Purification and cDNA cloning of HeLa cell p54nrb, a nuclear protein with two RNA recognition motifs and extensive homology to human splicing factor PSF and Drosophila NONA/BJ6.

While searching for a human homolog of the S.cerevisiae splicing factor PRP18, we found a polypeptide that reacted strongly with antibodies against PRP18. We purified this polypeptide from HeLa cells using a Western blot assay, and named it p54nrb (for nuclear RNA-binding protein, 54 kDa). cDNAs encoding p54nrb were cloned with probes derived from partial sequence of the purified protein. These cDNAs have identical coding sequences but differ as a result of alternative splicing in the 5' untranslated region. The cDNAs encode a 471 aa polypeptide that contains two RNA recognition motifs (RRMs). Human p54nrb has no homology to yeast PRP18, except for a common epitope, but is instead 71% identical to human splicing factor PSF within a 320 aa region that includes both RRMs. In addition, both p54nrb and PSF are rich in Pro and Gln residues outside the main homology region. The Drosophila puff-specific protein BJ6, one of three products encoded by the alternatively spliced no-on-transient A gene (nonA), which is required for normal vision and courtship song, is 42% identical to p54nrb in the same 320 aa region. The striking homology between p54nrb, PSF, and NONA/BJ6 defines a novel phylogenetically conserved protein segment, termed DBHS domain (for Drosophila behavior, human splicing), which may be involved in regulating diverse pathways at the level of pre-mRNA splicing.     

Identification of synaptic targets of Drosophila pumilio

Drosophila Pumilio (Pum) protein is a translational regulator involved in embryonic patterning and germline development. Recent findings demonstrate that Pum also plays an important role in the nervous system, both at the neuromuscular junction (NMJ) and in long-term memory formation. In neurons, Pum appears to play a role in homeostatic control of excitability via down regulation of para, a voltage gated sodium channel, and may more generally modulate local protein synthesis in neurons via translational repression of eIF-4E. Aside from these, the biologically relevant targets of Pum in the nervous system remain largely unknown. We hypothesized that Pum might play a role in regulating the local translation underlying synapse-specific modifications during memory formation. To identify relevant translational targets, we used an informatics approach to predict Pum targets among mRNAs whose products have synaptic localization. We then used both in vitro binding and two in vivo assays to functionally confirm the fidelity of this informatics screening method. We find that Pum strongly and specifically binds to RNA sequences in the 3′UTR of four of the predicted target genes, demonstrating the validity of our method. We then demonstrate that one of these predicted target sequences, in the 3′UTR of discs large (dlg1), the Drosophila PSD95 ortholog, can functionally substitute for a canonical NRE (Nanos response element) in vivo in a heterologous functional assay. Finally, we show that the endogenous dlg1 mRNA can be regulated by Pumilio in a neuronal context, the adult mushroom bodies (MB), which is an anatomical site of memory storage.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Soluble ICAM-1 in breast cancer: clinical significance and biological implications

OBJECTIVES: In previous experiments, we demonstrated a decreased expression of intercellular adhesion molecule I (ICAM-1) on both tumour cells and antigen-presenting cells derived from patients with breast cancer, resulting in an abrogation of antigen presentation and tumour cell lysis. Recently, increased levels of a soluble isoform of ICAM-1 (sICAM-1) have been detected in the sera of breast cancer patients. The present investigation was performed in order to investigate the biological relevance of serum concentrations and the effects of sICAM-1 in patients with breast cancer. PATIENTS AND METHODS: sICAM-1 was determined using a sandwich enzyme immunoassay on sera from 88 patients with various stages of breast cancer and correlated with clinical parameters. The effect of sICAM-1 present in the sera of patients with breast cancer upon unspecific and anti-Her-21/neu antibody-mediated cytotoxicity (ADCC), as well as upon antigen presentation, was determined using a 51Cr-release assay and [3H]thymidine-uptake of T cells after co-incubation with tetanus-toxoid-pulsed antigen-presenting cells. RESULTS: In patients with early breast cancer, serum levels of sICAM-1 were significantly lower compared to patients with metastatic disease, but did not correlate with usual clinical parameters. In patients with metastatic breast cancer, a significant correlation of sICAM-1 with tumour markers CEA and CA 15-3 was observed. No influence of sICAM-1 upon unspecific cytotoxicity, ADCC, or the ability to present antigen was observed. DISCUSSION: The origin of sICAM-1 in the sera of patients with breast cancer remains unknown. In contrast to its membrane-bound isoform, sICAM-1 was increased in the sera of patients with various stages of breast cancer, but its presence did not influence unspecific cytotoxicity, ADCC, or antigen-induced T cell proliferation.     

