p16Ink4a is overexpressed in H. pylori-associated gastritis and is correlated with increased epithelial apoptosis

Background. Cell cycle regulatory proteins may be critical targets during carcinogenesis. We have previously shown that chronic H. pylori infection is associated with decreased expression of the cyclin dependent kinase inhibitor (CDI) p27^kip1. Loss of p27^kip1 and p16^Ink4a (p16) expression, another CDI, has been reported during the progression of gastric tubular adenomas to advanced gastric cancer. The aim of the current study was to examine whether H. pylori infection also affects the expression of p16 in the gastric mucosa of H. pylori‐infected patients. Methods. p16 expression was evaluated in gastric antral biopsies by immunohistochemistry in 50 patients with nonulcer dyspepsia (n = 18 uninfected, n = 32 H. pylori infected, 24 by cagA⁺ strains). Adjacent sections were stained for proliferating epithelial cells (by Ki67) and for apoptotic cells (by TUNEL assay). Results. Both in H. pylori infected and uninfected patients the expression of p16 was higher in the neck and base of the gland than in the foveolar region. Epithelial staining for p16 was increased with H. pylori infection (31.3% vs. 11.1% in the foveolar region, 68.8% vs. 27.8% in the neck and 75% vs. 50% in the glandular base). There was no correlation between the expression of 16 and proliferation but there was a significant positive correlation between apoptosis and 16 immunostaining. Conclusions. The tumor suppressor gene 16 is over expressed in gastric epithelial cells of H. pylori infected patients and this is associated with an increase in apoptosis. These findings suggest a possible role for this cell cycle regulator in the increase in gastric cell turnover that is associated with H. pylori infection.     

A c-ras-Ki oncogene is activated, amplified and overexpressed in a human osteosarcoma cell line

We present a characterization of an activated oncogene which we found to be present in DNA of the OHA osteosarcoma cell line. We identify this tumor oncogene which transforms Swiss mouse 3T3-cells, with c-ras-Ki 2, one of two known members of the Kirsten ras family of human proto-oncogenes. Its structural outlines are given and we show that: 1) a single point mutation causing a substitution of valine for glycine in codon 12 was found by DNA sequencing; 2) the c-ras-Ki gene is amplified and overexpressed in the original OHA tumor cells and its transformants and 3) the gene product is an abnormal form of the p21 protein.     

Methotrexate in rheumatoid arthritis is frequently effective, even if re-employed after a previous failure

Effectiveness of therapy with individual disease-modifying antirheumatic drugs (DMARDs) in rheumatoid arthritis (RA) is limited, and the number of available DMARDs is finite. Therefore, at some stage during the lengthy course of RA, institution of traditional DMARDs that have previously been applied may have to be reconsidered. In the present study we investigated the effectiveness of re-employed methotrexate in patients with a history of previous methotrexate failure (original course). A total of 1,490 RA patients (80% female, 59% rheumatoid factor positive) were followed from their first presentation, yielding a total of 6,470 patient-years of observation. We identified patients in whom methotrexate was re-employed after at least one intermittent course of a different DMARD. We compared reasons for discontinuation, improvement in acute phase reactants, and cumulative retention rates of methotrexate therapy between the original course of methotrexate and its re-employment. Similar analyses were peformed for other DMARDs. Methotrexate was re-employed in 86 patients. Compared with the original courses, re-employment was associated with a reduced risk for treatment termination because of ineffectiveness (P = 0.02, by McNemar test), especially if the maximum methotrexate dose of the original course had been low (<12.5 mg/week; P = 0.02, by logistic regression). In a Cox regression model, re-employed MTX was associated with a significantly reduced hazard of treatment termination compared with the original course of methotrexate, adjusting for dose and year of employment (hazard ratio 0.64, 95% confidence interval 0.42-0.97; P = 0.04). These findings were not recapitulated in analyses of re-employment of other DMARDs. Re-employment of MTX despite prior inefficacy, but not re-employment of other DMARDs, is an effective therapeutic option, especially in those patients in whom the methotrexate dose of the original course was low.     

Induction of the p16INK4a senescence gene as a new therapeutic strategy for the treatment of rheumatoid arthritis.

