequence for negative regulation. We have identified a nuclear factor that binds to nt -63 to -24 including two cis-acting sites.

Animals↗

PubMed2001

Endocardial expression and functional characterization of endothelin-1.

Endothelin-1 (ET-1), a 21 amino acid peptide exerts a wide range of biological activities including vasoconstriction, mitogenesis and inotropic effects on the heart. In this study, we examined whether endocardial endothelial cells express ET-1 and evaluated its functional properties. Using immunofluorescence localization method, we demonstrated cytoplasmic staining of ET-1 in the human endocardial endothelial cells from the right atrium and left ventricle. Employing reverse transcriptase polymerase chain reaction (RT-PCR) expression of ET-1 mRNA and its receptors ET(A) and ET(B) mRNAs were found in human myocardial as well as in endocardial endothelial cells. Biological activity of endocardial endothelial cells derived ET-1 was established as the conditioned media obtained from cultured porcine endocardial endothelial cells induced a slowly developing, strong and long-lasting contraction of circular rat aortic segments, with similar characteristics to that obtained with exogenous ET-1. Furthermore, the selective endothelin-A receptor antagonist, FR 139317, blocked the conditioned media induced contractions. Our results suggest that endocardial endothelial cells express and release biologically active ET-1 which could play a pivotal role in the regulation of myocardial contractility as well as a circulatory peptide may further act in other peripheral target organs.

Animals↗

PubMed2000

Molecular and functional characterization of a family of rat brain T-type calcium channels.

Voltage-gated calcium channels represent a heterogenous family of calcium-selective channels that can be distinguished by their molecular, electrophysiological, and pharmacological characteristics. We report here the molecular cloning and functional expression of three members of the low voltage-activated calcium channel family from rat brain (alpha(1G), alpha(1H), and alpha(1I)). Northern blot and reverse transcriptase-polymerase chain reaction analyses show alpha(1G), alpha(1H), and alpha(1I) to be expressed throughout the newborn and juvenile rat brain. In contrast, while alpha(1G) and alpha(1H) mRNA are expressed in all regions in adult rat brain, alpha(1I) mRNA expression is restricted to the striatum. Expression of alpha(1G), alpha(1H), and alpha(1I) subunits in HEK293 cells resulted in calcium currents with typical T-type channel characteristics: low voltage activation, negative steady-state inactivation, strongly voltage-dependent activation and inactivation, and slow deactivation. In addition, the direct electrophysiological comparison of alpha(1G), alpha(1H), and alpha(1I) under identical recording conditions also identified unique characteristics including activation and inactivation kinetics and permeability to divalent cations. Simulation of alpha(1G), alpha(1H), and alpha(1I) T-type channels in a thalamic neuron model cell produced unique firing patterns (burst versus tonic) typical of different brain nuclei and suggests that the three channel types make distinct contributions to neuronal physiology.

Alternative Splicing↗

PubMed1995

Lagging-strand origins of the promiscuous plasmid pMV158: physical and functional characterization.

The streptococcal plasmid pMV158 replicates by a rolling circle mechanism, which involves the generation of single-stranded plasmid DNA intermediates. This plasmid has the unique feature of having two lagging-strand origins of replication. One of these origins, termed ssoU, is functional in Streptococcus pneumoniae and in Bacillus subtilis in an orientation-dependent manner. The other origin, ssoA, is only functional in the former host. RNA polymerase seems to be involved in the initiation of the conversion of single- to double-stranded plasmid DNA from both ssoA and ssoU. Mutational and deletion analyses have allowed us to define ssoA as being within a highly structured, non-coding 199 bp region. Within this region, two elements which are conserved in several rolling-circle replicating plasmids are located, the recombination site RSB and a 6 base consensus sequence. Both elements may play a role in the conversion of single- to double-stranded plasmid DNA.

Bacillus subtilis↗

PubMed2005

Functional characterization of rat rho2 subunits expressed in HEK 293 cells.

