Genetic aspects of the tolerance to allografts induced at metamorphosis in the toadXenopus laevis

Genetic aspects of tolerance to allografts induced at metamorphosis in the toadXenopus laevis were studied in one sibship expressing four different major histocompatibility complex (MHC) haplotypes. Tolerance by skin grafting was induced during metamorphosis in thiourea-blocked individuals, a technique that allows prolonged observation of graft behavior at this stage. Four classes of mutually tolerant animals could be determined. The use of antisera recognizing red blood cell antigens segregating with mixed lymphocyte reaction (MLR) haplotypes revealed that the four abovementioned classes corresponded to the four MHC genotypes of the family. The tolerance is, therefore, preferentially induced to antigens not dependent on the MHC. Under certain circumstances tolerance can also be induced to MHC antigens, provided that the animals differ at the level of one MHC haplotype only. Study of MLR during ontogeny suggested that, between sibs, only the two MLR haplotype differences were stimulatory at metamorphosis, whereas in larval and adult animals a one-haplotype difference was enough for stimulation.     

What limits affinity maturation of antibodies in Xenopus--the rate of somatic mutation or the ability to select mutants?

Although the Xenopus immunoglobulin heavy chain locus is structurally and functionally similar to mammalian IgH loci, Xenopus antibodies are limited in heterogeneity, and they mature only slightly in affinity during immune responses. During the antibody response of isogenic frogs to DNP-KLH, mu and upsilon cDNA sequences using elements of the VH1 family were cloned, sequenced and compared with germline counterparts. There were zero to four mutations per sequence, mostly single base substitutions, in the framework and CDRs 1 and 2 of VH. No mutations were found in JH. Since the point mutation rate was only 4- to 7-fold lower than that calculated for mice, affinity maturation does not seem to be limited by mutant availability. Because of a relatively low ratio of replacement to silent mutations in the CDRs and a very high ratio of GC to AT base pairs altered by mutation, it is suggested that the problem results from the absence of an effective mechanism for selecting mutants, which in turn might be related to the absence of germinal centers in Xenopus.     

ciCD94-1, an ascidian multipurpose C-type lectin-like receptor expressed in Ciona intestinalis hemocytes and larval neural structures

Abstract C-type lectins play an important role in the immune system and are part of a large superfamily that includes C-type lectin-like domain (CTLD)-containing proteins. Divergent evolution, acting on the CTLD fold, has generated the Ca²⁺-dependent carbohydrate-binding lectins and molecules, as the lectin-like natural killer (NK) receptors that bind proteins, rather than sugars, in a Ca²⁺-independent manner. We have studied ciCD94-1, a CTLD-containing protein from the tunicate Ciona intestinalis , which is a homolog of the CD94 vertebrate receptor that is expressed on NK cells and modulates their cytotoxic activity by interacting with MHC class I molecules. ciCD94-1 shares structural features with the CTLD-containing molecules that recognize proteins, suggesting that it could be located along the evolutionary pathway leading to the NK receptors.
ciCD94-1 was up-regulated in response to inflammation induced by lipopolysaccharide (LPS) acting on a blood cell type present in both the tunic and circulating blood. Furthermore, an anti-ciCD94-1 antibody specifically inhibited the phagocytic activity of these cells. ciCD94-1 was also expressed during development in the larva and in the early stages of metamorphosis in structures related to the nervous system, and loss of its function affected the correct differentiation of these territories. These findings suggest that ciCD94-1 has different roles in immunity and in development, thus strengthening the concept of gene co-option during evolution and of an evolutionary relationship between the nervous and the immune systems.     

PRED-CLASS: cascading neural networks for generalized protein classification and genome-wide applications.

A cascading system of hierarchical, artificial neural networks (named PRED-CLASS) is presented for the generalized classification of proteins into four distinct classes-transmembrane, fibrous, globular, and mixed-from information solely encoded in their amino acid sequences. The architecture of the individual component networks is kept very simple, reducing the number of free parameters (network synaptic weights) for faster training, improved generalization, and the avoidance of data overfitting. Capturing information from as few as 50 protein sequences spread among the four target classes (6 transmembrane, 10 fibrous, 13 globular, and 17 mixed), PRED-CLASS was able to obtain 371 correct predictions out of a set of 387 proteins (success rate approximately 96%) unambiguously assigned into one of the target classes. The application of PRED-CLASS to several test sets and complete proteomes of several organisms demonstrates that such a method could serve as a valuable tool in the annotation of genomic open reading frames with no functional assignment or as a preliminary step in fold recognition and ab initio structure prediction methods. Detailed results obtained for various data sets and completed genomes, along with a web sever running the PRED-CLASS algorithm, can be accessed over the World Wide Web at http://o2.biol.uoa.gr/PRED-CLASS.     