A close look at late carboniferous algal mounds: Schulterkofel,Carnic Alps,Austria

Summary During the uppermost Carboniferous and lowermost Permian algal mounds were formed in inner shelf settings of the Carnic Alps (Austria/Italy). A specific mound type, characterized by the dominance of the dasyclad green alga Anthracoporella was studied in detail with regard to geometry, relationship between mound and intermound rocks, composition of the sediment, biota and diagenetic criteria. The two meter-sized mounds studied, occur within depositional sequences of transgressive systems tracts in the Lower Pseudoschwagerina Limestones (uppermost Gzhelian) at the flank of the Schulterkofel. The mounds consist of an Anthracoporella core facies with a spongecrust boundstone facies at the base and at the top. The massive limestones of the Anthracoporella core facies exhibit abundant algal tufts and bushes, frequently in life position. The limestones of the intermound facies represented by thin-bedded bioclastic wackestones and packstones with abundant phylloid algae underlie and overlie the mounds. Intercalations of intermound beds within the mound facies indicate sporadic disruption of mound growth. Onlapping of intermound beds on steep mound flanks indicate rapid stabilization and lithification of mound flanks and the existence of a positive paleorelief. Asymmetrical shape of the mounds may be current controlled. Mound and intermound biota differ in the prevailing algae but are relatively similar with regard to associated foraminifera. Conspicuous differences concern bioerosion and biogenic encrustations. Bothare, high in intermound areas but low in the Anthracoporella core facies. The mounds show no ecological zonation. The mounds grew by in-place accumulation of disintegrated algal material and trapped bioclastic material between erect algal thalli. The comparison of the various Anthracoporella mounds demonstrates that almost each mound had ist own history. Establishing a general model for these mounds is a hazardous venture.     

Determinants of the inherent strength of human 5' splice sites

We previously showed that the authentic 5' splice site (5'ss) of the first exon in the human beta-globin gene is intrinsically stronger than a cryptic 5'ss located 16 nucleotides upstream. Here we examined by mutational analysis the contribution of individual 5'ss nucleotides to discrimination between these two 5'ss. Based on the in vitro splicing efficiencies of a panel of 26 wild-type and mutant substrates in two separate 5'ss competition assays, we established a hierarchy of 5'ss and grouped them into three functional subclasses: strong, intermediate, and weak. Competition between two 5'ss from different subclasses always resulted in selection of the 5'ss that belongs to the stronger subclass. Moreover, each subclass has different characteristic features. Strong and intermediate 5'ss can be distinguished by their predicted free energy of base-pairing to the U1 snRNA 5' terminus (DeltaG). Whereas the extent of splicing via the strong 5'ss correlates well with the DeltaG, this is not the case for competition between intermediate 5'ss. Weak 5'ss were used only when the competing authentic 5'ss was inactivated by mutation. These results indicate that extensive complementarity to U1 snRNA exerts a dominant effect for 5'ss selection, but in the case of competing 5'ss with similarly modest complementarity to U1, the role of other 5'ss features is more prominent. This study reveals the importance of additional submotifs present in certain 5'ss sequences, whose characterization will be critical for understanding 5'ss selection in human genes.     

Intrinsic differences between authentic and cryptic 5' splice sites

Cryptic splice sites are used only when use of a natural splice site is disrupted by mutation. To determine the features that distinguish authentic from cryptic 5′ splice sites (5′ss), we systematically analyzed a set of 76 cryptic 5′ss derived from 46 human genes. These cryptic 5′ss have a similar frequency distribution in exons and introns, and are usually located close to the authentic 5′ss. Statistical analysis of the strengths of the 5′ss using the Shapiro and Senapathy matrix revealed that authentic 5′ss have significantly higher score values than cryptic 5′ss, which in turn have higher values than the mutant ones. β-Globin provides an interesting exception to this rule, so we chose it for detailed experimental analysis in vitro. We found that the sequences of the β-globin authentic and cryptic 5′ss, but not their surrounding context, determine the correct 5′ss choice, although their respective scores do not reflect this functional difference. Our analysis provides a statistical basis to explain the competitive advantage of authentic over cryptic 5′ss in most cases, and should facilitate the development of tools to reliably predict the effect of disease-associated 5′ss-disrupting mutations at the mRNA level.     

<Emphasis Type="Italic">Candida albicans</Emphasis>genome sequence: a platform for genomics in the absence of genetics

Publication of the complete diploid genome sequence of the yeast Candida albicans will accelerate research into the pathogenesis of Candida infections. Comparative genomic analysis highlights genes that may contribute to C. albicans survival and its fitness as a human commensal and pathogen.