Accumulation of a novel spliceosomal complex on pre-mRNAs containing branch site mutations.

Pre-mRNA assembles into spliceosomal complexes in the stepwise pathway E-->A-->B-->C. We show that mutations in the metazoan branchpoint sequence (BPS) have no apparent effect on E complex formation but block the assembly of the A complex and the UV cross-linking of U2 small nuclear ribonucleoprotein particle (snRNP) proteins. Unexpectedly, a novel complex, designated E*, assembles on pre-mRNAs containing BPS mutations. Unlike the E complex, the E* complex accumulates in the presence of ATP. U1 snRNP and U2AF, which are tightly bound to pre-mRNA in the E complex, are not tightly bound in the E* complex. Significantly, previous work showed that U1 snRNP and U2AF become destabilized from pre-mRNA after E complex assembly on normal pre-mRNAs. Thus, our data are consistent with a model in which there are two steps in the transition from the E complex to the A complex (E-->E*-->A). In the first step, U1 snRNP and U2AF are destabilized in an ATP-dependent, BPS-independent reaction. In the second step, the stable binding of U2 snRNP occurs in a BPS-dependent reaction.     

Macrophage colony-stimulating factor induces thrombospondin I production by cultured human macrophages

The role of colony-stimulating factors (CSFs) in regulating the synthesis of thrombospondin 1 (TSP1) by cultured human macrophages is investigated. Macrophage (M)-CSF is shown rapidly and transiently to induce two predominant species of TSP1 mRNA. One of these species was 3.2 kb in size and appeared to be specific to M-CSF-stimulated macrophages. Adherent M-CSF-treated macrophages are also shown to express abundant surface cell-associated TSP rapidly when examined by indirect immunofluorescence staining. Granulocyte-macrophage (GM)-CSF induced TSP1 mRNA at a later time point, and this was attributable to the effects of endogenous M-CSF induced by the GM-CSF; the GM-CSF-treated cells did not display surface-associated TSP after 3 hr of treatment. Analysis of the TSP1 protein synthesised by the M-CSF-treated macrophages revealed the expected trimeric form of the molecule. In addition, an unidentified 95-kDa protein was found to be covalently associated with immunoreactive TSP1, and this appeared to be specific to the macrophages as it was not found in TSP1 precipitated from other cell types. It is suggested that the induction of TSP1 by M-CSF may play an important role in the major physiological functions of macrophages. © 1995 Wiley-Liss, Inc.     

Design of metal ion binding peptides

Two cyclic and branched peptides (PLA and AZU) were synthesized with the aim of reproducing the active site of the blue copper proteins plastocyanin and azurin. Both peptides, designed on the basis of the x-ray structures of Poplar plastocyanin and Alcaligenes denitrificans azurin. contain the same coordinating residues of the parent native proteins. The visible spectra of PLA in the presence of equimolar amount of Cu(II) strongly support the interaction between the peptide and copper(II) ion. The CD titration of AZU with the Hg(II) ion indicates for the formation of two species. [A ZUHg]⁺ and [A ZUHg₂]³⁺ having binding constants (K_eq) of 3.10⁶ and 2–10⁴M^?1, respectively. © 1994 John Wiley & Sons, Inc.     

Parametric sensitivity of stoichiometric flux balance models applied to wild-type Escherichia coli metabolism

The esterification of lauric acid with geraniol catalyzed by the commercially immobilized lipase preparation from Mucor miehei, Lipozyme(R), was studied in well-stirred flasks. The enzyme support was characterized in terms of its internal and external surface area, protein location, and protein content. It was found that the enzyme was mainly located on the external surface of the support, therefore, internal diffusional limitations were not important. It was also shown that the protein content of the support depends on the size of the particle, with smaller particles containing higher amounts of protein per unit weight. Under the conditions studied, the reaction was not under external mass transfer limitations, and the initial reaction rate depended on the size of the support particles. This was mainly due to the different protein contents on the support as a function of particle size and not to internal or external mass transfer limitations. Also, it was found that the inhibition exerted by water was predominantly a physical effect due to its accumulation around the enzyme. It was also found that the reaction was substrate inhibited by lauric acid, but not by geraniol. (c) 1995 John Wiley & Sons, Inc.     

