Differential regulation of central nervous system autoimmunity by T(H)1 and T(H)17 cells

Multiple sclerosis is an inflammatory, demyelinating disease of the central nervous system (CNS) characterized by a wide range of clinical signs. The location of lesions in the CNS is variable and is a crucial determinant of clinical outcome. Multiple sclerosis is believed to be mediated by myelin-specific T cells, but the mechanisms that determine where T cells initiate inflammation are unknown. Differences in lesion distribution have been linked to the HLA complex, suggesting that T cell specificity influences sites of inflammation. We demonstrate that T cells that are specific for different myelin epitopes generate populations characterized by different T helper type 17 (T(H)17) to T helper type 1 (T(H)1) ratios depending on the functional avidity of interactions between TCR and peptide-MHC complexes. Notably, the T(H)17:T(H)1 ratio of infiltrating T cells determines where inflammation occurs in the CNS. Myelin-specific T cells infiltrate the meninges throughout the CNS, regardless of the T(H)17:T(H)1 ratio. However, T cell infiltration and inflammation in the brain parenchyma occurs only when T(H)17 cells outnumber T(H)1 cells and trigger a disproportionate increase in interleukin-17 expression in the brain. In contrast, T cells showing a wide range of T(H)17:T(H)1 ratios induce spinal cord parenchymal inflammation. These findings reveal critical differences in the regulation of inflammation in the brain and spinal cord.     

The kinetochore protein Cenp‐F is a potential novel target for zoledronic acid in breast cancer cells

The anti‐resorptive agent zoledronic acid inhibits key enzymes in the mevalonate pathway, disrupting post‐translational modification and thereby correct protein localization and function. Inhibition of prenylation may also be responsible for the reported anti‐tumour effects of zoledronic acid, but the specific molecular targets have not been identified. Cenp‐F/mitosin, a kinetochore‐associated protein involved in the correct separation of chromosomes during mitosis, has been shown to undergo post‐translational prenylation and may therefore be a novel target contributing to the anti‐tumour effects of zoledronic acid. We investigated whether zoledronic acid causes loss of Cenp‐F from the kinetochore in breast cancer cells, to determine if the reported anti‐tumour effects may be mediated by impairing correct chromosome separation. MDA‐MB‐436, MDA‐MB‐231 and MCF‐7 breast cancer cells and MCF‐10A non‐malignant breast epithelial cells were treated with zoledronic acid in vitro, and the effect on Cenp‐F localization was analysed by immunoflourescence microscopy. Zoledronic acid caused loss of Cenp‐F from the kinetochore, accompanied by an increase in the number of cells in pro‐, /prometa‐ and metaphase in all of the cancer cell lines. There was also a significant increase in the number of lagging chromosomes in mitotic cells. The effects of zoledronic acid could be reversed by inclusion of an intermediary of the mevalonate pathway, showing that the loss of Cenp‐F from the kinetochore was caused by the inhibition of farnesylation. In contrast, no effect was seen on Cenp‐F in non‐malignant MCF‐10A cells. This is the first report showing a specific effect of zoledronic acid on a protein involved in the regulation of chromosome segregation, identifying Cenp‐F as a potential new molecular target for NBPs in tumour cells.     

Enumeration of Archaea and Bacteria in seafloor basalt using real-time quantitative PCR and fluorescence microscopy

A SYBR Green real-time quantitative PCR (Q-PCR) assay for the detection and quantification of Bacteria and Archaea present in the glassy rind of seafloor basalts of different ages and water depths is presented. Two sets of domain-specific primers were designed and validated for specific detection and quantification of bacterial and archaeal 16S rRNA genes in DNA extracted from basaltic glass. Total cell numbers were also estimated by fluorescence microscopy analysis of SYBR Gold-stained samples. The results from the two different approaches were concurrent, and Q-PCR results showed that the total number of cells present in basalts was in the range from 6 x 10(5) to 4 x 10(6) cells g(-1) basaltic glass. Further, it was demonstrated that these cells were almost exclusively from the domain Bacteria. When applying the same methods on samples of different ages (22 years-0.1 Ma) and water depths (139-3390 mbsl), no significant differences in cell concentrations or in the relative abundance of Archaea and Bacteria were detected.     

