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At least 487 records · Page 27Linked to original sources
PubMed2006

Prospective evaluation of clonal evolution during long-term follow-up of patients with untreated early-stage chronic lymphocytic leukemia.

PURPOSE: Retrospective studies suggest cytogenetic abnormalities detected by interphase fluorescent in situ hybridization (FISH) can identify patients with chronic lymphocytic leukemia (CLL) who will experience a more aggressive disease course. Other studies suggest that patients may acquire chromosome abnormalities during the course of their disease. There are minimal prospective data on the clinical utility of the widely used hierarchical FISH prognostic categories in patients with newly diagnosed early-stage CLL or the frequency of clonal evolution as determined by interphase FISH. PATIENTS AND METHODS: Between 1994 and 2002, we enrolled 159 patients with previously untreated CLL (83% Rai stage 0/I) on a prospective trial evaluating clonal evolution by FISH. Patients provided baseline and follow-up specimens for FISH testing during 2 to 12 years. RESULTS: Chromosomal abnormalities detected by FISH at study entry predicted overall survival. Eighteen patients experienced clonal evolution during follow-up. The rate of clonal evolution increased with duration of follow-up with only one occurrence in the first 2 years (n = 71; 1.4%) but 17 occurrences (n = 63; 27%) among patients tested after 5+ years. Clonal evolution occurred among 10% of ZAP-70-negative and 42% of ZAP-70-positive patients at 5+ years (P = .008). CONCLUSION: This clinical trial confirms prospectively that cytogenetic abnormalities detected by FISH can predict overall survival for CLL patients at the time of diagnosis, but also suggests that many patients acquire new abnormalities during the course of their disease. Patients with higher ZAP-70 expression may be more likely to experience such clonal evolution. These findings have important implications for both clinical management and trials of early treatment for patients with high-risk, early-stage CLL.

Adult↗
PubMed2006

[Molecular biology of CLL].

Recently, major advances have occurred in our understanding of the biology of chronic lymphocytic leukemia (CLL). CD5-positive CLL cells were once assumed to originate from immature, immunologically incompetent B lymphocytes, and to passively accumulate due to increased life time. In 1999, two research groups demonstrated that CLL, which is a morphologically uniform but clinically heterogenous disease, can be classified into two major subgroups on the basis of the mutational status of the immunoglobulin heavy chain (IgH) genes. It was also suggested that these two groups both originate from mature cells that have passed the antigen selection process. This hypothesis was confirmed by gene expression studies indicating a uniform pattern characteristic to memory cells, as well as specific B-cell receptor (BCR) structures supporting the existence of a functional antigen selection. The differences in the BCR signal transduction mechanisms may underlie the different clinical behavior in which zeta-associated tyrosine kinase (ZAP-70) may play a pivotal role, since elevated ZAP-70 expression is likely to be an unfavorable prognostic factor in CLL. The diagnostic testing for ZAP-70 expression plays an important role in the therapeutic decisions.

Gene Expression Regulation, Enzymologic↗
PubMed1999

Surface expression and functional competence of CD3-independent TCR zeta-chains in immature thymocytes.

In recombinase-deficient (RAG-2-/-) mice, double-negative thymocytes can be stimulated to proliferate and differentiate by anti-CD3 Abs. CD3 molecules are expressed on the surface of these cells in association with calnexin. In this study, we show that zeta-chains can be recovered as phosphorylated proteins in association with phosphorylated ZAP-70 from anti-CD3-stimulated RAG-2-/- thymocytes, even though they are not demonstrably associated with the CD3/calnexin complex. The lack of a physical association of zeta dimers with the CD3 complex in RAG-2-/- thymocytes and also in a pre-TCR-expressing cell line, as well as the efficient association of zeta dimers with ZAP-70 in the RAG-2-/- thymocytes, suggest that these zeta-chain dimers could contribute to pre-TCR signaling. This idea is supported by the finding that in RAG-2-/- zeta-deficient thymocytes, ZAP-70 and p120cbl were only weakly phosphorylated.

Animals↗
PubMed2004

Clinical significance of soluble p53 protein in B-cell chronic lymphocytic leukemia.

