Ek beta mutant antigen-presenting cell lines expressing altered Ak alpha molecules

Antigen-presenting cells (APC) expressing mutant Ek beta and Ak alpha proteins were isolated after chemical mutagenesis of TA3 cells and negative immunoselection for altered Ek beta molecules. Mutant clones were analyzed for biosynthesis, assembly, and cell surface expression of altered Ia molecules, and were assayed for antigen-presenting function by using a variety of T cell clones. Three types of mutants were detected: type 1, which had lost expression of the Ek beta chain and produced altered Ak alpha chains; type 2, which also expressed altered Ak alpha chains, and which expressed Ek beta proteins that had lost reactivity to the 17.3.3 and 74D monoclonal antibodies (mAb), but retained reactivity to other anti-Ek beta mAb; and type 3, which had lost expression of both Ek beta and Ak beta: Ak alpha surface molecules. Thus, all of the mutant clones that produced modified Ak alpha proteins also displayed either total loss or serologic modification of the Ek beta molecule. Ek beta:E alpha-reactive T cell clones were not stimulated when type 1 or type 3 cells were used as APC, but all such T cells were fully reactive with type 2 mutant APC. Most Ak beta:Ak alpha-reactive T cell clones could respond to type 1 and 2 APC, and none were responsive to type 3 APC. However, two autoreactive Ak beta:Ak alpha-specific T cell hybridomas were stimulated only very weakly by type 1 and type 2 cells expressing modified Ak alpha proteins. These results demonstrate that Ia mutations can have highly selective effects on antigen presentation to T cells as well as on mAb binding, and thus suggest that individual Ia molecules may be composed of many different functional subsites.     

Structural analysis of anti-DR1 allorecognition by using DR1/H-2Ek hybrid molecules. Influence of the beta 2-domain correlates with CD4 dependence.

A segmental analysis of the key regions of HLA-DR1 that control T cell allorecognition was performed by using a series of transfected cell lines expressing the products of recombinant DRB/H-2Eb genes, paired with either DR alpha or H-2E alpha. Four of eight human T cell clones tolerated substitution of the H-2E alpha chain, but only one clone showed any response to the DR alpha/H-2E beta k dimer. Both the membrane-proximal and the membrane-distal domains of the beta-chain played an important part in stimulating these clones. The response of four of eight clones was markedly inhibited by substitution of the H-2E beta 2 for the DR beta 2 domain. This inhibition showed a complete correlation with the sensitivity of the clones to inhibition by anti-CD4 mAb. Taken together, these results suggest that the interaction site for CD4 may include residues on the beta 2-domain. Introduction of H-2Ek sequence into either half of the beta 1-domain led to a complete loss of response by all but two of the clones. This is consistent with these clones having dual specificity for exposed DR1-specific polymorphisms and for DR1-bound peptides. The pattern of response of one of the clones suggested that indirect conformational effects on the alpha 1-domain may also contribute to the influence of the amino-terminal half of the beta 1-domain on T cell recognition. In the presence of H-2E alpha, this clone responded more strongly when the amino-terminal half of the beta 1-domain was of H-2Ek rather than DR1 sequence. This implies that species matching of the floor of the beta 1-domain with the alpha-chain is more important than the presence of the alpha-chain of the parental species.     

Haplotype-specific suppression of T cell response to lactate dehydrogenase B in (responder x nonresponder)F1 mice

Mouse strains that express the Ek (Ek beta E-1k alpha) molecule are nonresponders (NR) to the enzyme lactate dehydrogenase B (LDHB) in terms of T cell proliferation. Nonresponsiveness is caused by T suppressor (Ts) cells recognizing LDHB in the context of Ek molecules on the antigen-presenting cells. The data presented here demonstrate that the Ek-restricted Ts cells function in (R x NR)F1 mice in a remarkable haplotype-specific fashion: they selectively interfere with the Ak (ANR)-restricted response, and do not affect the response channeled through the A molecules of the responder parent. This haplotype-specificity of suppression provides an explanation of the dominance of responsiveness in (R x NR)F1 mice.     

Interactions between the amino-terminal domains of MHC class II molecules have a profound effect on serologic and T cell recognition: an analysis using recombinant HLA-DR/H-2E molecules.

