Cryptosporidium bollandi n. sp. (Apicomplexa: Cryptosporidiiae) from angelfish (Pterophyllum scalare) and Oscar fish (Astronotus ocellatus).

The species name Cryptosporidium bollandi n. sp. is proposed for Cryptosporidium piscine genotype 2 based on morphological, biological and molecular characterisation. Phylogenetic analyses of 18S rRNA (18S) sequences revealed that C. bollandi n. sp. was most closely related to piscine genotype 4 (5.1% genetic distance) and exhibited genetic distances of 10.0%, 12.2% and 25.2% from Cryptosporidium molnari, Cryptosporidium huwi and Cryptosporidium scophthtalmi, respectively. At the actin locus, C. bollandi n. sp. was again most closely related to piscine genotype 4 (6.8% genetic distance) and exhibited 15.5% (C. molnari), 18.4% (C. huwi), 22.9% (C. scophthalmi) and up to 27.5% genetic distance from other Cryptosporidium spp. (Cryptosporidium felis). Phylogenetic analysis of concatenated 18S and actin sequences showed that C. bollandi n. sp. exhibited 12.9% (C. molnari) to 21.1% (C. canis) genetic distance from all other Cryptosporidium spp. Genetic data as well as previous histological analysis clearly supports the validity of C. bollandi n. sp. as a separate species.

Naturally occurring Bruton's tyrosine kinase mutations have no dominant negative effect in an X-linked agammaglobulinaemia cellular model.

X-linked agammaglobulinaemia (XLA) is characterized by absence of mature B cells because of mutations in the Bruton's tyrosine kinase (Btk) gene. Btk-deficient early B cell precursors experience a block in their differentiation potentially reversible by the addition of an intact Btk gene. Btk expression was measured in 69 XLA patients with 47 different mutations and normal expression was detected in seven. We characterized these Btk mutant forms functionally by transfection into a lymphoma cell line that lacks endogenous Btk expression (Btk-/- DT40 cells) and analysed the calcium flux in response to B cell receptor stimulation. To test whether co-expression of a mutated form could compromise the function of the intact Btk transfection, studies in wild-type (WT) DT40 cells were also performed. Study reveals that none of the seven Btk mutants analysed was able to revert the absence of calcium mobilization upon IgM engagement in Btk-/- DT40 cells, as does intact Btk. In addition, calcium mobilization by anti-IgM stimulation in DT40 Btk+/+ cells was unaffected by co-expression with Btk mutants. These results suggest that gene addition would be feasible not only for patients with XLA and mutations that prevent Btk expression, but for those with expression of a mutant Btk.

Genetic organization of the conjugal DNA processing region of the IncW plasmid R388.

The region of the IncW plasmid R388 involved in conjugal DNA metabolism and mobilization (MOBw) has been analyzed by Tn5tac1 insertion mutagenesis, genetic complementation and DNA sequencing. Three genes (trwA, trwB and trwC) were mapped within MOBw. They are transcribed from the same strand and away from oriT. The predicted products of trwA, trwB and trwC are proteins of 121, 507 and 966 amino acids, respectively. The three proteins were visualized in a minicell expression system, showing apparent molecular masses of 13.5, 55 and 105 kDa, respectively. The deduced amino acid sequence of TrwA shows significant similarity to TraJ of the IncP plasmids RP4 and R751, to NikA of the IncI plasmid R64 and to MobB of plasmid pTF-FC2. The amino acid sequence of TrwB predicts an integral membrane protein which contains an NTP-binding motif. It shows 28% to 29% identity with TraD of plasmids F and R100, 23% identity with TraG of plasmids RP4 and R751 and 20% identity with VirD4 of the Ti plasmids of Agrobacterium tumefaciens. The amino acid sequence of TrwC shows the characteristic motifs of the Rep family of DNA helicases. It shows 33% identity with the sequence of helicase I (TraI) of plasmid F. The similarity is highest in the N-terminal segments of the proteins, which show conservation of characteristic amino acid motifs of a family of DNA-relaxases, including VirD2 of the Ti plasmid. The conserved features of these three proteins among the different transfer systems suggest that a very widespread conjugal DNA mobilization mechanism is shared by the transfer apparatuses of IncF, IncI, IncP, IncW and Ti plasmids.