BACKGROUND AND OBJECTIVES: p53 status and CD38 antigen are biological factors influencing response to therapy and clinical course in B-cell chronic lymphocytic leukemia (B-CLL). This study tests the hypothesis that soluble p53 alone and in association with CD38 can enucleate B-CLL subsets at worse prognosis. DESIGN AND METHODS: Wild and mutant forms of p53 protein were evaluated in 197 B-CLL patients at diagnosis or before progression by an immunoenzymatic method in plasma using an anti-p53 monoclonal antibody. CD38 expression was analyzed by a multicolor flow cytometric assay. RESULTS: Higher levels of both soluble p53 (sp53) and CD38 were significantly correlated with intermediate and high Rai stages, with higher beta2-microglobulin and soluble CD23 values, determined at diagnosis. Shorter overall survival (OS) and progression-free survival (PFS) were both observed in sp53+ and CD38+ patients (p<0.0001). Simultaneous positivity or negativity for sp53 and CD38 identified two subsets of patients, the former with a worse prognosis and the latter with a better prognosis with regard to PFS (p<0.0001) and OS (p<0.0001). The predictive value of sp53 and CD38 was retained among the patients within the intermediate Rai risk group. INTERPRETATION AND CONCLUSIONS: sp53 and CD38 together with ZAP-70 were confirmed to be independent prognostic factors in multivariate analysis. With regard to PFS, ZAP-70, sp53 and CD38 were confirmed to be independent prognostic factors. Concerning OS, ZAP-70, CD38 and age (< or > 60 years) were independent prognostic factors whereas sp53 showed only a tendency towards statistical significance.

ADP-ribosyl Cyclase 1↗
PubMed1996

Molecular basis of T cell dysfunction in cancer is influenced by the paracrine secretion of tumor-derived IL-2.

Previously we reported that T cells from mice bearing parental tumors have a variety of functional and biochemical defects when compared with T cells from mice bearing IL-2-secreting tumors. The biochemical defects included reduced levels of several cytoplasmic proteins such as p56lck, p59fyn, and zeta, all of which are known to be critical for signal transduction through the TCR. Based upon these results, we determined the consequences of these alterations on downstream signaling events. First, we showed that T cells of parental tumor-bearing mice have a reduction of total in vitro kinase activity associated with the TCR/CD3 compared with T cells from mice with IL-2-secreting tumors. Second, we observed that following activation, only T cells from IL-2-secreting tumor-bearing mice had completely phosphorylated CD3-associated zeta-chains and recruited ZAP-70 to cell surface-associated TCR. In contrast, T cells from mice with parental tumors contained incompletely phosphorylated CD3-associated zeta-chains with little or no TCR-associated ZAP-70. Third, the recruitment of ZAP-70 to the TCR/CD3 complex was seen only in animals with an increase in in vitro p56lck kinase activity after T cell activation. Finally, we report that these defects in T cell signaling were associated with generalized anergy in vivo. Our findings provide a molecular basis to explain T cell suppression of mice with parental tumors and offer a hypothesis to explain the protective effect of tumor-derived IL-2.

Animals↗
PubMed1998

Regulation of TCR signal transduction in murine thymocytes by multiple TCR zeta-chain signaling motifs.

The alphabeta TCR is a multimeric protein complex comprising ligand-binding and signal-transducing subunits. The signal transduction processes are mediated by the immunoreceptor tyrosine-based activation motifs (ITAMs), and up to 10 ITAMs are present within a single TCR complex. This multiplicity may allow for signal amplification and/or the formation of qualitatively distinct intracellular signals. Notably, the TCR-zeta subunit contains three ITAMs, and exists as a disulfide-linked homodimer in the TCR complex. In normal murine thymocytes and peripheral T cells, a proportion of TCR-zeta molecules is constitutively tyrosine phosphorylated and associated with the ZAP-70 protein tyrosine kinase. We examined the contribution of the different TCR-zeta ITAMs in regulating the constitutive phosphorylation of the TCR-zeta subunit in thymocytes by analyzing TCR-zeta-deficient mice that had been reconstituted with either full-length or single ITAM-containing TCR-zeta subunits. We report in this work that in the absence of a full-length TCR-zeta subunit, there is no apparent constitutive phosphorylation of the remaining TCR/CD3 ITAMs. Following TCR ligation, all of the CD3 ITAMs become inducibly phosphorylated and associate with the ZAP-70 protein tyrosine kinase. Regardless of the number of TCR-zeta ITAMs present in the TCR complex, we report that a number of molecules involved in downstream signaling events, such as ZAP-70, SLP-76, and pp36, are all inducibly tyrosine phosphorylated following TCR ligation. These results support the notion that the different TCR ITAMs function in a quantitative rather than qualitative manner.