A series of transfected L cell lines were generated expressing the products of wild-type or recombinant HLA-DR1/H-2Ek beta-chain-encoding genes paired to DR alpha or E alpha. The recombinant genes were created by reciprocal exchange of the gene segments encoding the amino (NH2)-terminal and carboxy (COOH)-terminal halves of the beta 1 domain and the beta 2 domain. The majority of the serologic determinants, predicted from the genetic composition of the class II dimers, were expressed indicating that no gross conformational changes were induced by the creation of the interspecies recombinant molecules. Subtle conformational variation was detected by the anti-H-2Eb,k,s mAb Y17. Epitope expression was dependent on the presence of the E alpha-chain and NH2-terminal sequence from the beta 1 domain of H-2Ek. Substitution of DR1 sequence in either region led to loss of recognition by Y17. This pattern of reactivity maps the Y17 epitope either to the E alpha-chain or to an exposed sequence on the fourth strand of the beta sheet of the beta 1 domain. If the Y17 epitope is located on the E alpha-chain this raises the interesting possibility that the conformation of this chain, which is invariant by sequence, may vary according to the beta-chain with which it is coexpressed. The ability of the recombinant class II dimers to present Ag to the pigeon cytochrome c-specific, H-2Ek-restricted T cell hybridoma 2B4 was assessed. Transfected L cells expressing E beta k paired to E alpha or DR alpha presented Ag with equal efficiency, and the beta 2 domain of H-2Ek could be substituted with the equivalent region from DR1 without any loss of response. Wild-type DR1 failed to function as a restriction element, however, substitution of the COOH-terminal portion of the beta 1 domain with the equivalent sequence from H-2Ek was sufficient to produce a partial recovery of Ag recognition. Cells expressing a recombinant beta 1 domain comprising the COOH-terminal sequence from H-2Ek and the NH2-terminal sequence from DR1 presented Ag when paired to DR alpha but failed to do so when paired to E alpha. This indicates that a subtle conformational disturbance caused by mismatching of the NH2-terminal region of the beta-chain and the alpha-chain can have pronounced effects on T cell recognition.(ABSTRACT TRUNCATED AT 400 WORDS)     

The molecular basis of alloreactivity in antigen-specific, major histocompatibility complex-restricted T cell clones

We have studied the relationship between major histocompatibility complex (MHC)-restricted antigen recognition and alloreactivity by examining T cell receptor (TCR) alpha and beta gene expression in cytochrome c-specific, Ek alpha:Ek beta (Ek)-restricted helper T cell clones derived from B10.A mice. The clones could be segregated on the basis of four distinct alloreactivity patterns. Clones cross-reactive for three different allogeneic la molecules (As alpha:As beta [As], Ab alpha:Ab beta [Ab], Ek alpha: Eb beta [Eb]) expressed the same V alpha and V beta gene segments, generating the distinct alloreactive specificities via unique V alpha-J alpha and V beta-D beta-J beta joining events. Ek alpha:Es beta (Es)-alloreactive B10.A clones expressed the same V alpha, J alpha, and V beta segments as an Es-restricted, Ek-alloreactive, cytochrome c-specific, H-2-congenic B10.S(9R) clone. This homology between TCRs mediating allorecognition of la molecules and recognition of the same la molecules as restriction elements associated with nominal antigen suggests that MHC-restricted recognition and allorecognition represent differences in the affinity of the TCR-MHC molecule interaction.     

A generalized information function applied to the genetic code

The problem of the partitioning of the degeneracy of the codons in the genetic code is considered in the framework of a generalized information function IG = c sigma kpk(ln pk + G(Ek] where k represents the number of codons in a specific degeneracy class and G(Ek) is an arbitrary real valued function. For G(Ek) = 0 the Shannon information function is recovered. For a particular choice of G(Ek) that takes the dominance of even degeneracies into account, it is found by direct numerical calculations that the correct degeneracy partitioning appears as optimal values of the Ig function. This results is also supported by optimization calculations in which the generalized information function is regarded as a continuous function in the degeneracy variables.     

Stimulation of normal inducer T cell clones with antigen presented by purified Ia molecules in planar lipid membranes: specific induction of a long-lived state of proliferative nonresponsiveness

Culture of normal inducer T cell clones with antigen and purified Ek beta:Ek alpha incorporated into planar lipid membranes resulted in specific T cell activation as determined by cell volume increase and IL 3 production. However, in contrast to results obtained with T cell hybridomas, antigen presentation by planar membranes did not induce measurable IL 2 production, and proliferative responses were not detected. Rather, recognition of only Ek beta:Ek alpha and antigen resulted in the specific induction of a long-lived state of proliferative nonresponsiveness to subsequent stimulation by conventional APC and antigen. Induction of nonresponsiveness required protein synthesis, and was not simply due to the absence of IL 2. The antigen-nonresponsive cells could respond to either PMA plus ionomycin or IL 2, and they expressed normal levels of surface antigen-receptor molecules. These results demonstrate that recognition by normal T cell clones of antigen and Ia molecules in the absence of other accessory cell molecules and signals results in a prolonged state of proliferative nonresponsiveness, possibly similar to a state of T cell tolerance in vivo.     

