B cells expressing a natural polyreactive autoantibody have a distinct phenotype and are overrepresented in immunoglobulin heavy chain transgenic mice

Despite stringent regulation of disease-associated autoantibodies, a substantial proportion of circulating Abs in sera of healthy individuals exhibit self-reactivity. These Abs are referred to as naturally occurring or natural autoantibodies (NAAs). To understand the origin and function of NAAs, we have generated a new site-directed transgenic mouse model in which a prerearranged VDJ gene coding for the H chain of a typical polyreactive NAA, ppc1-5, is inserted into the IgH locus. This H chain, when combined with its original L chain, the lambda1 L chain, yields a NAA that characteristically binds a variety of self and non-self Ags including ssDNA, actin, ubiquitin, and nitrophenyl phosphocholine. Despite their autoreactivity, B cells expressing ppc1-5H/lambda1 NAA are not negatively selected, but rather are overrepresented in the transgenic mice. The shift toward lambda1 expression mainly occurs during the transition of immature to mature B cells in the spleen, suggesting a BCR selection process. The ppc1-5H/lambda1 B cells exhibit a phenotype that is different from those of the known mature B cell populations, and they are located predominantly in the lymphoid follicles of the spleen and the lymph nodes. These B cells are functionally active, producing high levels of Abs in vivo and responding well to BCR stimulation in vitro. The findings indicate that the ppc1-5/lambda1 natural autoantibodies originate from a distinct B cell subset that may be positively selected by virtue of its poly/autoreactivity.     

Differential response of iron metabolism to oxidative stress generated by antimycin A and nitrofurantoin

The close interrelationship of oxidative stress and iron is evident by the influence of intracellular reactive oxygen species on iron metabolism. Oxygen radicals can lead to release of iron from iron-sulfur proteins and ferritin, and can damage iron-containing enzymes such as mitochondrial aconitase. Treatment of HepG2 human hepatoma cells with antimycin A has two effects relating to iron depending on the concentrations of antimycin A: increase of the labile iron pool and stimulation of non-transferrin-bound iron uptake. Whereas the first could also be generated with nitrofurantoin, the stimulation of non-transferrin-bound iron uptake was only seen with antimycin A and needed considerably higher concentrations. Pretreatment of the cells with ebselen, which scavenges peroxides, reverted only the effect of nitrofurantoin on the labile iron pool. Depletion with iron chelators before or after treatment with antimycin A diminished the stimulation of non-transferrin-bound iron uptake. We conclude that the generation of oxygen radicals in the mitochondria leads to the liberation of iron from mitochondrial enzymes, which enters the labile iron pool. But high concentrations of antimycin A leading to the stimulation of non-transferrin-bound iron uptake is possibly not related to the inhibition of the respiratory chain.     

Binding to the high-affinity M-type receptor for secreted phospholipases A(2) is not obligatory for the presynaptic neurotoxicity of ammodytoxin A

R180, isolated from porcine brain cortex, is a high-affinity membrane receptor for ammodytoxin A (AtxA), a secreted phospholipase A(2) (sPLA(2)) and presynaptically active neurotoxin from venom of the long-nosed viper (Vipera ammodytes ammodytes). As a member of the M-type sPLA(2) receptors, present on the mammalian plasma membrane, R180 has been proposed to be responsible for one of the first events in the process of presynaptic neurotoxicity, the binding of the toxin to the nerve cell. To test this hypothesis, we prepared and analyzed three N-terminal fusion proteins of AtxA possessing a 12 or 5 amino acid residue peptide. The presence of such an additional "propeptide" prevented interaction of the toxin with the M-type receptor but not its lethality in mouse and neurotoxic effects on a mouse phrenic nerve-hemidiaphragm preparation. In addition, antibodies raised against the sPLA(2)-binding C-type lectin-like domain 5 of the M-type sPLA(2) receptor were unable to abolish the neurotoxic action of AtxA on the neuromuscular preparation. The specific enymatic activities of the fusion AtxAs were two to three orders of magnitude lower from that of the wild type, yet resulting in a similar but less pronounced neurotoxic profile on the neuromuscular junction. This is in accordance with other data showing that a minimal enzymatic activity suffices for presynaptic toxicity of sPLA(2)s to occur. Our results indicate that the interaction of AtxA with the M-type sPLA(2) receptor at the plasma membrane is not essential for presynaptic activity of the toxin. Interaction of AtxA with two intracellular proteins, calmodulin and the R25 receptor, was affected but not prevented by the presence of the N-terminal fusion peptides, implying that these proteins may play a role in the sPLA(2) neurotoxicity.     