Adaptor Proteins, Signal Transducing↗
PubMed1986

The effect of intravascular neutrophil chemotactic factors on blood neutrophil and platelet kinetics.

Intravenous infusion of an analogue (f-met-leu-phe [FMLP]) of a bacterial-derived polymorphonuclear leukocyte (PMNL) chemotactic factor, or of the complement-derived chemotactic stimulus, zymosan-activated plasma (ZAP, containing C5ades Arg) into rabbits induces acute PMNL margination in the pulmonary vasculature. This process also occurs during hemodialysis and the adult respiratory distress syndrome. The pulmonary PMNL sequestration is accompanied by thrombocytopenia. Because of the role platelets and PMNLs play in hemostasis and defense against infection, we studied the fate of these blood elements following sequestration induced by chemotactic factors. By employing 111In-labelled platelets and external radioisotope scanning, platelets were found to sequester in the pulmonary vasculature during FMLP infusion. Simultaneous 51Cr PMNL and 111In-platelet studies showed that following sequestration, PMNLs returned to the circulation and disappeared with a normal half-life (T1/2) whereas the T1/2 of the platelets was markedly shortened (T1/2 of control = 49 +/- 3.0 hr; FMLP or ZAP infused T1/2 = 27 +/- 2.7 hr). Infusion of platelet-activating factor (PAF) induced PMN and platelet sequestration with similar abnormalities in platelet kinetics. Studies with 51Cr- and 14C-serotonin-labelled platelets showed that platelets did not release serotonin during FMLP, ZAP, or low dose PAF-induced sequestration. In contrast to platelet survival, platelet size, platelet aggregation responses, and platelet glycoproteins were not affected by transient sequestration. These results indicate that during PMNL margination induced by relatively "pure" PMNL stimuli such as FMLP, platelets may reversibly marginate and subsequently be cleared at an accelerated rate. The reason for accelerated platelet clearance is not a result of circulating platelet aggregates or detectable proteolytic modification of membrane glycoproteins. Such altered platelet kinetics may contribute to thrombocytopenia during sepsis, the adult respiratory distress syndrome, and other states in which excess PMNL margination occurs.

Animals↗
PubMed2005

Decreased CD4 expression by polarized T helper 2 cells contributes to suboptimal TCR-induced phosphorylation and reduced Ca2+ signaling.

Polarized Th1 and Th2 cells expressing the same TCR produce distinct biochemical responses to ligand engagement. Compared to Th1 cells, Th2 cells show altered substrate tyrosine phosphorylation and a diminished or transient Ca2+ response. Here we demonstrate that agonist stimulation of Th1 cells leads to the predominant appearance of fully phosphorylated (p23) TCR zeta, substantial phosphorylation of zeta-associated protein 70 (ZAP-70), and strong elevation of intracellular Ca2+, whereas agonist stimulation of Th2 cells expressing an identical TCR results in an elevated p21:p23 TCR zeta ratio, little or no detectable ZAP-70 phosphorylation, and a more limited elevation in intracellular Ca2+. Th2 cells consistently had twofold lower surface CD4 expression as compared to Th1 cells with the same TCR. When CD4 levels in Th2 cells were raised to Th1 levels using retroviral gene transfer, the transduced cells showed greater generation of p23 phospho-zeta, measurable phosphorylation of ZAP-70, and increased Ca2+ responses. These findings suggest that the apparent qualitative differences in TCR signaling characterizing Th1 versus Th2 cells are largely the result of modest quantitative variation in CD4 expression, with decreased CD4 expression playing a significant role in attenuating the proximal signaling responsiveness of Th2 cells to TCR ligands.

Amino Acid Sequence↗
PubMed2004

Implementation and testing of stable, fast implicit solvation in molecular dynamics using the smooth-permittivity finite difference Poisson-Boltzmann method.

A fast stable finite difference Poisson-Boltzmann (FDPB) model for implicit solvation in molecular dynamics simulations was developed using the smooth permittivity FDPB method implemented in the OpenEye ZAP libraries. This was interfaced with two widely used molecular dynamics packages, AMBER and CHARMM. Using the CHARMM-ZAP software combination, the implicit solvent model was tested on eight proteins differing in size, structure, and cofactors: calmodulin, horseradish peroxidase (with and without substrate analogue bound), lipid carrier protein, flavodoxin, ubiquitin, cytochrome c, and a de novo designed 3-helix bundle. The stability and accuracy of the implicit solvent simulations was assessed by examining root-mean-squared deviations from crystal structure. This measure was compared with that of a standard explicit water solvent model. In addition we compared experimental and calculated NMR order parameters to obtain a residue level assessment of the accuracy of MD-ZAP for simulating dynamic quantities. Overall, the agreement of the implicit solvent model with experiment was as good as that of explicit water simulations. The implicit solvent method was up to eight times faster than the explicit water simulations, and approximately four times slower than a vacuum simulation (i.e., with no solvent treatment).