GABA(C) receptors are thought to be homo- or heteropentamers composed of rho1, rho2 and rho3 subunits. Previous work on rat rho2 subunits expressed in Xenopus oocytes has suggested that they do not form functional homo-oligomeric GABA(C) receptors, but do combine with rho1 or rho3 subunits to form hetero-oligomers. These findings are difficult to interpret because both human and mouse rho2 subunits do form functional homo-oligomeric receptors. Also, many regions of the rat brain express solely rho2 subunit transcripts which, according to presently available evidence, would not result in expression of functional GABA(C) receptors. We show here that homomeric rat rho2 receptors can be expressed in HEK 293 cells. Homo-oligomeric rat rho2 receptors expressed in mammalian cells matured slowly and displayed small but detectable GABA-induced currents with slow kinetics. Rat rho2 receptors also had a decreased sensitivity to picrotoxin and a marked sensitivity to the GABA(C) receptor agonist cis-aminocrotonic acid. Our results demonstrate for the first time the expression of functional homomeric rat rho2 receptors, and suggest that rho(2) subunits may contribute to brain function, including in areas not expressing other rho subunits.

Animals↗

PubMed2005

Molecular, structural, and functional characterization of Alzheimer's disease: evidence for a relationship between default activity, amyloid, and memory.

Alzheimer's disease (AD) and antecedent factors associated with AD were explored using amyloid imaging and unbiased measures of longitudinal atrophy in combination with reanalysis of previous metabolic and functional studies. In total, data from 764 participants were compared across five in vivo imaging methods. Convergence of effects was seen in posterior cortical regions, including posterior cingulate, retrosplenial, and lateral parietal cortex. These regions were active in default states in young adults and also showed amyloid deposition in older adults with AD. At early stages of AD progression, prominent atrophy and metabolic abnormalities emerged in these posterior cortical regions; atrophy in medial temporal regions was also observed. Event-related functional magnetic resonance imaging studies further revealed that these cortical regions are active during successful memory retrieval in young adults. One possibility is that lifetime cerebral metabolism associated with regionally specific default activity predisposes cortical regions to AD-related changes, including amyloid deposition, metabolic disruption, and atrophy. These cortical regions may be part of a network with the medial temporal lobe whose disruption contributes to memory impairment.

Adolescent↗

PubMed2003

The UV responsive elements in the human mimecan promoter: a functional characterization.

PURPOSE: A major environmental stress encountered by humans is solar UV light, which can cause a spectrum of eye diseases, such as photokeratitis, cataract, pterygia, and ocular neoplasms. Mammalian defense mechanisms in response to adverse effects of UV light result in induction of a number of genes. Studies on the transcriptional regulation of genes that are expressed in the eye will increase understanding of both the physiological functions of these genes in the mammalian UV response, and the molecular bases for abnormalities associated with the above diseases. Mimecan is an extracellular matrix proteoglycan that is abundantly expressed in the cornea. The purpose of this study was to determine and characterize the UV responsive regulatory elements of the human mimecan promoter. METHODS: Transcriptional activity of the promoter was evaluated, before and after UV irradiation, using transient transfection of human mimecan promoter/luciferase reporter constructs into corneal keratocytes and non-corneal cells. Site directed mutagenesis and corresponding functional assays were used to determine the contribution of UV responsive regions to human mimecan transcription. Co-transfection experiments were used to investigate the role of transcription factors that bind these elements in the promoter and mediate the UV response. mRNA expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The shortest promoter construct that was strongly activated following UV irradiation contained three initiator elements, an E-box element that is conserved between species, and the entire first intron of the human mimecan gene. Deletion of the intronic p53 binding site from this construct considerably diminished transcription and the UV response of the promoter. Surprisingly, deletion of the E-box sequence from this construct completely abolished both transcription and UV response of the promoter. These results demonstrated that the E-box is essential to transcription of the human mimecan gene and also is required for activation by p53. The role of the E-box, and the E-box binding protein, USF-1, in transcription and UV responses of the human mimecan promoter were confirmed by co-transfection experiments using dominant negative transcription factor, A-USF. In addition to these positive regulators, we demonstrate that the region between nucleotides -1314 and -1907 contains a transcriptional repressor site that is active in a time dependent manner following UV irradiation. Finally, we show that UV irradiation results in changes in mimecan mRNA levels in bovine corneal keratocytes in a time-dependent manner. CONCLUSIONS: The human mimecan promoter contains several UV responsive regulatory elements that are conserved between human and bovine species and include the intronic p53 DNA binding site, the E-box in the proximal promoter, and the region between nucleotides -1314 and -1907. The E-box plays an important role in transcription and UV response of the human mimecan promoter. UV irradiation modulates expression of mimecan mRNA in bovine corneal keratocytes and non-corneal cells.