An intronic splicing enhancer binds U1 snRNPs to enhance splicing and select 5' splice sites

Intronic G triplets are frequently located adjacent to 5' splice sites in vertebrate pre-mRNAs and have been correlated with splicing efficiency and specificity via a mechanism that activates upstream 5' splice sites in exons containing duplicated sites (26). Using an intron dependent upon G triplets for maximal activity and 5' splice site specificity, we determined that these elements bind U1 snRNPs via base pairing with U1 RNA. This interaction is novel in that it uses nucleotides 8 to 10 of U1 RNA and is independent of nucleotides 1 to 7. In vivo functionality of base pairing was documented by restoring activity and specificity to mutated G triplets through compensating U1 RNA mutations. We suggest that the G-rich region near vertebrate 5' splice sites promotes accurate splice site recognition by recruiting the U1 snRNP.     

Lithium leads to an increased FRQ protein stability and to a partial loss of temperature compensation in the Neurospora circadian clock

In many organisms, the presence of lithium leads to an increase of the circadian period length. In Neurospora crassa, it was earlier found that lithium results in a decrease of overall growth and increased circadian periods. In this article, the authors show that lithium leads to a reduction of FRQ degradation with elevated FRQ levels and to a partial loss of temperature compensation. At a concentration of 13 mM lithium, FRQ degradation is reduced by about 60% while, surprisingly, the activity of the 20S proteasome remains unaffected. Experiments and model calculations have shown that the stability of FRQ is dependent on its phosphorylation state and that increased FRQ protein stabilities lead to increased circadian periods, consistent with the observed increase of the period when lithium is present. Because in Neurospora the proteasome activity is unaffected by lithium concentrations that lead to significant FRQ stabilization, it appears that lithium acts as an inhibitor of kinases that affect phosphorylation of FRQ and other proteins. A competition between Li(+) and Mg(2+) ions for Mg(2+)-binding sites may be a mechanism to how certain kinases are inhibited by Li(+). A possible kinase in this respect is GSK-3, which in other organisms is known to be inhibited by lithium. The partial loss of temperature compensation in the presence of lithium can be understood as an increase in the overall activation energy of FRQ degradation. This increase in activation energy may be related to a reduction in FRQ phosphorylation so that more kinase activity, that is, higher temperature and longer times, is required to achieve the necessary amount of FRQ phosphorylation leading to turnover. Using a modified Goodwin oscillator as a semiquantitative model for the Neurospora clock, the effects of lithium can be described by adding lithium inhibitory terms of FRQ degradation to the model.     

Human U1 and U2 small nuclear ribonucleoproteins contain common and unique polypeptides.

Immunoprecipitation of human small nuclear ribonucleoproteins (snRNPs) containing the small nuclear RNAs U1, U2, U4, U5, and U6 with two antibodies produced in certain patients suffering from systemic lupus erythematosus was used to identify the polypeptides present on human U1 and U2 snRNPs. U1 and U2 snRNPs contain both common and unique polypeptides; visualization of the differences was possible through the use of non-methionine protein labeling and partial fractionation of snRNP populations. To facilitate comparisons with results from other laboratories, we have designated the snRNP polypeptides by their molecular weights. Four small polypeptides, P8, P9, P10, and P12, of 8,000 to 12,000 daltons, are each present in equal amounts on both U1 and U2 snRNPs. U1 snRNPs also contain a unique 30,000-dalton polypeptide, P30, whereas U2 snRNPs contain a unique 27,000-dalton, methionine-deficient polypeptide, P27. A closely migrating pair of polypeptides, P23 and P22, of 23,000 and 21,500 daltons, respectively, is present on both snRNPs; U2 snRNPs are enriched in the former, and U1 snRNPs are enriched in the latter.     