Biliary glycoprotein (BGPa, CD66a, CEACAM1) mediates inhibitory signals.

Biliary glycoprotein (BGP, CD66a, CEACAM1) is a member of the carcinoembryonic antigen family (CEA, CD66), a group of transmembrane proteins belonging to the immunoglobulin superfamily. The structural features surrounding the tyrosine residues in the cytoplasmic domain of BGP share similarity with the consensus sequence of the immunoreceptor tyrosine-based inhibition motif (ITIM), the docking site for SHIP, SHP-1, and SHP-2 molecules. Using the well-characterized inhibitory receptor, FcgammaRIIB, we constructed a FcgammaRIIB-BGPa chimeric molecule that contained the extracellular and transmembrane domain of FcgammaRIIB and the cytoplasmic tail of BGPa and expressed it in DT40 B cells. Our results showed that FcgammaRIIB-BGPa, just like the unmodified FcgammaRIIB molecule, inhibited calcium influx in activated DT40 B cells. Substitution of tyrosine with phenylalanine (Y459F) in FcgammaRIIB-BGPa completely abrogated its ability to inhibit calcium influx, indicating that the motif surrounding Y459 is ITIM. The presence of ITIM was also supported by showing that the FcgammaRIIB-BGPa-mediated inhibitory effect was reduced in SHP-1and SHP-2 mutant DT40 B cells and further diminished in a SHP-1/-2 double-deficient mutant line. The results suggest that SHP-1 and SHP-2 are required for the FcgammaRIIB-BGPa-mediated inhibitory signals.

The two single-strand cleavages at each end of Tn10 occur in a specific order during transposition.

During Tn10 transposition, the element is excised from the donor site by double-strand cleavages at the two transposon ends. Double-strand cleavage is a central step in the nonreplicative transposition reaction of many transposons in both prokaryotes and eukaryotes. Evidence is presented to show that the Tn10 double-strand cut is made by an ordered, sequential cleavage of the two strands. The transferred strand is cut first, and then the nontransferred strand is cleaved. The single-strand nicked intermediate is seen to accumulate when Mn2+ is substituted for Mg2+ in the reaction or when certain mutant transposases are used. The fact that the transferred strand is cleaved before the non-transferred strand implies that the order of strand cleavages is not the determining factor that precludes a replicative mechanism of transposition.

The three chemical steps of Tn10/IS10 transposition involve repeated utilization of a single active site.

Nonreplicative transposition by Tn10/IS10 involves three chemical steps at each transposon end: cleavage of the two strands plus joining of one strand to target DNA. These steps occur within a synaptic complex comprising two transposon ends and monomers of IS10 transposase. We report four transposase mutations that individually abolish each of the three chemical steps without affecting the synaptic complex. We conclude that a single constellation of residues, the "active site," directly catalyzes each of the three steps. Analyses of reactions containing mixtures of wild-type and catalysis-defective transposases indicate that a single transposase monomer at each end catalyzes the cleavage of two strands and that strand transfer is carried out by the same monomers that previously catalyzed cleavage. These and other data suggest that one active site unit carries out all three reactions in succession at one transposon end.

IgG Fc receptors.

Since the description of the first mouse knockout for an IgG Fc receptor seven years ago, considerable progress has been made in defining the in vivo functions of these receptors in diverse biological systems. The role of activating Fc gamma Rs in providing a critical link between ligands and effector cells in type II and type III inflammation is now well established and has led to a fundamental revision of the significance of these receptors in initiating cellular responses in host defense, in determining the efficacy of therapeutic antibodies, and in pathological autoimmune conditions. Considerable progress has been made in the last two years on the in vivo regulation of these responses, through the appreciation of the importance of balancing activation responses with inhibitory signaling. The inhibitory FcR functions in the maintenance of peripheral tolerance, in regulating the threshold of activation responses, and ultimately in terminating IgG mediated effector stimulation. The consequences of deleting the inhibitory arm of this system are thus manifested in both the afferent and efferent immune responses. The hyperresponsive state that results leads to greatly magnified effector responses by cytotoxic antibodies and immune complexes and can culminate in autoimmunity and autoimmune disease when modified by environmental or genetic factors. Fc gamma Rs offer a paradigm for the biological significance of balancing activation and inhibitory signaling in the expanding family of activation/inhibitory receptor pairs found in the immune system.