Animals↗

PubMed1999

Functional characterization of soluble and membrane-bound forms of vaccinia virus complement control protein (VCP).

Vaccinia virus secretes a 35 kD protein, vaccinia virus complement control protein (VCP), that inhibits the classical and alternative pathways of complement at several points, indicating that it may be a viral analogue of human complement receptor type 1 (CR1; CD35). Structurally, however, CR1 is composed of 30 short consensus repeats (SCRs), whereas VCP consists entirely of four SCRs. We have begun a structure-function analysis of VCP to define the minimum number of SCRs necessary for function, the functional differences between VCP and CR1, and the potential therapeutic roles for VCP. We addressed these questions by creating and characterizing recombinant soluble and membrane-bound forms of VCP. We have determined that (1) VCP requires all four SCRs to bind C3b, (2) whereas CR1 binds C3b and iC3b, VCP binds C3b but not iC3b, and (3) although normally secreted, if expressed on the membrane of mammalian cells, VCP effectively protects the cells from complement-mediated lysis. Thus, VCP appears to be a compact and unique complement regulatory protein with the ability to inhibit both arms of the complement cascade, but lacking affinity for iC3b. By releasing rather than capturing iC3b-bearing complexes following inactivation of C3b, VCP may 'recycle' its active site locally among infected cells, and thereby enable the virus to evade more efficiently host immune and inflammatory responses. The unique function, compact structure, and capacity of VCP to protect mammalian cells from complement-mediated attack, suggests that it could be used both to better understand the structure-function relationship of complement regulatory proteins, in general, and also to rationally design and develop novel therapeutic agents.

Animals↗

PubMed2002

Overexpression and functional characterization of an ABC (ATP-binding cassette) transporter encoded by the genes drrA and drrB of Mycobacterium tuberculosis.

The genes encoding ATP-binding cassette (ABC) transporters occupy 2.5% of the genome of Mycobacterium tuberculosis. However, none of these putative ABC transporters has been characterized so far. We describe the development of expression systems for simultaneous expression of the ATP-binding protein DrrA and the membrane integral protein DrrB which together behave as a functional doxorubicin efflux pump. Doxorubicin uptake in Escherichia coli or Mycobacterium smegmatis expressing DrrAB was inhibited by reserpine, an inhibitor of ABC transporters. The localization of DrrA to the membrane depended on the simultaneous expression of DrrB. ATP binding was positively regulated by doxorubicin and daunorubicin. At the same time, DrrB appeared to be sensitive to proteolysis when expressed alone in the absence of DrrA. Simultaneous expression of the two polypeptides was essential to obtain a functional doxorubicin efflux pump. Expression of DrrAB in E. coli conferred 8-fold increased resistance to ethidium bromide, a cationic compound. 2',7'-bis-(2-Carboxyethyl)-5(6)-carboxyfluorescein (BCECF), a neutral compound, also behaved as a substrate of the reconstituted efflux pump. When expressed in M. smegmatis, DrrAB conferred resistance to a number of clinically relevant, structurally unrelated antibiotics. The resistant phenotype could be reversed by verapamil and reserpine, two potent inhibitors of ABC transporters.