Algorithms↗
PubMed1997

Activation-associated changes in blood and bone marrow neutrophils.

Accelerated granulocytopoiesis and polymorphonuclear leukocyte (PMN) activation via the complement system are important events in the inflammatory response. This study tests the hypothesis that PMN of the peripheral blood and the bone marrow behave differently when stimulated with zymosan-activated plasma (ZAP). PMN were treated with ZAP and processed to determine the content and distribution of F-actin, the expression of CD18-integrins, and the cell area/shape with the use of a combination of flow cytometry, fluorescence, and light microscopy. The results show that untreated bone marrow PMN display similar F-actin content and cell area but express less CD18 than peripheral blood PMN. ZAP-activated peripheral blood PMN show a marked increase in the F-actin content, CD18 expression, cell area, and length. Highly elongated PMN develop a polar shape in which microfilaments are redistributed in cell protrusions and CD18 is located in the uropod and the lamellipodium. In comparison, activated bone marrow PMN show a lower and more transient increase in the F-actin content, express less CD18, show a reduced increase in the cell area, and display reduced polarity and microfilament redistribution. We conclude that bone marrow PMN show lower complement-activation response than peripheral blood PMN and suggest that the lower expression of surface membrane integrins and related cytoskeletal filaments may account for their reduced ability to develop the polar shape required for diapedesis and migration.

Actins↗
PubMed2001

Phase specific analysis of herpes zoster associated pain data: a new statistical approach.

Herpes zoster or shingles is a frequent occurrence in both elderly individuals and immunocompromised hosts. The pain associated with herpes zoster is the most debilitating complication of the disease. It can be described as acute pain and post-herpetic neuralgia or zoster associated pain (ZAP). The latter definition encompasses pain from the onset of disease through its resolution and provides a convenient analytic tool for evaluation of antiviral therapy. A heuristic examination of ZAP historical data suggests the existence of three phases of pain resolution: the acute, subacute and chronic phases. The subacute and chronic phases comprise the post-herpetic neuralgia (PHN) stage. Common analytic methods, such as a Kaplan-Meier survival function or a Cox's model, have been used to assess the pain. However, such approaches do not adequately allow for phase comparison. Notably, in the clinical trial setting the comparison of specific treatment effects on the latter stages of pain are of the greatest medical relevance since this is the most debilitating phase of the illness. In order to incorporate the phase-specific information in the modelling of time to cessation of ZAP, we assumed the hazard function was a stepwise constant. Utilizing the full likelihood function, we obtained the maximum likelihood estimate for the transition times (that is, change-points), and other parameters of medical importance. The standard error of the change-point estimates were obtained through a bootstrapping method. The asymptotic properties of the parameter estimates are also discussed. Hence, the rates of pain resolution across all phases can be examined in order to precisely define the existence of multiple phases. In addition, the covariates effect can be examined across phases and populations, thereby allowing us to translate potential efficacy of a standard therapy to different populations. These results can be utilized in the design of clinical trials or in targeting the outcome for a specific phase while controlling for the effect of other variables.

Acute Disease↗
PubMed1998

Structural basis for Syk tyrosine kinase ubiquity in signal transduction pathways revealed by the crystal structure of its regulatory SH2 domains bound to a dually phosphorylated ITAM peptide.

The Syk family of kinases, consisting of ZAP-70 and Syk, play essential roles in a variety of immune and non-immune cells. This family of kinases is characterized by the presence of two adjacent SH2 domains which mediate their localization to the membrane through receptor encoded tyrosine phosphorylated motifs. While these two kinases share many structural and functional features, the more ubiquitous nature of Syk has suggested that this kinase may accommodate a greater variety of motifs to mediate its function. We present the crystal structure of the tandem SH2 domain of Syk complexed with a dually phosphorylated ITAM peptide. The structure was solved by multiple isomorphous replacement at 3.0 A resolution. The asymmetric unit comprises six copies of the liganded protein, revealing a surprising flexibility in the relative orientation of the two SH2 domains. The C-terminal phosphotyrosine-binding site is very different from the equivalent region of ZAP-70, suggesting that in contrast to ZAP-70, the two SH2 domains of Syk can function as independent units. The conformational flexibility and structural independence of the SH2 modules of Syk likely provides the molecular basis for the more ubiquitous involvement of Syk in a variety of signal transduction pathways.

