The RNA-binding protein Sex-lethal (Sxl) is a critical regulator of sexual differentiation and dosage compensation in Drosophila. This regulatory activity is a consequence of the ability of Sxl to ...bind uridine-rich RNA tracts involved in pre-mRNA splicing. Sxl contains two RNP consensus-type RNA-binding domains (RBDs). A structural study of a portion of Sxl (amino acids 199-294) containing the second RNA-binding domain (RBD-2) using multidimensional heteronuclear NMR is presented here. Nearly complete 1H, 13C, and 15N resonance assignments have been obtained from 15N- and 13C/15N-uniformly labeled protein. These assignments were used to analyze 3D 15N-separated NOESY and 13C/13C-separated 4D NOESY spectra which produced 494 total and 169 long-range NOE-derived distance restraints. Along with 41 backbone dihedral restraints, these distance restraints were employed to generate an intermediate-resolution family of calculated structures, which exhibits the beta alpha beta-beta alpha beta tertiary fold found in other RBD-containing proteins. The RMSD to the average structure for the backbone atoms of residues 11-93 is 1.55 +/- 0.30 A, while the RMSD for backbone atoms involved in secondary structure is 0.76 +/- 0.14 A. A capping box Harper, E.T., & Rose, G.D. (1993) Biochemistry 32, 7605-7609 was identified at the N-terminus of the first helix and has been characterized by short- and medium-range NOEs. Finally, significant structural similarities and differences between Sxl RBD-2 and other RBD-containing proteins are discussed.
BackgroundObinutuzumab is an anti-CD20 monoclonal antibody approved in combination with chlorambucil for patients with chronic lymphocytic leukaemia (CLL) and comorbidities, making them unsuitable ...for full-dose fludarabine-based therapy.PurposeTo analyse the safety and effectiveness of obinutuzumab combined with chlorambucil as first-line treatment in our hospital.Material and methodsObservational, retrospective, descriptive study. Inclusion criteria: adults (>18 years) that initiate treatment with obinutuzumab-chlorambucil. Study period: January 2017 to September 2018. Demographic variables: gender, age; clinical variables: diagnose and cumulative illness rating scale (CIRS); and therapy-related: adverse events and suspension. Safety was evaluated in all patients that received at least one obinutuzumab dose by analysing adverse events (AE) from clinical records, treatment delays and/or concomitant medication required. AE are classified following the 5.0 version of National Institute Cancer: Common Terminology Criteria for Adverse Events. Effectiveness was evaluated following International Workshop CLL; Halleck 2008 criteria a minimum of 3 months after the end of treatment.ResultsSeven patients were included (four male and three female), median age: 72 years (rank 67–82). Median CIRS punctuation: 9 (rank 6–11). All patients received premedication with corticosteroids, antipyretic and antihistaminic to avoid infusion-related reactions (IRRs) and allopurinol as prophylaxis for tumour lysis syndrome.During the first infusion, two patients presented hypertension, abdominal pain and cold as grade 1–2 IRRs requiring temporary interruption. IRRs were not recorded in the following perfusions.Four patients presented haematologic toxicity, grade 3 neutropenia, requiring G-CSF, treatment delay was only required in one of them.Other AE: grade 2 anaemia treated with erythropoietin (n=1), grade 2 thrombocytopenia (n=1), respiratory infections (n=2; one patient with hypogammaglobulinaemia previous to treatment required hospital admission and treatment suspension).By the time the study was finished, effectiveness was evaluated in four up to six patients that finished treatment: complete response (n=3) and partial response (n=1).ConclusionIn our experience, the obinuzumab-chlorambucil scheme presented a good safety profile in patients with comorbidities. The main AE were IRRs: limited to first administration that did not require treatment suspension; and neutropaenia, which was the most frequent haematologic toxicity.Regarding response, a continuous monitoring is necessary to confirm long-term effectiveness.References and/or acknowledgementsNo conflict of interest.
Background. It has long been proposed that secreted proteinases, including the matrix metalloproteinases, play an important part in tumor progression in mediating extracellular matrix remodeling. ...More recently, it has been suggested that extracellular proteinases also regulate growth factors and cytokines that may contribute to tumor progression.
Methods. RNA in situ hybridization and immunohistochemistry were used to study the expression, in breast and other types of human carcinomas, of the stromelysin‐3 (ST3) gene, which encodes a putative new member of the matrix metalloproteinase family.
Results. The ST3 gene is overexpressed in most types of human carcinomas, including breast carcinoma where ST3 RNA was detected in 95% (99 of 104) of invasive primary tumors. Both ST3 protein and RNA are detected in fibroblastic cells immediately surrounding the cancer cells, but not in the malignant cells or in stromal cells at a distance from them. The ST3 gene also is expressed in some in situ breast carcinomas, where ST3 expression correlates with the known risk of these tumors to become invasive.
Conclusions. ST3 is the paradigm of tumor proteinases that are not expressed in the malignant cells of human carcinomas but in fibroblastic cells of tumor stroma. ST3 represents a potential new prognostic parameter to identify subpopulations of aggressive tumors, particularly to evaluate the likelihood of in situ breast carcinoma progression to invasive cancer. Furthermore, the specific expression of the ST3 gene in fibroblastic cells immediately surrounding cancer cells suggests that ST3 may be involved in tumor progression and that it represents a potential target for cancer treatment.