Synovial tissue affected by rheumatoid arthritis is characterized by proliferation, which leads to irreversible cartilage and bone destruction. Current and experimental treatments have been aimed mainly at correcting the underlying immune abnormalities, but these treatments often prove ineffective in preventing the invasive destruction. We studied the expression of cyclin-dependent kinase inhibitors in rheumatoid synovial cells as a means of suppressing synovial cell proliferation. Synovial cells derived from hypertrophic synovial tissue readily expressed p16INK4a when they were growth-inhibited. This was not seen in other fibroblasts, including those derived from normal and osteoarthritis-affected synovial tissues. In vivo adenoviral gene therapy with the p16INK4a gene efficiently inhibited the pathology in an animal model of rheumatoid arthritis. Thus, the induction of p16INK4a may provide a new approach to the effective treatment of rheumatoid arthritis.     

LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing

Little is known about the protein constituents of autophagosome membranes in mammalian cells. Here we demonstrate that the rat microtubule-associated protein 1 light chain 3 (LC3), a homologue of Apg8p essential for autophagy in yeast, is associated to the autophagosome membranes after processing. Two forms of LC3, called LC3-I and -II, were produced post-translationally in various cells. LC3-I is cytosolic, whereas LC3-II is membrane bound. The autophagic vacuole fraction prepared from starved rat liver was enriched with LC3-II. Immunoelectron microscopy on LC3 revealed specific labelling of autophagosome membranes in addition to the cytoplasmic labelling. LC3-II was present both inside and outside of autophagosomes. Mutational analyses suggest that LC3-I is formed by the removal of the C-terminal 22 amino acids from newly synthesized LC3, followed by the conversion of a fraction of LC3-I into LC3-II. The amount of LC3-II is correlated with the extent of autophagosome formation. LC3-II is the first mammalian protein identified that specifically associates with autophagosome membranes.     

The significance of p16INK4a in cell defenses against transformation

Human cells, including fibroblast strains that have been immortalized by telomerase, are much more resistant to transformation than rodent cells. Most of the experimental evidence suggests that transformation of human fibroblasts requires inactivation of both the retinoblastoma (pRb) and p53 tumor suppressors as well as the addition of one or more dominant oncogenes. By starting with strains of primary fibroblast (Leiden and Q34 cells) that are genetically deficient for p16INK4a, we have been able to generate anchorage independent colonies simply by addition of telomerase (hTERT) and either Ras or Myc. Importantly, the transformed cells appear to retain pRb and p53 functions and are essentially diploid. Whereas Leiden cells expressing the individual oncogenes did not form tumors in mice, the combination of hTERT, Myc and Ras enabled them to become tumorigenic, albeit at a frequency suggestive of an additional genetic event. Significantly, we have obtained karyotypically stable tumors without the need to use DNA tumor virus oncoproteins and without deliberate ablation of p53.     

Pig-n, a mammalian homologue of yeast Mcd4p, is involved in transferring phosphoethanolamine to the first mannose of the glycosylphosphatidylinositol

Many cell surface proteins are anchored to the membrane via a glycosylphosphatidylinositol (GPI) moiety, which is attached to the C terminus of the proteins. The core of the GPI anchor is conserved in all eukaryotes but is modified by various side chains. We cloned a mouse phosphatidylinositol glycan-class N (Pig-n) gene that encodes a 931amino acid protein expressed in the endoplasmic reticulum, which is homologous to yeast Mcd4p. We disrupted the gene in F9 embryonal carcinoma cells. In the Pig-n knockout cells, the first mannose in the GPI precursors was not modified by phosphoethanolamine. Nevertheless, further biosynthetic steps continued with the addition of the third mannose and the terminal phosphoethanolamine. The surface expression of Thy-1 was only partially affected, indicating that modification of the first mannose by phosphoethanolamine is not essential for attachment of GPI anchors in mammalian cells. An inhibitor of GPI biosynthesis, YW3548/BE49385A, inhibited transfer of phosphoethanolamine to the first mannose in mammalian cells but only slightly affected the surface expression of GPI-anchored proteins. Biosynthesis of GPI in the Pig-n knockout cells was not affected by YW3548/BE49385A, and yeast overexpressing MCD4 was highly resistant to YW3548/BE49385A, suggesting that Pig-n and Mcd4p are targets of this drug.     

