Discovery of 342 putative new genes from the analysis of 5'-end-sequenced full-length-enriched cDNA human transcripts

In this work we describe the process that, starting with the production of human full-length-enriched cDNA libraries using the CAP-Trapper method, led us to the discovery of 342 putative new human genes. Twenty-three thousand full-length-enriched clones, obtained from various cell lines and tissues in different developmental stages, were 5'-end sequenced, allowing the identification of a pool of 5300 unique cDNAs. By comparing these sequences to various human and vertebrate nucleotide databases we found that about 40% of our clones extended previously annotated 5' ends, 662 clones were likely to represent splice variants of known genes, and finally 342 clones remained unknown, with no or poor functional annotation. cDNA-microarray gene expression analysis showed that 260 of 342 unknown clones are expressed in at least one cell line and/or tissue. Further analysis of their sequences and the corresponding genomic locations allowed us to conclude that most of them represent potential novel genes, with only a small fraction having protein-coding potential.     

Genetic dissection of herbicide tolerance in maize by molecular markers

In order to detect the genetic architecture of maize tolerance to Alachlor, a widely used chloroacetanilide, linkage analysis between the expression of the trait and allelic composition of molecular markers was performed. The experiment was carried out on a population of 142 recombinant inbred lines, developed starting from the F₁ between two lines with different reactivity to the herbicide, and self-fertilized for 10 generations; the lines were typed by 48 RFLP markers and 66 microsatellites (SSR). Besides seedling tolerance, evaluated as proportion of normal (non-injured) plants after herbicide treatment, other minor components of tolerance were studied: seed germination ability, pollen germination and tube growth in the presence of the herbicide. The analysis, performed by three statistical methods, revealed the presence of factors controlling seedling tolerance on seven chromosomal regions. Five QTLs appeared to be involved in seed germination ability in the presence of Alachlor, four QTLs in pollen tolerance in terms of germination and four in tube growth under stress were detected. Three loci, on chromosomes 1, 7 and 10, explained most of the variation of seedling tolerance, thus being interesting candidate for marker-assisted selection.     

Enhanced clearing of Helicobacter pylori after omeprazole plus roxithromycin treatment

The in vitro antibacterial activity of omeprazole against eight strains of Helicobacter pylori was evaluated. Minimum inhibitory concentration (MIC) values were 32 micrograms/ml and 64 micrograms/ml (MIC50 and MIC90 respectively). We performed a randomized single blind study comparing the efficacy of omeprazole alone (for 4 weeks) or combined with roxithromycin (for 2 weeks) in the treatment of duodenal ulcer and chronic active gastritis associated with H. pylori infection, H. pylori was eradicated in 75% of patients treated with omeprazole alone whereas the patients treated with the combination of these drugs were completely free from H. pylori at the end of the therapy.     

Expression of interferon genes in murine macrophages: Possible role of endogenous interferon in the modulation of cell differentiation

The expression of interferon (IFN)- gene was studied in mouse peritoneal macrophages (PM) harvested from normal mice (lps ⁿ ) or LPS-hyporesponsive mice (lps ^d ). A strong direct correlation between the LPS response of PM and their capacity to expressing basal levels of IFN was found. The results suggest that the constitutive expression of IFN- gene can play an important role not only in the resistance to viral infection but also in the modulation of cell differentiation.     

Interaction of DNA with cationic liposomes: ability of transfecting lentil protoplasts.

The vesicle made of dipalmitoylphosphatidylcholine and stearylamine (9:1) were multilamellar and rather homogeneous in shape as seen by transmission electron microscopy. Upon addition of circular DNA plasmids of different lengths to the liposomes, the formation of vesicle clusters around the DNA filament was observed, with dimensions dictated by the ratio DNA/lipid. These liposomes were able to transfect lentil (Lens culinaris) protoplasts inside the cells two different reporter genes, chloramphenicol-acetyltransferase and beta-glucuronidase. The activity of these two enzymes could be found in the cell lysates after 24 h from the incubation of protoplasts with the lipid-DNA complexes.     