Immunohistochemical detection of tankyrase 2 in human breast tumors and normal renal tissue

Tankyrase, which functions at telomeres and other cellular compartments, is thought to be a positive regulator of telomerase; its isoenzyme tankyrase 2 has been cloned as a putative cancer antigen. This pilot immunohistochemical study was designed to examine whether tumors overexpress tankyrase 2. An antibody was generated by using synthetic peptide specific for tankyrase 2 and was tested by Western blot and immunocytochemically; no cross-reaction with isoenzyme 1 was revealed. Among tissue sections, two tumors of 18 specimens were positive for tankyrase 2. Others were negative or contained barely detectable protein. The surrounding normal tissues were negative. Tankyrase 2 was also revealed in epithelial cells of a limited number of normal renal tubules, whereas other renal tissues were negative. These data suggest that tankyrase 2 is not expressed ubiquitously in human tissues. To determine whether the up-regulation of tankyrase 2 is associated with tissue regeneration and cell proliferation, we compared the activity and concentration of the enzyme in a model human embryonic kidney cell line 293 arrested by serum deprivation and restimulated with serum. The serum-starved quiescent cell culture exhibited detectable protein as did the proliferating cells; enzyme activity dramatically increased in the latter. We conclude that pathologic overexpression of tankyrase 2 in some tumors may be a result of the cancer-related adaptation of the malignant cells dependent on tankyrase activity. Under normal conditions, the protein might be up-regulated during cell differentiation and also posttranslationally in proliferating cells. This study was supported by the Russian Foundation for Basic Research (grant no. 03-04-48835, principal investigator A.N. Kuimov)     

Molecular dynamics simulations of human tRNA Lys,3 UUU: the role of modified bases in mRNA recognition

Accuracy in translation of the genetic code into proteins depends upon correct tRNA-mRNA recognition in the context of the ribosome. In human tRNA(Lys,3)UUU three modified bases are present in the anticodon stem-loop--2-methylthio-N6-threonylcarbamoyladenosine at position 37 (ms2t6A37), 5-methoxycarbonylmethyl-2-thiouridine at position 34 (mcm5s2U34) and pseudouridine (psi) at position 39--two of which, ms2t6A37 and mcm5s2U34, are required to achieve wild-type binding activity of wild-type human tRNA(Lys,3)UUU [C. Yarian, M. Marszalek, E. Sochacka, A. Malkiewicz, R. Guenther, A. Miskiewicz and P. F. Agris (2000) Biochemistry, 39, 13390-13395]. Molecular dynamics simulations of nine tRNA anticodon stem-loops with different combinations of nonstandard bases were performed. The wild-type simulation exhibited a canonical anticodon stair-stepped conformation. The ms2t6 modification at position 37 is required for maintenance of this structure and reduces solvent accessibility of U36. Ms2t6A37 generally hydrogen bonds across the loop and may prevent U36 from rotating into solution. A water molecule does coordinate to psi39 most of the simulation time but weakly, as most of the residence lifetimes are <40 ps.     

An overview of statistical approaches for adaptive designs and design modifications

With adaptive design methods for clinical trials, design elements such as sample size or further interim sample sizes may be changed during the course of the trial depending on all previously collected data. The focus of the overview is on group sequential approaches where the types of adaptations need not be specified in advance. An overview of the different statistical approaches for adaptive design methods proposed in the literature is given, relations between these methods are described and some perspectives of application and for future research are discussed.     