Interactions between highly conserved U2 small nuclear RNA structures and Prp5p, Prp9p, Prp11p, and Prp21p proteins are required to ensure integrity of the U2 small nuclear ribonucleoprotein in Saccharomyces cerevisiae.

Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA is an early and important step in spliceosome assembly. We searched for evidence of cooperative function between yeast U2 small nuclear RNA (snRNA) and several genetically identified splicing (Prp) proteins required for the first chemical step of splicing, using the phenotype of synthetic lethality. We constructed yeast strains with pairwise combinations of 28 different U2 alleles with 10 prp mutations and found lethal double-mutant combinations with prp5, -9, -11, and -21 but not with prp3, -4, -8, or -19. Many U2 mutations in highly conserved or invariant RNA structures show no phenotype in a wild-type PRP background but render mutant prp strains inviable, suggesting that the conserved but dispensable U2 elements are essential for efficient cooperative function with specific Prp proteins. Mutant U2 snRNA fails to accumulate in synthetic lethal strains, demonstrating that interaction between U2 RNA and these four Prp proteins contributes to U2 snRNP assembly or stability. Three of the proteins (Prp9p, Prp11p, and Prp21p) are associated with each other and pre-mRNA in U2-dependent splicing complexes in vitro and bind specifically to synthetic U2 snRNA added to crude splicing extracts depleted of endogenous U2 snRNPs. Taken together, the results suggest that Prp9p, -11p, and -21p are U2 snRNP proteins that interact with a structured region including U2 stem loop IIa and mediate the association of the U2 snRNP with pre-mRNA.     

Prp16p, Slu7p, and Prp8p interact with the 3' splice site in two distinct stages during the second catalytic step of pre-mRNA splicing.

For the second catalytic step of pre-mRNA splicing to occur, a 3' splice site must be selected and juxtaposed with the 5' exon. Four proteins, Prp16p, Slu7p, Prp17p, Prp18p, and an integral spliceosomal protein, Prp8p, are known to be required for the second catalytic step. prp8-101, an allele of PRP8 defective in 3' splice site recognition, exhibits specific genetic interactions with mutant alleles of the other second step splicing factors. The prp8-101 mutation also results in decreased crosslinking of Prp8p to the 3' splice site. To determine the role of the step-two-specific proteins in 3' splice site recognition and in binding of Prp8p to the 3' splice site, we performed crosslinking studies in mutant and immunodepleted extracts. Our results suggest an ordered pathway in which, after the first catalytic step, Prp16p crosslinks strongly to the 3' splice site and Prp8p and Slu7p crosslink weakly. ATP hydrolysis by Prp16p affects a conformational change that reduces the crosslinking of Prp16p with the 3' splice site and allows stronger crosslinking of Prp8p and Slu7p. Thus, the 3' splice site appears to be recognized in two stages during the second step of splicing. Strong 3' splice site crosslinking of Prp8p and Slu7p also requires the functions of Prp17p and Prp18p. Therefore, Prp8p and Slu7p interact with the 3' splice site at the latest stage of splicing prior to the second catalytic step that can currently be defined, and may be at the active site.     

Absence of mutagenicity of praziquantel, a new, effective, anti-schistosomal drug, in bacteria, yeasts, insects and mammalian cells.

Praziquantel (Embay 8440, Droncit) a new, effective anti-schistosomal drug, was tested in various short-term assays that have shown a predictive value for the detection of potential carcinogens. Indicator organisms S. typhimurium strains, S. pombe, S. cerevisiae, cultured V79 Chinese hamster cells or human heteroploid cells and Drosophila melanogaster were treated with Praziquantel. The induction of reverse and forward mutations, mitotic gene conversions, X-linked recessive lethals, sister-chromatid exchanges and unscheduled DNA-repair synthesis was scored; rodent-liver microsome-, cell- and host-mediated assays were also performed. Hycanthone, another schistosomicide was included as a positive control. The absence of a genetic activity of Praziquantel uniformly observed in such a battery of tests (i) confirms the assumption that the anti-schistosomal effectiveness of this drug is not related to the mutagenic activity and (ii) should encourage the implementation of extended clinical and field trials.     