Spontaneous autoimmune disease in Fc(gamma)RIIB-deficient mice results from strain-specific epistasis.

By virtue of its ability to couple the BCR to an inhibitory pathway, FcgammaRIIB can potentially determine the fate of B cells upon IgG immune complex engagement. We now provide evidence for FcgammaRIIB as a component of a peripheral tolerance pathway with the observation that RIIB-/- mice develop autoantibodies and autoimmune glomerulonephritis in a strain-dependent fashion. Transfer of the autoimmune phenotype is associated with the presence of donor RIIB-/- B cells, with the RIIB+/+ myeloid cells primarily derived from the recipient. These results suggest that deficiency of RIIB on B cells leads to autoimmune disease in specific genetic backgrounds, thus identifying it as a susceptibility factor under the influence of epistatic modifiers for the development of autoimmunity.

Role of the inositol phosphatase SHIP in negative regulation of the immune system by the receptor Fc(gamma)RIIB.

Immune complexes are potent activators of inflammatory cells, triggering effector responses through the crosslinking of Fc receptors (FcRs) such as Fc(epsilon)RI or Fc(gamma)RIII. On B cells and mast cells, immune complexes are also negative regulators of activation triggered by antigen and Fc receptors, a consequence of coligation of the B-cell antigen receptor or Fc(epsilon)RI, respectively, and the inhibitory receptor Fc(gamma)RIIB. Here we show that inhibitory signalling by Fc(gamma)RIIB does not require the SH2-domain-containing protein tyrosine phosphatase, SHP-1, in mast cells and results in the recruitment of the SH2-domain-containing inositol polyphosphate 5-phosphatase, SHIP, to the tyrosine-phosphorylated 13-amino-acid inhibitory motif of Fc(gamma)RIIB in both B cells and mast cells. SHIP, by hydrolysing the 5-phosphate of phosphatidylinositol(3,4,5)P3 and inositol(1,3,4,5)P4, suggests a mechanism by which Fc(gamma)RIIB can inhibit calcium influx and downstream responses triggered by immune receptors.

Structural and functional analysis of the origin of conjugal transfer of the broad-host-range IncW plasmid R388 and comparison with the related IncN plasmid R46.

We cloned and sequenced a 402 bp DNA segment containing the origin of conjugal transfer (oriT) of the IncW plasmid R388. Progressive deletions from each end of the sequence were assayed for oriT activity. Stepwise reductions in mobilization frequencies, representing the loss of functional elements, correlated with deletion of structural motifs in the sequence. A sequence of 330 bp of oriT was sufficient for efficient mobilization. The first 86 bp of the sequence contains five tandemly repeated DNA sequences of 11 bp, followed by a 10 bp perfect inverted repeat. Deletion of the first 95 bp reduced the frequency of transfer by a hundred-fold. The sequence between bp 183 and 218 was necessary and sufficient for low frequency mobilization and, thus, it was assumed to contain the nick site. This basis core was cloned as a 60 bp segment (from bp 176-236) that could be mobilized at low frequency. It includes two inverted repeats and a perfect integration host factor (IHF) consensus binding site. A third functionally important segment in oriT was located between bp 260 and 330. The DNA sequence of the oriT of R388 could be aligned with that of the broad-host-range IncN plasmid R46. Moreover, the relative positions of the three inverted repeats are also conserved. Overall sequence similarity was 52%, but was significantly higher in particular regions, which coincided with the functionally important segments mapped by deletion analysis. Conservation of these segments provided independent support for their essential role in oriT function.

General organization of the conjugal transfer genes of the IncW plasmid R388 and interactions between R388 and IncN and IncP plasmids.