The splicing factor Prp43p, a DEAH box ATPase, functions in ribosome biogenesis

Biogenesis of the small and large ribosomal subunits requires modification, processing, and folding of pre-rRNA to yield mature rRNA. Here, we report that efficient biogenesis of both small- and large-subunit rRNAs requires the DEAH box ATPase Prp43p, a pre-mRNA splicing factor. By steady-state analysis, a cold-sensitive prp43 mutant accumulates 35S pre-rRNA and depletes 20S, 27S, and 7S pre-rRNAs, precursors to the small- and large-subunit rRNAs. By pulse-chase analysis, the prp43 mutant is defective in the formation of 20S and 27S pre-rRNAs and in the accumulation of 18S and 25S mature rRNAs. Wild-type Prp43p immunoprecipitates pre-rRNAs and mature rRNAs, indicating a direct role in ribosome biogenesis. The Prp43p-Q423N mutant immunoprecipitates 27SA2 pre-rRNA threefold more efficiently than the wild type, suggesting a critical role for Prp43p at the earliest stages of large-subunit biogenesis. Consistent with an early role for Prp43p in ribosome biogenesis, Prp43p immunoprecipitates the majority of snoRNAs; further, compared to the wild type, the prp43 mutant generally immunoprecipitates the snoRNAs more efficiently. In the prp43 mutant, the snoRNA snR64 fails to methylate residue C2337 in 27S pre-rRNA, suggesting a role in snoRNA function. We propose that Prp43p promotes recycling of snoRNAs and biogenesis factors during pre-rRNA processing, similar to its recycling role in pre-mRNA splicing. The dual function for Prp43p in the cell raises the possibility that ribosome biogenesis and pre-mRNA splicing may be coordinately regulated.     

Forebrain-specific inactivation of Gq/G11 family G proteins results in age-dependent epilepsy and impaired endocannabinoid formation

Metabotropic receptors coupled to Gq/G11 family G proteins critically contribute to nervous system functions by modulating synaptic transmission, often facilitating excitation. We investigated the role of Gq/G11 family G proteins in the regulation of neuronal excitability in mice that selectively lack the alpha-subunits of Gq and G11, G alpha q and G alpha 11, respectively, in forebrain principal neurons. Surprisingly, mutant mice exhibited increased seizure susceptibility, and the activation of neuroprotective mechanisms was impaired. We found that endocannabinoid levels were reduced under both basal and excitotoxic conditions and that increased susceptibility to kainic acid could be normalized by the enhancement of endocannabinoid levels with an endocannabinoid reuptake inhibitor, while the competitive cannabinoid type 1 receptor antagonist SR141716A did not cause further aggravation. These findings indicate that Gq/G11 family G proteins negatively regulate neuronal excitability in vivo and suggest that impaired endocannabinoid formation in the absence of Gq/G11 contributes to this phenotype.     

Screening for PTB domain binding partners and ligand specificity using proteome-derived NPXY peptide arrays

Modular interaction domains that recognize peptide motifs in target proteins can impart selectivity in signaling pathways. Phosphotyrosine binding (PTB) domains are components of cytoplasmic docking proteins that bind cell surface receptors through NPXY motifs. We have employed a library of human proteome-derived NXXY sequences to explore PTB domain specificity and function. SPOTS peptide arrays were used to create a comprehensive matrix of receptor motifs that were probed with a set of 10 diverse PTB domains. This approach confirmed that individual PTB domains have selective and distinct recognition properties and provided a means to explore over 2,500 potential PTB domain-NXXY interactions. The results correlated well with previously known associations between full-length proteins and predicted novel interactions, as well as consensus binding data for specific PTB domains. Using the Ret, MuSK, and ErbB2 receptor tyrosine kinases, we show that interactions of these receptors with PTB domains predicted to bind by the NXXY arrays do occur in cells. Proteome-based peptide arrays can therefore identify networks of receptor interactions with scaffold proteins that may be physiologically relevant.