The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line.

The Saccharomyces cerevisiae DPM1 gene product, dolichol-phosphate-mannose (Dol-P-Man) synthase, is involved in the coupled processes of synthesis and membrane translocation of Dol-P-Man. Dol-P-Man is the lipid-linked sugar donor of the last four mannose residues that are added to the core oligosaccharide transferred to protein during N-linked glycosylation in the endoplasmic reticulum. We present evidence that the S. cerevisiae gene DPM1, when stably transfected into a mutant Chinese hamster ovary cell line, B4-2-1, is able to correct the glycosylation defect of the cells. Evidence for complementation includes (i) fluorescence-activated cell sorter analysis of differential lectin binding to cell surface glycoproteins, (ii) restoration of Dol-P-Man synthase enzymatic activity in crude cell lysates, (iii) isolation and high-performance liquid chromatography fractionation of the lipid-linked oligosaccharides synthesized in the transfected and control cell lines, and (iv) the restoration of endoglycosidase H sensitivity to the oligosaccharides transferred to a specific glycoprotein synthesized in the DPM1 CHO transfectants. Indirect immunofluorescence with a primary antibody directed against the DPM1 protein shows a reticular staining pattern of protein localization in transfected hamster and monkey cell lines.     

The mouse SKD1, a homologue of yeast Vps4p, is required for normal endosomal trafficking and morphology in mammalian cells

The mouse SKD1 is an AAA-type ATPase homologous to the yeast Vps4p implicated in transport from endosomes to the vacuole. To elucidate a possible role of SKD1 in mammalian endocytosis, we generated a mutant SKD1, harboring a mutation (E235Q) that is equivalent to the dominant negative mutation (E233Q) in Vps4p. Overexpression of the mutant SKD1 in cultured mammalian cells caused defect in uptake of transferrin and low-density lipoprotein. This was due to loss of their receptors from the cell surface. The decrease of the surface transferrin receptor (TfR) was correlated with expression levels of the mutant protein. The mutant protein displayed a perinuclear punctate distribution in contrast to a diffuse pattern of the wild-type SKD1. TfR, the lysosomal protein lamp-1, endocytosed dextran, and epidermal growth factor but not markers for the secretory pathway were accumulated in the mutant SKD1-localized compartments. Degradation of epidermal growth factor was inhibited. Electron microscopy revealed that the compartments were exaggerated multivesicular vacuoles with numerous tubulo-vesicular extensions containing TfR and endocytosed horseradish peroxidase. The early endosome antigen EEA1 was also redistributed to these aberrant membranes. Taken together, our findings suggest that SKD1 regulates morphology of endosomes and membrane traffic through them.     

Functional analysis of the fission yeast Prp4 protein kinase involved in pre-mRNA splicing and isolation of a putative mammalian homologue.

The prp4 gene of Schizosaccharomyces pombe encodes a protein kinase. A physiological substrate is not yet known. A mutational analysis of prp4 revealed that the protein consists of a short N-terminal domain, containing several essential motifs, which is followed by the kinase catalytic domain comprising the C-terminus of the protein. Overexpression of N-terminal mutations disturbs mitosis and produces elongated cells, Using a PCR approach, we isolated a putative homologue of Prp4 from human and mouse cells. The mammalian kinase domain is 53% identical to the kinase domain of Prp4. The short N-terminal domains share <20% identical amino acids, but contain conserved motifs. A fusion protein consisting of the N-terminal region from S. pombe followed by the mammalian kinase domain complements a temperature-sensitive prp4 mutation of S. pombe. Prp4 and the recombinant yeast/mouse protein kinase phosphorylate the human SR splicing factor ASF/SF2 in vitro in its RS domain.     

Autoantibodies to BRAF,a new family of autoantibodies associated with rheumatoid arthritis

Introduction  

BRAF (v raf murine sarcoma viral oncogene homologue B1) is a serine-threonine kinase involved in the mitogen-activated protein kinase (MAPK) signalling pathway, known to be implicated in the production of pro-inflammatory cytokines.     

spp42, identified as a classical suppressor of prp4-73, which encodes a kinase involved in pre-mRNA splicing in fission yeast, is a homologue of the splicing factor Prp8p.