Comparative chromosome painting between two marsupials: origins of an XX/XY1Y2 sex chromosome system

Cross-species chromosome painting was used to investigate genome rearrangements between tammar wallaby Macropus eugenii (2n = 16) and the swamp wallaby Wallabia bicolor (2n = 10♀/11♂), which diverged about 6 million years ago. The swamp wallaby has an XX female:XY₁Y₂ male sex chromosome system thought to have resulted from a fusion between an autosome and the small original X, not involving the Y. Thus, the small Y₁ should represent the original Y and the large Y₂ the original autosome. DNA paints were prepared from flow-sorted and microdissected chromosomes from the tammar wallaby. Painting swamp wallaby spreads with each tammar chromosome-specific probe gave extremely strong and clear signals in single-, two-, and three-color FISH. These showed that two tammar wallaby autosomes are represented unchanged in the swamp wallaby, two are represented by different centric fusions, and one by a tandem fusion to make the very long arms of swamp wallaby Chromosome (Chr) 1. The large swamp wallaby X comprises the tammar X as its short arm, and a tandemly fused 7 and 2 as the long arm. The acrocentric swamp wallaby Y₂ is a 2/7 fusion, homologous with the long arm of the X. The small swamp wallaby Y₁ is confirmed as the original Y by its painting with the tammar Y. However, the presence of sequences shared between the microdissected tammar Xp and Y on the swamp wallaby Y₂ implies that the formation of the compound sex chromosomes involved addition of autosome(s) to both the original X and Y. We propose that this involved fusion with an ancient pseudoautosomal region followed by fission proximal to this shared region. Received: 16 October 1996/Accepted: 30 January 1997     

Model for prediction of immobilizedAcetobacter cell number in calcium alginate gel droplets

The geometry of calcium alginate gel spheres is studied by fractal interpretation for prediction of number of cells to be immobilized. For alginate concentrations from 1 to 5% the simulated results are of 6.65×10⁸, 1.44×10⁸ and 5.27×10⁷ N cell mL⁻¹ for respectively 1.5, 2.5 and 3.5 mm gel sphere diameters. Simulated data are compared with those resulting from literature and particularly with experimental trials where from 10⁷ to 10⁸ N cell mL⁻¹, in 2% alginate beads with a diameter of 1.5 mm, is able to fulfil mechanical and swelling characteristics of alginate gel beads besides to supply good oxygenation.     

Efficacy of the alcohol use disorders identification test as a screening tool for hazardous alcohol intake and related disorders in primary care: a validity study.

OBJECTIVE: To determine the properties of the alcohol use disorders identification test in screening primary care attenders for alcohol problems. DESIGN: A validity study among consecutive primary care attenders aged 18-65 years. Every third subject completed the alcohol use disorders identification test (a 10 item self report questionnaire on alcohol intake and related problems) and was interviewed by an investigator with the composite international diagnostic interview alcohol use module (a standardised interview for the independent assessment of alcohol intake and related disorders). SETTING: 10 primary care clinics in Verona, north eastern Italy. PATIENTS: 500 subjects were approached and 482 (96.4%) completed evaluation. RESULTS: When the alcohol use disorders identification test was used to detect subjects with alcohol problems the area under the receiver operating characteristic curve was 0.95. The cut off score of 5 was associated with a sensitivity of 0.84, a specificity of 0.90, and a positive predictive value of 0.60. The screening ability of the total score derived from summing the responses to the five items minimising the probability of misclassification between subjects with and without alcohol problems provided an area under the receiver operating characteristic curve of 0.93. A score of 5 or more on the five items was associated with a sensitivity of 0.79, a specificity of 0.95, and a positive predictive value of 0.73. CONCLUSIONS: The alcohol use disorders identification test performs well in detecting subjects with formal alcohol disorders and those with hazardous alcohol intake. Using five of the 10 items on the questionnaire gives reasonable accuracy, and these are recommended as questions of choice to screen patients for alcohol problems.     

Effects of water level fluctuations on phytoplankton in a river-floodplain lake system (Paraná River,Argentina)

Several aspects of community organization wereanalyzed comparatively in a small side-arm of theParaná River (Correntoso) and a shallowfloodplain lake (El Tigre) (31° 41 S and60° 42 W), in relation to the hydrology of thesystem. Taxonomic and morphological composition inthe river differed from that in the lake: the riverhad lower species richness (151 vs 218),different contributions of some Classes to totalspecies number (higher Cyano-, Zygo- andDiatomophyceae vs higher Chlorophyceae), anddiffent proportions of nannoplanktonic algae (67.5%vs 80.7%) and netplanktonic filamentousspecies (18.2% vs 4.2%). Phytoplanktonbiomass, higher in the lake than in the river due tothe retention time, was mostly dominated bynannoplankton and netplankton. Loticphytoplankton was dominated by typical fluvialspecies of Diatomophyceae (R-strategists). Riverconditions seem to maintain a subclimacticcommunity, which was little impacted by the flushingof populations from floodplain lakes. Water levelwas the main factor controlling phytoplanktonbiomass, species diversity (H), evenness (E) andcommunity change rate () in the river. Inthe lake, phytoplankton had an autogenicsuccessional sequence during the isolation phase (C-to S-strategists) and other responses todisturbance, mainly during the flood(R-strategists). Frequent changes in phytoplanktoncomposition, biomass, H, E and , revealed aenvironmental instability in the lake, which may beexplained by interactions of external factors(hydrology and climatology) and those of internalorigin, such as nutrients and grazing.