A Role for SRp54 during Intron Bridging of Small Introns with Pyrimidine Tracts Upstream of the Branch Point

One of the earliest steps in pre-mRNA recognition involves binding of the splicing factor U2 snRNP auxiliary factor (U2AF or MUD2 in Saccharomyces cerevisiae) to the 3′ splice site region. U2AF interacts with a number of other proteins, including members of the serine/arginine (SR) family of splicing factors as well as splicing factor 1 (SF1 or branch point bridging protein in S. cerevisiae), thereby participating in bridging either exons or introns. In vertebrates, the binding site for U2AF is the pyrimidine tract located between the branch point and 3′ splice site. Many small introns, especially those in nonvertebrates, lack a classical 3′ pyrimidine tract. Here we show that a 59-nucleotide Drosophila melanogaster intron contains C-rich pyrimidine tracts between the 5′ splice site and branch point that are needed for maximal binding of both U1 snRNPs and U2 snRNPs to the 5′ and 3′ splice site, respectively, suggesting that the tracts are the binding site for an intron bridging factor. The tracts are shown to bind both U2AF and the SR protein SRp54 but not SF1. Addition of a strong 3′ pyrimidine tract downstream of the branch point increases binding of SF1, but in this context, the upstream pyrimidine tracts are inhibitory. We suggest that U2AF- and/or SRp54-mediated intron bridging may be an alternative early recognition mode to SF1-directed bridging for small introns, suggesting gene-specific early spliceosome assembly.Pre-mRNA splicing is a conserved process occurring in a wide variety of eucaryotes with differing exon/intron architectures (reviewed in references 4, 6, 9, 15, 20, and 26). Vertebrates typically have small exons and large introns. Nonmetazoans frequently have the opposite genetic organization, with introns smaller than the minimum permissible for splicing of a vertebrate intron. Drosophila melanogaster possesses a mixture of these two classes of intron sizes (16, 23). In addition, more than half of the small introns in Drosophila are missing a prominent vertebrate splicing signal, the 3′ polypyrimidine tract (23). For these reasons, Drosophila provides a model system in which to study potential mechanistic variations operating during recognition of splicing signals.In the general model of early vertebrate spliceosome complex assembly, U1 snRNP binds to the 5′ splice site and U2 snRNP auxiliary factor (U2AF) binds to the 3′ polypyrimidine tract, thereby facilitating U2 snRNP interaction with the branch point. Various members of the serine/arginine (SR) family of proteins may participate by promoting or stabilizing these interactions (reviewed in references 13, 22, and 31). This family of proteins may also act as exon or intron bridging factors via their SR-mediated interaction with SR domains on the small subunit of U2AF (U2AF³⁵) and the U1 70K protein (32, 33, 38). SF1, originally discovered as an essential splicing factor in reconstitution assays (19), has also been observed to bind to the branch point (7, 8). In yeast, BBP (branch point bridging protein), the ortholog to SF1, functions as an intron bridging factor via interactions with U1 snRNP-associated proteins and the large subunit of U2AF (U2AF⁶⁵) (1, 2). It is assumed that vertebrate SF1 can play a similar role, although the mammalian equivalents to the yeast U1 snRNP proteins that interact with BBP have not yet been identified. Furthermore, the relationship between bridging by SR proteins and that afforded by SF1 is unclear.We have previously examined the cis-acting sequences required for efficient splicing of a constitutively spliced small (59-nucleotide [nt]) intron from the D. melanogaster mle gene that lacks a well-defined pyrimidine tract between the branch point and 3′ splice site (18, 29). Assembly of initial ATP-dependent spliceosomes (complex A) on the mle intron requires both the 5′ and 3′ splice sites, suggesting concerted recognition of the entire intron (29). Instead of a classic pyrimidine tract, the mle intron contains two C-rich tracts located between the 5′ splice site and branch point that are necessary for efficient splicing of this intron (18). In addition to a requirement for maximal splicing efficiency, the pyrimidine stretches are also necessary for binding of U2AF, interaction of factors with the 5′ splice site, and proper assembly of the active spliceosome, suggesting that these sequences affect early assembly events at both ends of this small intron. Interestingly, the upstream C-rich tracts are inhibitory if a classical 3′ pyrimidine tract is introduced between the branch point and 3′ splice site (18). This observation suggests competing pathways of factor binding to this substrate and also raises the possibility of alternative gene-specific modes of association of constitutive factors with introns.Here we demonstrate that both U2AF and an SR protein, SRp54, interact with the C-rich tracts in the mle intron. The central location of the pyrimidine tracts, their importance for maximal splicing, and the ability of human SRp54 to interact with U2AF⁶⁵ instead of U2AF³⁵ (37) suggested that the binding of SRp54 to the tracts could replace SF1 in bridging this intron. Immunoprecipitation studies using an antibody specific for SF1 indicated that SF1 did not contact mle precursor RNA unless a pyrimidine tract was introduced downstream of the branch point. Furthermore, antibodies against either SRp54 or U2AF immunoprecipitated both halves of a precleaved mle splicing substrate, suggesting that these factors either directly or indirectly interact with both the 5′ and 3′ splice sites. We suggest that SRp54 participates in bridging the small mle intron via its ability to bind both the C-rich tracts and the large subunit of U2AF.     