Marine stratocumulus clouds of the eastern Pacific play an essential role in the earth's energy and radiation budget. Parts of these clouds off the western coast of South America form the major ...source of water to the hyperarid Atacama Desert coastal region at the northern coast of Chile. For the first time, a full year of vertical structure observations of the coastal stratocumulus and their environment is presented and analyzed. Installed at Iquique Airport in northern Chile in 2018/2019, three state-of-the-art remote sensing instruments provide vertical profiles of cloud macro- and micro-physical properties, wind, turbulence, and temperature as well as integrated values of water vapor and liquid water.
Distinct diurnal and seasonal patterns of the stratocumulus life cycle are observed. Embedded in a land–sea circulation with a superimposed southerly wind component, maximum cloud occurrence and vertical extent occur at night but minima at local noon. Nighttime clouds are maintained by cloud-top cooling, whereas afternoon clouds reappear within a convective boundary layer driven through local moisture advection from the Pacific. During the night, these clouds finally re-connect to the maritime clouds in the upper branch of the land–sea circulation. The diurnal cycle is much more pronounced in austral winter, with lower, thicker, and more abundant (5×) clouds than in summer. This can be associated with different sea surface temperature (SST) gradients in summer and winter, leading to a stable or neutral stratification of the maritime boundary layer at the coast of the Atacama Desert in Iquique.
U1 snRNP is required at an early stage during assembly of
the spliceosome, the dynamic ribonucleoprotein (RNP) complex
that performs nuclear pre-mRNA splicing. Here, we report the
purification of U1 ...snRNP particles from Drosophila
nuclear extracts and the characterization of their biochemical
properties, polypeptide contents, and splicing activities.
On the basis of their antigenicity, apparent molecular
weight, and by peptide sequencing, the Drosophila
70K, SNF, B, U1-C, D1, D2, D3, E, F, and G proteins are
shown to be integral components of these particles. Sequence
database searches revealed that both the U1-specific and
the Sm proteins are extensively conserved between human
and Drosophila snRNPs. Furthermore, both species
possess a conserved intrinsic U1-associated kinase activity
with identical substrate specificity in vitro. Finally,
our results demonstrate that a second type of functional
U1 particle, completely lacking the U1/U2-specific protein
SNF and the associated protein kinase activity, can be
isolated from cultured Kc cell or Canton S embryonic nuclear
extracts. This work describes the first characterization
of a purified Drosophila snRNP particle and reinforces
the view that their activity and composition, with the
exception of the atypical bifunctional U1-A/U2-B″
SNF protein, are highly conserved in metazoans.
In human carcinomas, stromelysin-3/matrix metalloproteinase 11 (ST3, MMP11) expression by nonmalignant fibroblastic cells located in the immediate vicinity of cancer cells is a bad prognostic factor. ...Using mouse models of primary tumors, it has been demonstrated that ST3 is a key player during local invasion, favoring cancer cell survival in connective tissue through an antiapoptotic function. To investigate the ST3 impact on additional phases of cancer cell invasion, we developed mammary gland cancer prone MMTV-ras transgenic mice in wild-type (ras+/+;ST3+/+) or ST3-deficient (ras+/+;ST3-/-) genotype and studied their whole natural cancer history. The tumor-free survival and delay between the first ras oncogenic hit and primary tumor appearance increased in ras+/+;ST3-/- mice (P < 0.000001 and <0.0000007, respectively). A systematic search for occult primary tumors and metastases revealed, in addition to a lower total number and size of primary tumors (P < 0.02), an unexpected higher number of metastases (P < 0.01) in ras+/+;ST3-/- mice. Moreover, for a similar number and size of primary invasive tumors, ras+/+;ST3-/- mice developed more metastases, indicating that the cancer cells evolving in ST3-deficient stroma have an increased potential to hematogenous dissemination. We conclude that the ST3 microenvironment is a consistently active partner of invading cancer cells but that its function differs throughout cancer progression, being tumor enhancer or repressor in processes leading to local or distal invasion. Such a dual effect for an MMP might shed light, at least partially, for the absence of survival benefit for patients included in anti-MMP clinical trials.
Synthetic mRNA precursors from the Drosophila fushi tarazu (ftz) gene were shown to be accurately and efficiently spliced in Drosophila nuclear extracts derived from Kc tissue culture cells or 0- to ...12-hr embryos. Splicing the ftz pre-mRNA requires ATP and low levels of Mg2+. The reaction proceeds with a lag of 20-30 min prior to appearance of spliced mRNA and appears to proceed in two steps. The first step is cleavage at the 5′ splice site to generate a 5′ exon (E1) fragment and an intron-3′ exon (IVS-E2) species. The second step involves cleavage at the 3′ splice site, ligation of the two exons (E1-E2), and intron (IVS) release. The excised intron (IVS) and intron-3′ exon (IVS-E2) exhibit anomalous electrophoretic mobility, suggesting that they contain branched structures. Nuclease analysis using two-dimensional thin-layer chromatography indicates that both the IVS and IVS-E2 species possess branched trinucleotides in which a guanosine residue at the 5′ end of the intron is linked in a 2′-5′ phosphodiester bond to the 2′ hydroxyl group of an adenosine residue in the intron. The site of branchpoint formation was localized by debranching the Drosophila Iariat with mammalian (HeLa) cell debranching enzyme and by P1 and T2 nuclease analysis. These findings indicate that nuclear extracts derived from Drosophila cultured cells or embryos can accurately splice mRNA precursors and that the reaction mechanism is the same as has been observed in yeast and mammalian cells. This system provides an initial step toward the biochemical analysis of developmentally regulated pre-mRNA splicing events in Drosophila.
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