ATP-Binding Cassette Transporters↗

PubMed1997

Structural and functional characterization of a recombinant PorB class 2 protein from Neisseria meningitidis. Conformational stability and porin activity.

An outer membrane PorB class 2 protein from Neisseria meningitidis has been overexpressed in Escherichia coli, isolated from inclusion bodies, and refolded in the presence of zwitterionic detergent. The purified recombinant and native (strain M986) counterpart exhibit most of the typical functional and structural properties that are characteristic of bacterial porins. Channel forming activity has been monitored by incorporating class 2 into reconstituted liposomes and measuring the permeation rates of various oligosaccharides through the proteoliposomes to derive a pore diameter of approximately 1.6 nm. Structural studies employing a combination of spectroscopic and electrophoretic techniques reveal that recombinant and native class 2 are identical in terms of overall conformational stability. Both proteins form stable trimers in zwitterionic detergent and retain significant secondary and tertiary structure in the presence of SDS. The thermal unfolding of zwittergen-solubilized class 2 trimers (Tm = 88 degrees C) is reversible and characterized by solvent exposure of aromatic residues with concomitant disruption of tertiary and partial loss of secondary structures. SDS-induced destabilization and irreversible unfolding of the native trimeric assembly occurs at temperatures above 60 degrees C. Our physicochemical studies of PorB class 2 protein furnish significant insight regarding the structural and functional properties of this meningococcal outer membrane protein within the porin superfamily.

Amino Acid Sequence↗

PubMed2004

Reconstruction and functional characterization of the human mitochondrial metabolic network based on proteomic and biochemical data.

Diverse datasets including genomic, proteomic, isotopomer, and DNA sequence variation are becoming available for human mitochondria. Thus there is a need to integrate these data within an in silico modeling framework where mitochondrial biology and related disorders can be studied and analyzed. This paper reports a reconstruction and characterization of the human mitochondrial metabolic network based on proteomic and biochemical data. The 189 reactions included in this reconstruction are both elementally and charge-balanced and are assigned to their respective cellular compartments (mitochondrial, cytosol, or extracellular). The capabilities of the reconstructed network to fulfill three metabolic functions (ATP production, heme synthesis, and mixed phospholipid synthesis) were determined. Network-based analysis of the mitochondrial energy conversion process showed that the overall ATP yield per glucose is 31.5. Network flexibility, characterized by allowable variation in reaction fluxes, was evaluated using flux variability analysis and analysis of all of the possible optimal flux distributions. Results showed that the network has high flexibility for the biosynthesis of heme and phospholipids but modest flexibility for maximal ATP production. A subset of all of the optimal network flux distributions, computed with respect to the three metabolic functions individually, was found to be highly correlated, suggesting that this set may contain physiological meaningful fluxes. Examinations of optimal flux distributions also identified correlated reaction sets that form functional modules in the network.

Amino Acids↗

PubMed1993

Preparation and functional characterization of a catalytically active fragment of phosphorylase kinase.