Amino Acid Sequence↗
PubMed1990

Chemotactic competence of neutrophils from neonatal calves. Functional comparison with neutrophils from adult cattle.

Neonates demonstrate an increased susceptibility to infection. Defects in locomotory functions of newborn neutrophils may play a crucial role in this context. We therefore compared the migratory response of newborn (N-PMN) and adult (A-PMN) bovine neutrophils in a microwell filter assay. Stimulation with four different endotoxins (E. coli O128B:4 and O55B:5; S. abortus equi; S. typhimurium), with zymosan-activated plasma (ZAP) and with C5a induced dose-dependent migration of A-PMNs and N-PMNs. Migration of unstimulated cells and of cells stimulated with diluted ZAP or C5a was higher (P less than 0.05) in N-PMNs. Migration of A- and N-PMNs towards C5a was inhibited (P less than 0.001) by preincubation with either a steroidal (122 microM flumethasone) or nonsteroidal (3.3 microM phenylbutazone) antiinflammatory drug. Migratory responses of N-PMNs were inhibited less by SAIDs than were responses of A-PMNs (P less than 0.05); indeed dexamethasone slightly enhanced N-PMN responses towards C5a, and 510 microM flunixin meglumine enhanced C5a-induced migration in both age groups. Endotoxins from E. coli O55:B4, S. abortus equi, and S. typhimurium induced a higher rate of migration (P less than 0.05) in N-PMNs. In contrast to the above findings, measurement of the maximal distance of migration by the leading-front method did not reveal age-related differences. Migration speed of PMNs was lower after stimulation with C5a than with ZAP, but could be restored partly by adding human vitamin D-binding protein (Gc-globulin). The demonstrated hyperirritability of bovine N-PMNs represents a major functional difference to neonatal neutrophils from other species, including man. It may additionally be related to altered PMN functions and neonatal disease susceptibility.

Aging↗
PubMed1988

Two patterns of early neutrophil accumulation in acute inflammatory lesions.

A model of acute inflammation in the skin of sheep was developed using [111In]oxine-labeled neutrophils to quantify the accumulation of neutrophils in inflammatory lesions. Peak accumulation occurred during the first hour in lesions induced by zymosan-activated sheep plasma (ZAP, 100%) and during the second hour in lesions induced by endotoxin (10(-11) moles) and casein (2 x 10(-8) moles). The accumulation of neutrophils after the final stimulus diminished when lesions were restimulated every 2 h on two or more occasions. Thus desensitization of inflammatory lesions previously observed in rabbit skin is not restricted to that species. The early accumulation of neutrophils in primary and restimulated lesions was measured in lesions from 10 min to 2 h old. In primary lesions, ZAP induced significant neutrophil accumulation by 10 min, whereas 30 to 45 min elapsed before significant neutrophil accumulation occurred in lesions induced with endotoxin and casein. The initial responses in restimulated lesions were not diminished, but there was a failure of neutrophil accumulation to continue for as long as occurred in primary lesions. This finding indicates that regulation of the neutrophil influx into dermal inflammatory lesions may occur at a point after occupancy of the putative tissue receptors in the pathway which leads to accumulation of neutrophils at the injection site. The results indicate that there are at least two patterns of neutrophil accumulation in primary inflammatory lesions. The delayed pattern of neutrophil accumulation induced by casein and endotoxin resembles the enhanced binding of neutrophils to stimulated endothelial cultures in vitro, whereas the basis for the immediate response induced by ZAP remains conjectural. It is proposed that the primary action of chemotactic agents may be to provoke changes in the adhesive properties of endothelium and resident connective tissue cells thus permitting the migration of neutrophils into inflammatory lesions.

Acute Disease↗
PubMed1991

In vitro modulation of canine polymorphonuclear leukocyte function by granulocyte-macrophage colony stimulating factor.