We have identified two classical extragenic suppressors, spp41 and spp42, of the temperature sensitive (ts) allele prp4-73. The prp4(+) gene of Schizosaccharomyces pombe encodes a protein kinase. Mutations in both suppressor genes suppress the growth and the pre-mRNA splicing defect of prp4-73(ts) at the restrictive temperature (36 degrees ). spp41 and spp42 are synthetically lethal with each other in the presence of prp4-73(ts), indicating a functional relationship between spp41 and spp42. The suppressor genes were mapped on the left arm of chromosome I proximal to the his6 gene. Based on our mapping data we isolated spp42 by screening PCR fragments for functional complementation of the prp4-73(ts) mutant at the restrictive temperature. spp42 encodes a large protein (p275), which is the homologue of Prp8p. This protein has been shown in budding yeast and mammalian cells to be a bona fide pre-mRNA splicing factor. Taken together with other recent genetic and biochemical data, our results suggest that Prp4 kinase plays an important role in the formation of catalytic spliceosomes.     

Absence of mutagenicity of praziquantel,a new,effective, anti-schistosomal drug,in bacteria,yeasts, insects and mammalian cells

Praziquantel (Embay 8440, Droncit) a new, effective anti-schistosomal drug, was tested in various short-term assays that have shown a predictive value for the detection of potential carcinogens. Indicator organisms S. typhimurium strains, S. pombe, S. cerevisiae, cultured V79 Chinese hamster cells or human heteroploid cells and Drosophila melanogaster were treated with Praziquantel. The induction of reverse and forward mutations, mitotic gene conversions, X-linked recessive lethals, sister-chromatid exchanges and unscheduled DNA-repair synthesis was scored; rodent-liver microsome-, cell- and host-mediated assays were also performed. Hycanthone, another schistosomicide was included as a positive control. The absence of a genetic activity of Praziquantel uniformly observed in such a battery of tests (i) confirms the assumption that the anti-schistosomal effectiveness of this drug is not related to the mutagenic activity and (ii) should encourage the implementation of extended clinical and field trials.     

Impaired regulation of Epstein-Barr virus-induced lymphocyte proliferation in rheumatoid arthritis is due to a T cell defect

The rate of outgrowth of EBV-infected B lymphocytes is regulated by normal T lymphocytes. Removal of T cells from normal whole lymphoid populations (PBM) markedly shortens the outgrowth time of the remaining B lymphocytes. There is little difference in the much more rapid outgrowth of rheumatoid PBM after the removal of T cells, which suggests that RA lymphoid cells are unable to regulate this process. To determine whether RA T cells are defective, or EBV-infected RA B cells are unresponsive to regulatory signals, EBV-induced outgrowth in autologous and allogeneic mixtures of RA and normal B and T cells was evaluated, employing morphologic criteria and 3H-thymidine incorporation. The difference in outgrowth between RA and normal PBM was reproduced by reconstitution of EBV-infected B cells with mitomycin-treated autologous T cells. In cell-mixing experiments, normal T cells appropriately regulated both normal and RA B cells similarly, whereas RA T cells were defective in regulating either B cell population. Thus, the rapid outgrowth of EBV-infected rheumatoid lymphoid cells is due to defective T cell regulation. Moreover, normal regulation does not require cell proliferation.     

DjPum, a homologue of Drosophila Pumilio, is essential to planarian stem cell maintenance

As stem cells are rare and difficult to study in vivo in adults, the use of classical models of regeneration to address fundamental aspects of the stem cell biology is emerging. Planarian regeneration, which is based upon totipotent stem cells present in the adult--the so-called neoblasts--provides a unique opportunity to study in vivo the molecular program that defines a stem cell. The choice of a stem cell to self-renew or differentiate involves regulatory molecules that also operate as translational repressors, such as members of PUF proteins. In this study, we identified a homologue of the Drosophila PUF gene Pumilio (DjPum) in the planarian Dugesia japonica, with an expression pattern preferentially restricted to neoblasts. Through RNA interference (RNAi), we demonstrate that gene silencing of DjPum dramatically reduces the number of neoblasts, thus supporting the intriguing hypothesis that stem cell maintenance may be an ancestral function of PUF proteins.