The complete conjugal transfer gene region of the IncW plasmid R388 has been cloned in multicopy vector plasmids and mapped to a contiguous 14.9-kilobase segment by insertion mutagenesis. The fertility of the cloned region could still be inhibited by a coresident IncP plasmid. The transfer region has been dissected into two regions, one involved in pilus synthesis and assembly (PILW), and the other involved in conjugal DNA metabolism (MOBW). They have been separately cloned. PILW also contains the genes involved in entry exclusion. MOBW contains oriT and the gene products required for efficient mobilization by PILW. MOBW plasmids could also be mobilized efficiently by PILN, the specific pilus of the IncN plasmid pCU1, but not by PILP, the specific pilus of the IncP plasmid RP1.

The CGM1a (CEACAM3/CD66d)-mediated phagocytic pathway of Neisseria gonorrhoeae expressing opacity proteins is also the pathway to cell death.

Phagocytosis of Opa+ Neisseria gonorrhoeae (gonococcus, GC) by neutrophils is in part dependent on the interaction of Opa proteins with CGM1a (CEACAM3/CD66d) antigens, a neutrophil-specific receptor. However, the signaling pathways leading to phagocytosis have not been characterized. Here we show that interaction of OpaI bacteria with neutrophils or CGM1a-transfected DT40 cells induces calcium flux, which correlates with phagocytosis of bacteria. We identified an immunoreceptor tyrosine-based activation motif (ITAM) in CGM1a, and showed that the ability of CGM1a to transduce signals and mediate phagocytosis was abolished by mutation of the ITAM tyrosines. We also demonstrated that CGM1a-ITAM-mediated bacterial phagocytosis is dependent on Syk and phospholipase C activity in DT40 cells. Unexpectedly, the activation of the CGM1a-ITAM phagocytic pathway by Opa+ GC results in induction of cell death.

Deletion of SHIP or SHP-1 reveals two distinct pathways for inhibitory signaling.

Two signaling molecules have been implicated in the modulation of immune receptor activation by inhibitory coreceptors: an inositol polyphosphate 5'-phosphatase, SHIP, and a tyrosine phosphatase, SHP-1. To address the necessity, interaction, or redundancy of these signaling molecules, we have generated SHP-1- or SHIP-deficient B cell lines and determined their ability to mediate inhibitory signaling. Two distinct classes of inhibitory responses are defined, mediated by the selective recruitment of SHP-1 or SHIP. The Fc gammaRIIB class of inhibitory signaling is dependent on SHIP and not SHP-1; conversely, the KIR class requires SHP-1 and not SHIP. The consequence of this selective recruitment by inhibitory receptor engagement is seen in BCR-triggered apoptosis. SHP-1-mediated inhibitory signaling blocks apoptosis, while SHIP recruitment attenuates a proapoptotic signal initiated by Fc gammaRIIB.

TrwD, a protein encoded by the IncW plasmid R388, displays an ATP hydrolase activity essential for bacterial conjugation.

A 1.7-kilobase pair segment from the conjugative transfer region of plasmid R388 DNA was cloned and sequenced. It contained trwD, a gene essential for plasmid R388 conjugation, for expression of the conjugative W-pilus and for sensitivity to phage PRD1. The deduced amino acid sequence of TrwD showed homology to the PulE/VirB11 superfamily of potential ATPases involved in various types of transport processes. A fusion of trwD with the glutathione S-transferase (GST) was constructed, and the resulting fusion protein was purified from overproducing bacteria. Factor Xa hydrolysis of GST-TrwD and further purification rendered TrwD protein with more than 95% purity. Antibodies raised against TrwD localized it both in the soluble fraction and in the outer membrane of Escherichia coli. TrwD is probably a peripheral outer membrane protein because it could be solubilized by increasing salt concentration to 0.5 M NaCl in the lysis buffer. Both purified GST-TrwD and TrwD could hydrolize ATP. ATPase activity increased 2-fold in the presence of detergent-phospholipid mixed micelles. To study the importance of the nucleotide-binding site, Walker box A (GXXGXGK(T/S)), present in TrwD, the conserved lysine residue was replaced by glutamine. The mutant protein, expressed and purified under the same conditions as the wild type, did not exhibit ATPase activity. TrwD(K203Q) was not able to complement the mutation in trwD of the R388 mutant plasmid, suggesting the essentiality of the ATPase activity of the protein in the conjugative process. Furthermore, the dominant character of this mutation suggested that GST-TrwD(K432Q) was still able to interact either with itself or with other component(s) of the conjugative machinery.