Granulocyte-macrophage colony stimulating factor (GMCSF) promotes the growth of granulocytes and macrophages from undifferentiated bone marrow cells and modulates the oxidative responses of polymorphonuclear leukocytes (PMN) to endogenous chemoattractants. We found that, in vitro, naturally occurring glycolsylated human GMCSF does not disturb the resting canine PMN membrane potential, may attentuate PMN oxidative responses to PMA, and is, to a small degree, chemotaxigenic. GMCSF, however, inhibits PMN chemotaxis to zymosan-activated plasma (ZAP). Compared to temperature controls, GMCSF (1-100 U/ml) produced up to 1.5-fold increases in H2O2 production after 15 minutes, while phorbol myristate acetate (PMA) treated cells increased H2O2 production 8-12-fold after 15 minutes. Preincubation of cells with GMCSF (1-100 U/ml) prior to PMA stimulation significantly reduced the H2O2 levels induced by PMA. H2O2 production was inhibited up to 15% after 15 minutes of GMCSF preincubation and up to 40% after 60 minutes of preincubation. As a chemotaxigenic agent, GMCSF (10-1000 U/ml) was able to elicit 49%-102% increases in quantitative cellular migration, compared to random migration. Total cellular chemotaxis to GMCSF was less than 30% of the response to ZAP. Preincubation of PMNs with GMCSF for 15 minutes significantly inhibited ZAP-induced cellular migration. Human GMCSF does not appear to activate canine PMN in vitro and may actually down-regulate PMN inflammatory responses.

Animals↗
PubMed1990

A chamberless field exposure system for determining the effects of gaseous air pollutants on crop growth and yield.

A modification of the chamberless Zonal Air Pollution System (ZAPS) has been developed to permit the exposure of crops to various regimes of exposure to gaseous air pollutants under true field conditions. It provides 12 different levels and patterns of pollutant exposure, simulating a wide range of ambient conditions. The different levels and patterns of exposure are achieved through different rates of discharge of pollutant-enriched gas through manifolds suspended above individual plots. A feature of the layout is that, since the plots are grouped together in blocks of four, wind speed and direction dictate the actual exposure patterns received in any plot. For studies with ozone, O(3), enrichment of the ambient air occurs daily between 0700 and 2059 h (PDT). The overall O(3) concentration supplied to the manifolds is maintained proportional to that in the ambient air, except during the first 3 and the last 3 h of each enrichment period when enrichment is slowly increased and decreased, respectively, to provide smooth transitions from and to the ambient level. Additional control progressively reduces enrichment at low wind speeds. Over a season, the ZAPS results in typical unimodal distributions of concentrations on each plot, with good vertical mixing and reasonable horizontal distributions achieved by the large numbers of discharge orifices in the manifolds. Summary results from two seasons' experiments with processing peas are presented to provide examples of the use of the ZAPS to determine crop yield responses to ozone, based on simple linear regressions of yield against two different exposure indices.

Journal Article↗
PubMed2002

The Tec family of tyrosine kinases in T cells, amplifiers of T cell receptor signals.

ITK and Rlk/Txk are the predominant Tec family of tyrosine kinases expressed in T cells, and are involved in T cell antigen receptor mediated activation of T cells. These kinases require prior activation of Lck, Zap-70 and PI3-kinase for efficient activation. They share major substrates with both Lck and Zap-70, however the pathways they regulate are unclear. Recent evidence suggests that these kinases may not activate unique pathways, but instead serve as amplifiers for the upstream kinases Lck and Zap-70. This review will discuss the evidence for this view.

Enzyme Activation↗
PubMed2006

Construction and screening of a cDNA library from the triactinomyxon spores of Myxobolus cerebralis, the causative agent of salmonid whirling diseases.

The ZAP Express cDNA library was constructed using mRNA extracted from the triactinomyxon spores of Myxobolus cerebralis. First-strand cDNA was synthesized using Moloney Murine leukaemia virus reverse transcriptase. Following second-strand cDNA synthesis, the double-stranded cDNA was digested with Xho I restriction enzyme, cDNA fragments less than 400 bp were removed and the remaining cDNA was ligated with the lambda ZAP Express vector. The recombinants were packaged in vitro using Gigapack III gold packaging extract. The primary cDNA library titre contained 0.5 x 10(6) clones, with 97% recombinant and only 3% non-recombinant clones. The cDNA library was then screened using the anti-triactinomyxon antibodies. Positive clones were selected and re-screened twice more to give a final selection of 526 clones. One clone (46-5) was selected randomly and subjected to in vivo excision of the pBK-CMV phagemid from the ZAP express vector. The sequence of the entire clone was obtained using rapid amplification of the cDNA ends. A search of the clone sequence against GenBank revealed that it related to ribosomal protein L23 and it had a high percentage similarity to this protein from different species. A conserved domain for ribosomal protein L23 was also identified in the clone sequence.

Amino Acid Sequence↗