Polo-like kinase confers MPF autoamplification competence to growing Xenopus oocytes

During oogenesis, the Xenopus oocyte is blocked in prophase of meiosis I. It becomes competent to resume meiosis in response to progesterone at the end of its growing period (stage VI of oogenesis). Stage IV oocytes contain a store of inactive pre-MPF (Tyr15-phosphorylated Cdc2 bound to cyclin B2); the Cdc25 phosphatase that catalyzes Tyr15 dephosphorylation of Cdc2 is also present. However, the positive feedback loop that allows MPF autoamplification is not functional at this stage of oocyte growth. We report that when cyclin B is overexpressed in stage IV oocytes, MPF autoamplification does not occur and the newly formed cyclin B-Cdc2 complexes are inactivated by Tyr15 phosphorylation, indicating that Myt1 kinase remains active and that Cdc25 is prevented to be activated. Plx1 kinase (or polo-like kinase), which is required for Cdc25 activation and MPF autoamplification in full grown oocytes is not expressed at the protein level in small stage IV oocytes. In order to determine if Plx1 could be the missing regulator that prevents MPF autoamplification, polo kinase was overexpressed in stage IV oocytes. Under these conditions, the MPF-positive feedback loop was restored. Moreover, we show that acquisition of autoamplification competence does not require the Mos/MAPK pathway.     

JC virus-specific cytotoxic T lymphocytes in individuals with progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by a reactivation of the polyomavirus JC (JCV) within a setting of immunosuppression. The nature of the immune response that contains replication of this virus is unknown. We have explored JCV-specific cellular immune responses in patients with PML and control subjects. JCV antigen-stimulated peripheral blood mononuclear cells (PBMC) of four human immunodeficiency virus (HIV)-infected patients who were survivors of PML and one HIV-uninfected patient recently diagnosed with PML lysed autologous B-lymphoblastoid cell lines expressing either the JCV T regulatory protein or the VP1 major capsid protein. This lysis was mediated by CD8(+) T lymphocytes and was major histocompatibility complex class I restricted. These cells were therefore cytotoxic T lymphocytes (CTL). JCV-specific CTL could not be detected in PBMC of three HIV-infected PML patients who had progressive neurologic disease and an eventual fatal outcome. These data suggest that the JCV-specific cellular immune response may play a crucial role in the containment of PML. This finding may also prove useful as a favorable prognostic marker in the clinical management of these patients.     

Alternative endocytic pathway for immunoglobulin A Fc receptors (CD89) depends on the lack of FcRgamma association and protects against degradation of bound ligand

IgA is the most abundant immunoglobulin in mucosal areas but is only the second most common antibody isotype in serum because it is catabolized faster than IgG. IgA exists in monomeric and polymeric forms that function through receptors expressed on effector cells. Here, we show that IgA Fc receptor(s) (FcalphaR) are expressed with or without the gamma chain on monocytes and neutrophils. gamma-less FcalphaR represent a significant fraction of surface FcalphaR molecules even on cells overexpressing the gamma chain. The FcalphaR-gamma2 association is up-regulated by phorbol esters and interferon-gamma. To characterize gamma-less FcalphaR functionally, we generated mast cell transfectants expressing wild-type human FcalphaR or a receptor with a point mutation (Arg --> Leu at position 209) which was unable to associate with the gamma chain. Mutant gamma-less FcalphaR bound monomeric and polymeric human IgA1 or IgA2 but failed to induce exocytosis after receptor clustering. The two types of transfectant showed similar kinetics of FcalphaR-mediated endocytosis; however, the endocytosis pathways of the two types of receptor differed. Whereas mutant FcalphaR were localized mainly in early endosomes, those containing FcalphaR-gamma2 were found in endo-lysosomal compartments. Mutant gamma-less FcalphaR recycled the internalized IgA toward the cell surface and protected against IgA degradation. Cells expressing the two forms of FcalphaR, associated or unassociated with gamma chains, may thus have differential functions either by degrading IgA antibody complexes or by recycling serum IgA.     

Determination of age at death: assessment of an algorithm of age prediction using numerical three-dimensional CT data from pubic bones

The determination of age at death is an important part of physical and forensic anthropology. Because of resistance to decomposition and the simplicity/accuracy ratio, the direct observation of the os pubis by Suchey-Brooks phase analysis is the preferred reference system for determination of age at death. We propose an age-prediction system using a pubic symphysis numerical database obtained from CT x-ray through quantification of age-linked parameters. Our system increases the accuracy of age estimation and at the same time preserves the integrity of the archeological material.     

Attract and kill: a new method for control of the codling moth Cydia pomonella

The attract and kill technique has been formulated in a product under the trade name `Sirene CM®'. It consists of a viscous paste containing 0.16% codlemone to attract the male moths and 6.0% permethrin to kill them. The formulation is applied by hand twice per season using a specially developed system which can be calibrated for application of the paste to the host plant in small droplets of either 100 l or 50 l. Between 1995 and 1997, 15 trials on control of the codling moth were conducted in isolated orchards in the Lake Geneva region. In each plot, depending on tree size, two applications varying between 52 and 537 g ha^–1 of Sirene CM were made. In 14 trials, the larval attack of the codling moth on fruit was below the economic threshold of 1% and the hibernating population stayed at a low level. One single plot (0.4 ha) had to be treated with a curative spray in 1995, because the initial population was much too high. According to the reductions in trap catch and of mating frequency measured by tethered codling moth females, efficiency of the attract and kill droplets lasted 5–7 weeks, after which it decreased slowly.