Conjugation-independent, site-specific recombination at the oriT of the IncW plasmid R388 mediated by TrwC.

Plasmids containing a direct repeat of plasmid R388 oriT are capable of site-specific recombination, which results in deletion of the intervening DNA. This reaction occurs in the presence, but not in the absence, of the region of R388 implicated in DNA processing during conjugation. This region contains three genes, trwA, trwB, and trwC. By using mutants of each of the three genes, it was demonstrated that only trwC is required for the oriT-specific recombination. Further analysis showed that the N-terminal 272 amino acids of the protein are sufficient to catalyze recombination. TrwC is also capable of promoting intermolecular recombination between two plasmids containing oriT, suggesting that double-strand breaks in both plasmid DNAs are involved in the process. Additionally, intramolecular recombination between R388 oriT and R46 oriT did not occur in the presence of both nickases. This suggests that the half-reactions at each oriT are not productive if they occur separately; therefore, an interaction between the recombination complexes formed at each recombining site is required. This is the first report in which a nicking-closing enzyme involved in conjugal DNA transfer promotes oriT-specific recombination of double-stranded DNA in the absence of conjugation.

Target similarity effects: support for the parallel distributed processing assumptions.

Recent research has begun to provide support for the assumptions that memories are stored as a composite and are accessed in parallel (Tehan & Humphreys, 1998). New predictions derived from these assumptions and from the Chappell and Humphreys (1994) implementation of these assumptions were tested. In three experiments, subjects studied relatively short lists of words. Some of the lists contained two similar targets (thief and theft) or two dissimilar targets (thief and steal) associated with the same cue (robbery). As predicted, target similarity affected performance in cued recall but not free association. Contrary to predictions, two spaced presentations of a target did not improve performance in free association. Two additional experiments confirmed and extended this finding. Several alternative explanations for the target similarity effect, which incorporate assumptions about separate representations and sequential search, are rejected. The importance of the finding that, in at least one implicit memory paradigm, repetition does not improve performance is also discussed.

SHIP modulates immune receptor responses by regulating membrane association of Btk.

Membrane recruitment of SHIP is responsible for the inhibitory signal generated by FcgammaRIIB coligation to the BCR. By reducing the level of PIP3, SHIP regulates the association of the tyrosine kinase Btk with the membrane through PH domain-phosphoinositol lipid interactions. Inhibition of BCR signaling by either FcgammaRIIB coligation, membrane expression of SHIP, or inhibition of P13K, conditions which result in decreased levels of PIP3, is suppressed by the expression of Btk as a membrane-associated chimera. Conversely, increasing PIP3 levels by deletion of SHIP results in increased Btk association with the membrane and hyperresponsive BCR signaling. These results suggest a central role for PIP3 in regulating the B cell stimulatory state by modulating Btk localization and thereby calcium fluxes.

SHIP recruitment attenuates Fc gamma RIIB-induced B cell apoptosis.

Fc gammaRIIB is an inhibitory receptor that terminates activation signals initiated by antigen cross-linking of the BCR through the recruitment of SHIP. Fc gammaRIIB can also signal independently of BCR coligation to directly mediate an apoptotic response, requiring only an intact transmembrane domain. Failure to recruit SHIP, either by deletion of SHIP or mutation of Fc gammaRIIB, results in enhanced Fc gammaRIIB-triggered apoptosis. Thus, in the germinal center, where ICs are retained by FDCs, Fc gammaRIIB may be an active determinant in the negative selection of B cells whose BCRs have reduced affinity for antigen as a result of somatic hypermutation. Selection of B cells may represent the sum of opposing signals generated by the interaction of ICs with the BCR and Fc gammaRIIB through pathways modulated by SHIP.