Limited proteolysis of rabbit muscle phosphorylase kinase catalyzed by chymotrypsin generates a 33 kD product whose kinase activity is independent of both calcium and pH over the range of 6.8 to 8.3 (Malencik, D.A. & Fischer, E.H. Calcium and Cell Function III: 161-188, 1982). This active preparation consists of three related species containing residues 1-290, 1-296, and 1-298 of the 44.7 kD gamma-subunit of phosphorylase kinase (Harris, W.R., Malencik, D.A., Johnson, C.M., Carr, S.A., Roberts, G.D., Byles, C.E., Anderson, S.R., Heilmeyer, L.M.G., Fischer, E.H. & Crabb, J.W.J. Biol. Chem. 265:11740-11745, 1991). Good recoveries of catalytic activity--with varying degrees of calcium dependence--result upon the digestion of phosphorylase kinase with assorted proteases. However, especially high yields of the chymotryptic fragment are obtainable, with purification on an Ultrogel-34 column and a DEAE Sepharose CL-6B column giving 23% of the maximum possible protein. Physical characterization shows that the 33 kD chymotryptic fragment is globular, with S20,w = 2.9S, and that it has an isoelectric point of 5.3. Our continuous catalytic assay, based on differences in the binding of the fluorescent dye 1-anilinonaphthalene-8-sulfonate by phosphorylase a and b, shows that, on a molar basis, the activity of the fragment is 2.8 fold greater than that of phosphorylase kinase (Malencik, D.A., Zhao, Z. and Anderson, S.R. Biochem. Biophys. Res. Comm. 174: 344-350, 1991). The active fragment also undergoes autophosphorylation. Incubation with Mg[gamma-P32] ATP results in the reaction of 0.7 mol 32P/mol fragment. When the catalytic subunit of the cAMP-dependent protein kinase is also present, the amount of 32P incorporated increases to 1.1 mol/mol. In the former case, phosphorylation occurs primarily at Ser30 while in the latter an additional reaction takes place at Ser81. The phosphopeptides correspond to sequences occurring in the gamma-subunit of phosphorylase kinase.

Amino Acid Sequence↗

PubMed1999

Functional characterization of a novel hematopoietic stem cell and its place in the c-Kit maturation pathway in bone marrow cell development.

While the majority of purified pluripotential hematopoietic stem cells (PHSC) express c-Kit, the receptor for steel factor, we have phenotypically and functionally separated a distinct class of PHSC that does not express c-Kit. In contrast to c-Kit-positive (c-Kit(pos)) PHSC, the c-Kit-negative (c-Kit(neg)) PHSC do not proliferate in response to multiple hematopoietic growth factors in vitro and do not radioprotect or form macroscopic spleen colonies (CFU-s) when transplanted into lethally irradiated recipients. However, the c-Kit(neg) PHSC show delayed or slow reconstitution kinetics when cotransplanted with radioprotective bone marrow cells. c-Kit(neg) PHSCs cells can give rise to c-Kit(pos) cells with CFU-s activity, radioprotective activity, and PHSC activity. Thus, constitutive hematopoiesis is maintained by c-Kit(pos) PHSCS cells that are recruited from a more primitive quiescent c-Kit(neg) PHSC population, which represents a critical developmental stage in definitive hematopoiesis.

Animals↗

PubMed1983

Serologic and functional characterization of a panel of antigen-presenting cell lines expressing mutant I-A class II molecules.

An improved method is described for selecting mutant cells with an altered pattern of Ia antigenic determinants and antigen-presenting properties from an homogeneous population of functional antigen-presenting cells (APC). The APC line used, TA3, was a somatic cell hybrid obtained by fusing normal heterozygous H-2a/d B cells with a drug-marked variant of a BALB/c B lymphoma line. Two phenotypic groups of mutants were obtained by this method. Serologic analysis with a panel of anti-I-Ak monoclonal antibodies suggested that the change in the first group of mutants (type A mutants) involved the alteration of a portion of one epitope of the I-Ak molecule while in the second group of mutants (type B), an alteration of a different Ia epitope group had occurred. Functional studies using a panel of cloned antigen-specific and autoreactive T cell hybridomas demonstrated that the loss of a limited number of I-Ak determinants in the type A mutants correlated with the loss of some but not all I-Ak-encoded restriction elements, while the type B mutation(s) resulted in the ablation of all I-Ak-restricted APC functions tested. These mutations may occur in the region of the Ia molecule that interacts with the T cell receptor (the histope) or in a postulated region that interacts with antigen (the desetope). The finding that both type A and B mutations lead to loss in the capacity to be corecognized with many different antigens by I-Ak-restricted T cell hybridomas suggests that the Ia molecule may possess very few distinct histotopes and/or desetopes or that the tertiary structure of the Ia molecule is crucial in the formation of these sites. Alternatively, the mutations, particularly the type B mutations, may have led to the failure of expression of an entire alpha or beta chain.

Animals↗