Arsonate-specific murine T cell clones. IV. Properties of I-E- and I-A-restricted clones

The T cell antigen L-tyrosine-p-azobenzenearsonate is unique in being a simple determinant that can be presented in the context of both I-A and I-E. I-E-restricted T cell clones derived from B10.A(5R) mice were found to fall into three groups: Type I clones recognized antigen only in the context of syngeneic apcs, Type II clones recognized antigen with the same highly specific major histocompatibility complex restriction but in addition proliferated in response to allogeneic stimuli; Type III clones were "degenerate" in their major histocompatibility complex-restricted recognition of antigen and proliferated when antigen-presenting cells bearing Eb beta Ek alpha (syngeneic), Ek beta Ek alpha, or Ed beta Ed alpha were used. These observations allow some conclusions to be drawn about sites on the I-E molecule that may be functionally significant in the presentation of this antigen. By using the B cell hybridoma LK35.2 as target cells, some of these T cell clones act as cytotoxic cells in the Class II-restricted manner predicted from the results of proliferative assays. Class II-restricted cytotoxicity can therefore be controlled by both I-A and I-E mouse Ir gene loci.     

Neural cell fate in rca1 and cycA mutants: the roles of intrinsic and extrinsic factors in asymmetric division in the Drosophila central nervous system.

In the central nervous system (CNS) of Drosophila embryos lacking regulator of cyclin A (rca1) or cyclin A, we observe that several ganglion mother cells (GMCs) fail to divide. Whereas GMCs normally produce two sibling neurons that acquire different fates ('A/B'), non-dividing GMCs differentiate exclusively in the manner of one of their progeny ('B'). In zygotic numb mutants, sibling neuron fate alterations ('A/B' to 'A/A') occur infrequently or do not occur in some sibling pairs; we have determined that depletion of both maternal and zygotic numb causes sibling neurons to acquire equalized fates ('A/A') with near-complete expressivity. In rca1, numb mutant embryos, we observe binary cell fate changes ('B' to 'A') in several GMCs as well. Finally, we have demonstrated that expression of Delta in the mesoderm is sufficient to attain both sibling fates. Our results indicate that the intrinsic determinant Numb is absolutely required to attain differential sibling neuron fates. While the extrinsic factors Notch and Delta are also required to attain both fates, our results indicate that Delta signal can be received from outside the sibling pair.     

Electrophoretic Characterization of Members of the Crypthecodinium cohnii (Dinophyceae) Species Complex1

Eighty-seven axenic clones of the colorless inshore dinoflagellate Crypthecodinium cohnii were found by mating experiments to fall into 52 sibling species, seven wide ranging (two possibly global)—called major sibling species—and 45 found only once—minor sibling species. Electrophoretic analysis of three soluble enzymes from these strains revealed the following: 1) Despite some polymorphism most members of major sibling species closely resemble one another electrophoretically. 2) Major sibling species and most minor ones are electrophoretically distinct. 3) Sharing of electromorphs is sufficiently extensive, however, that no major sibling species is totally unrelated to all others. 4) Some minor sibling species are electrophoretically indistinguishable from a member of a major sibling species or from one another, suggesting recent origin by sexual isolation in situ. 5) Other minor sibling species differ from majors by one, two, or all three of the enzymes studied. A “model” of sexual isolation and diversification is offered.     

Requirements of class II-mediated B cell differentiation for class II cross-linking and cyclic AMP.