An intron enhancer containing a 5' splice site sequence in the human calcitonin/calcitonin gene-related peptide gene.

Regulation of calcitonin (CT)/calcitonin gene-related peptide (CGRP) RNA processing involves the use of alternative 3' terminal exons. In most tissues and cell lines, the CT terminal exon is recognized. In an attempt to define regulatory sequences involved in the utilization of the CT-specific terminal exon, we performed deletion and mutation analyses of a mini-gene construct that contains the CT terminal exon and mimics the CT processing choice in vivo. These studies identified a 127-nucleotide intron enhancer located approximately 150 nucleotides downstream of the CT exon poly(A) cleavage site that is required for recognition of the exon. The enhancer contains an essential and conserved 5' splice site sequence. Mutation of the splice site resulted in diminished utilization of the CT-specific terminal exon and increased skipping of the CT exon in both the mini-gene and in the natural CT/CGRP gene. Other components of the intron enhancer modified utilization of the CT-specific terminal exon and were necessary to prevent utilization of the 5' splice site within the intron enhancer as an actual splice site directing cryptic splicing. Conservation of the intron enhancer in three mammalian species suggests an important role for this intron element in the regulation of CT/CGRP processing and an expanded role for intronic 5' splice site sequences in the regulation of RNA processing.     

Temperature-Sensitive Mutants Blocked in the Folding or Subunit Assmbly of the Bacteriophage P22 Tailspike Protein. I. Fine-Structure Mapping

As part of a study of protein folding, we have constructed a fine-structure map of 9 existing and 29 newly isolated UV- and hydroxylamine-induced temperature-sensitive (ts) mutations in gene 9 of Salmonella bacteriophage P22. Gene 9 specifies the polypeptide chain of the multimeric tail spikes, six of which form the cell attachment organelle of the phage. The 38 ts mutants were mapped against deletion lysogens with endpoints in gene 9. They mapped in 10 of the 15 deletion intervals. Two- and three-factor crosses between mutants within each interval indicated that at least 31 ts sites are represented among the 38 mutants. To determine the distribution of ts sites within the physical map, we identified the protein fragments from infection of su^- hosts with 10 gene 9 amber mutants. Their molecular weights, ranging from 13,900 to 55,000 daltons, were combined with the genetic data to yield a composite map of gene 9. The 31 ts sites were distributed through most of the gene, but were most densely clustered in the central third.—None of the ts mutant pairs tested exhibited intragenic complementation. Studies of the defective phenotypes of the ts mutants (Goldenberg and King 1981; Smith and King 1981) revealed that most do not affect the thermostability of the mature protein, but instead prevent the folding or subunit assembly of the mutant chains synthesized at restrictive temperature. Thus, many of thes ts mutations identify sites in the polypeptide chain that are critical for the folding or maturation of the tail-spike protein.     

Identification of P48 and P54 as components of bacteriophage T4 baseplates.

The involvement of two bacteriophage T4 gene products in the initiation of T4 tail tube and sheath polymerization on mature baseplates has been studied by radioautography of acrylamide gels of various partially completed tail structures. The products of genes 48 and 54 (P48[the nomenclature P48 refers to the protein product of bacteriophage T4 gene 48] and P54), which are known to be required for the synthesis of mature baseplates, have been shown to be structural components of the baseplate. These gene products have molecular weights of 42,000 and 33,000, respectively. The addition of P54 to the baseplate not only permits the polymerization of the core protein, P19, onto the baseplate, but also caused the disappearance of a polypeptide of molecular weight about 15,000 from the supernatant fraction of infected cells. Another gene product, P27, has been identified in the crude extracts of infected cells. This gene product, which is required for the synthesis of baseplate structures, has the same mobility as one of the unidentified structural polypeptides of the baseplate and is therefore probably also a baseplate component.