Cells of the mouse B cell clone, CH12.LX, receive Ag-dependent differentiative signals through their surface membrane class II molecules. The present study was performed to determine the role of class II cross-linking and cAMP in the successful delivery of these signals. Delivery of differentiative signals by anti-Ek mAb was increased by further cross-linking with a secondary anti-isotype antibody. Intact or (Fab')2, but not Fab forms of anti-Ek successfully delivered the Ag-dependent differentiative signal. Inability of monovalent Fab fragments to deliver the signal could not be attributed to an inability to adequately bind Ek molecules. The requirement for cAMP for class II-mediated signaling was also examined, because previous studies have implicated elevated cAMP levels as necessary for class II signaling. Both Ag-dependent, Ek-mediated differentiation and the Ek-mediated inhibition of Ag-independent LPS-induced differentiation were inhibited by the adenyl cyclase inhibitor 2'5'ddA, although elevation of cAMP was not in itself sufficient to deliver the differentiative signal. Inhibition of LPS-induced differentiation could be mediated by mAb binding to either Ek, Abk, or Abb on CH12.LX or an Ab-bearing transfectant, CH12.ABB1. This inhibition was abrogated by 2'5'ddA in the case of Ek or Abb, both of which deliver Ag-dependent differentiative signals to CH12.LX cells. In the case of Abk, which does not deliver such signals to CH12.LX, 2'5'ddA did not abrogate anti-Abk-mediated inhibition of the LPS response. The effects of 2'5'ddA were reversed by the cAMP analog, dibutyryl cAMP, and Ag-dependent-induced differentiation of CH12.LX or CH12.ABB1 was accompanied by an increase in cAMP levels.     

Abnormal blood group galactosyltransferase in blood type A1B-subjects whose sera contain anti-B agglutinin

Summary A blood type A₁B female, whose plasma agglutinates B red blood cells from other subjects but not her own, was found. Her sister with blood type A₁B (sister-1) exhibited the same peculiarity. The agglutination titer of red cells from these two subjects is lower than that of normal B. Her father is blood type B, and his plasma did not agglutinate B red cells from other subjects and his own. Her mother and another sister (sister-2) were usual blood type A₁. Blood group galactosyltransferase (B-enzyme) activity of plasma from the propositus, sister-1, and their father was very low. Km for 2-fucosyllactose of B-enzyme of these subjects was 1.3 mM, which was more than two times higher than that of the normal value. Km for UDP-Gal was similar, but the pH-activity profile differed for the two enzymes.Red cell membranes from her father contained about 70% ungalactosylated H-sites, whereas, virtually all H-sites are galactosylated in the usual B red cell membranes. The blood group ABH components are known to be heterogeneous. Because of the abnormal B-enzyme with low activity and low substrate affinity, some H components might not be galactosylated, particularly in A₁B cases (i.e., the propositus and her sister-1), due to competition by A₁ enzyme. The lack of certain B components is likely to be the cause of the existence of the anti-B agglutinin in their sera.     

T-cell receptor expressed on an autoreactive T-cell clone, clone 4. I. Induction of various T-receptor functions by anti-T idiotypic antibodies

Three monoclonal antibodies (1G3, 2H11, and 3G12) specific for a syngeneic Ek-specific T-cell clone, clone 4, have been established. The antibodies specifically blocked not only the activation of the clone in response to the specific antigen Ek but also the activation by IL-2. Kinetic studies of the blocking activity revealed that the antibodies blocked activation not only through steric hindrance of the antigen-binding site of the receptor but also via inhibition of DNA synthesis. The antibodies induced unresponsiveness of the clone to the specific antigen Ek, but not to nonspecific activation by IL-2. The state of unresponsiveness induced by 1G3 continued for 14 days, the longest time so far examined. The recovery from the unresponsiveness (tolerance) was not observed unless the clone cells proliferated vigorously in response to IL-2. The idiotope recognised by 1G3 was different from that by 2H11 and/or 3G12. This might explain some functional differences elicited by the antibodies.     

萼花臂尾轮虫种复合体内三个姐妹种生活史特征的比较研究

应用生命表统计学方法,在13℃、18℃、23℃和28℃下对采自芜湖市莲塘湖和荷花塘水体中的萼花臂尾轮虫三个姐妹种HE1、HE3和LE9的生活史特征进行了比较研究。结果表明,三个姐妹种生命表参数间的差异因温度的不同而异。13℃下,姐妹种LE9的生命期望、平均寿命和世代时间都显著长于姐妹种HE3,LE9的净生殖率和种群内禀增长率均最大,三个姐妹种所产后代中的混交雌体百分率无显著的差异。18℃下,姐妹种LE9的生命期望和平均寿命显著长于姐妹种HE3,姐妹种LE9的世代时间显著长于姐妹种HE1和HE3,姐妹种 HE3的种群内禀增长率显著高于姐妹种LE9,姐妹种HE3所产后代中的混交雌体百分率最高,三个姐妹种的净生殖率无显著的差异。23℃下,姐妹种HE1的生命期望、平均寿命和世代时间均最长,姐妹种HE1所产后代中的混交雌体百分率显著低于HE3,三个姐妹种的净生殖率和种群内禀增长率均无显著的差异。28℃下,三个姐妹种的生命期望、平均寿命、世代时间和种群内禀增长率均无显著的差异;姐妹种HE1的净生殖率最低;姐妹种HE3所产后代中的混交雌体百分率最高。三个姐妹种的生命表参数对升高的温度的响应也不相同。温度、姐妹种及两者间的交互作用对轮虫的世代时间、平均寿命、出生时的生命期望和轮虫所产后代中的混交雌体百分率均有显著的影响;温度和姐妹种对轮虫的净生殖率和种群内禀增长率均有极显著影响,但两者的交互作用对其无显著性影响。     

Further clinical delineation in trisomy 1q32 syndrome.

A male newborn with multiple congenital abnormalities was studied. Clinically, he showed prominent forehead, facial dysmorphism, ear malformations, congenital heart defect and limb anomalies. The cytogenetic studies demonstrated a karyotype 46,XY, der(18) t(1;18)(q32;p11.3)pat with partial trisomy 1q32-qter and a monosomy 18p. The patient displayed clinical features of trisomy 1q but not of monosomy 18p. There are around 80 reports of trisomy 1q32. The purpose of this paper is to describe the first case of a translocation involving 1q and 18p chromosome breakpoints. Additional findings detected in the propositus permit us a further delineation of the trisomy 1q syndrome.     

Auto-antibody to an Ig VH region allotype: induction of anti-a1 antibody in an a1-suppressed a1a2 heterozygous rabbit.

Two a1a2 heterozygous sibling rabbits were first suppressed for the paternally inherited a1 VH region allotype and then immunized with a1 IgG. Anti-a1 antibody was detected in the serum of one of the rabbits. The anti-a1 auto-antibody reacted with the same amount of a1 IgG as did a conventional anti-a1 allo-antibody. Most of the IgG and IgM of this rabbit was of the a2 allotype and no significant amount of the a1 allotype was detected as would be expected for an a1 suppressed a1a2 heterozygous rabbit. However, allotype suppression in this rabbit is maintained by endogenous anti-allotype antibody. Rabbits with anti-allotype auto-antibody may be exploited to produce litters of heterozygous and homozygous rabbits efficiently suppressed for selected allotypes.     

Human Delta4-3-oxosteroid 5beta-reductase (AKR1D1) deficiency and steroid metabolism

Conclusive in vivo evidence regarding the enzyme responsible for steroid hormone 5beta-reduction has not been obtained, although studies have suggested it may be the same enzyme as that utilized for cholic acid and chenodeoxycholic bile-acid synthesis. We have recorded the steroid metabolome of a patient with a defect in the "bile-acid" 5beta-reductase (AKR1D1) and from this confirm that this enzyme is additionally responsible for steroid hormone metabolism. The 13-year old patient has been investigated since infancy because of a cholestasis phenotype caused by bile-acid insufficiency. Several years ago it was shown that she had a 662C>T missense mutation in AKR1D1 causing a Pro198Leu substitution. It was found that the patient had an almost total absence of 5beta-reduced metabolites of corticosteroids and severely reduced production of 5beta-reduced metabolites of other steroids. The patient is healthy in spite of her earlier hepatic failure and is on no treatment. All her vital signs were normal, as were results of many biochemical analyses. She had normal pubertal changes and experiences regular menstrual cycles. There was no evidence for any clinical condition that could be attributed to attenuated ability to metabolize steroids in normal fashion. Both parents were heterozygous for the mutation but the steroid excretion was entirely normal, although an older female sibling showed definitive evidence for attenuated 5beta-reduction of cortisol. A younger brother had a normal steroid metabolome. The sibling genotypes were not available.     

Identification of the sibling species of the Drosophila willistoni subgroup through the electrophoretical mobility of acid phosphatase-1

The Drosophila willistoni group consists of 23 species of which six are sibling species and belong to the D. willistoni subgroup: D. willistoni, Drosophila equinoxialis, Drosophila tropicalis, Drosophila insularis, Drosophila pavlovskiana and Drosophila paulistorum. These sibling species are abundant in the Neotropical region and can hardly be differentiated by the usual taxonomic traits. Four of them (D. willistoni, D. equinoxialis, D. tropicalis and D. paulistorum) cover extensive geographic distribution areas overlapping in places while two of them are endemic (D. insularis and D. pavlovskiana). In this study, we presented a method for the identification of five sibling species of the D. willistoni subgroup based on the allozyme variation of acid phosphatase‐1 (Acph‐1) in acrylamide gel electrophoresis. Our work showed that Acph‐1 allozyme differences can be used for species‐diagnostic characterization. This method was shown to be a more efficient tool for species identification than others because it is both quicker and produces reliable results.