Clinical and cytogenetic staging of chronic myeloid leukemia. Philadelphia positive

A cooperative study between clinical and cytogenetic steps in 44 patients with Ph'+ CGL is reported in order to verific the usefulness of the cytogenetic screening for the diagnosis and the right classification of the patients. The study of the clinical steps is carried out on the basis of the parameters suggested by Tura and coll.; in the one of the cytogenetic steps Sandberg classification modified by the Authors is adopted. In 40 cases the comparison shows a marrow correspondence between clinical and cytogenetic steps. In fact the overage survival in the classical true steps of the disease is almost the sance in the 1st and 2nd step. In the 3rd step the survival is strongly reduced meaning that the more chromosome alterations are observed the more survival is reduced.     

Mutational analysis of cell cycle arrest, nuclear localization and virion packaging of human immunodeficiency virus type 1 Vpr.

Human immunodeficiency virus type 1 Vpr is a virion-associated, regulatory protein that is required for efficient viral replication in monocytes/macrophages. The protein is believed to act in conjunction with the Gag matrix protein to allow import of the viral preintegration complex in nondividing cells. In cells, Vpr localizes to the nucleus. Recently, we showed that Vpr prevents the activation of p34cdc2-cyclin B. This results in arrest of Vpr-expressing cells in the G2/M phase of the cell cycle. Here, we use a panel of expression vectors encoding Vpr molecules mutated in the amino-terminal alpha-helical region, the central hydrophobic region, or the carboxy-terminal basic region to define the functional domains of the protein. The results showed cell cycle arrest was largely controlled by the carboxy-terminal basic domain of the protein. In contrast, the amino-terminal alpha-helical region of Vpr was required for nuclear localization and packaging into virions. The carboxy terminus appeared to be unnecessary for nuclear localization. In the alpha-helical region, mutation of Ala-30 to Pro resulted in a protein that localized to the cytoplasm. Surprisingly, fusion of Vpr to luciferase resulted in a molecule that failed to localize to the nucleus. In addition, we show that simian immunodeficiency virus Vpr, but not Vpx, induces G2 arrest. We speculate that Vpr has two sites for interaction with cellular factors: one in the alpha-helical region that specifies nuclear localization and one in the carboxy-terminal domain that is required for Cdc2 inhibition.     

Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity.

The Vpr accessory gene product of human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus is believed to play a role in permitting entry of the viral core into the nucleus of nondividing cells. A second role for Vpr was recently suggested by Rogel et al. (M. E. Rogel, L. I. Wu, and M. Emerman, J. Virol. 69:882-888, 1995), who showed that Vpr prevents the establishment in vitro of chronically infected HIV producer cell lines, apparently by causing infected cells to arrest in the G2/M phase of the cell cycle. In cycling cells, progression from G2 to M phase is driven by activation of the p34cdc2/cyclin B complex, an event caused, in part, by dephosphorylation of two regulatory amino acids of p34cdc2 (Thr-14 and Tyr-15). We show here that Vpr arrests the cell cycle in G2 by preventing the activation of the p34cdc2/cyclin B complex. Vpr expression in cells caused p34cdc2 to remain in the phosphorylated, inactive state, p34cdc2/cyclin B complexes immunoprecipitated from cells expressing Vpr were almost completely inactive in a histone H1 kinase assay. Coexpression of a constitutively active mutant p34cdc2 molecule with Vpr relieved the G2 arrest. These findings strongly suggest that Vpr arrests cells in G2 by preventing the activation of the p34cdc2/cyclin B complex that is required for entry into M phase. In vivo, Vpr might, by preventing p34cdc2 activation, delay or prevent apoptosis of infected cells. This would increase the amount of virus each infected cell produced.     

Respiratory gas monitoring in patients with chronic respiratory failure during intensive positive pressure ventilation vs aerosol therapy

Summary The aim of this study is to evaluate the variations of respiratory gas concentration with transcutaneous blood gas measurements (tcm PO₂; tcm PCO₂) during intermittent positive pressure ventilation (I.P.P.V.) and aerosol therapy in patients (5 female, 5 male; 43–75 years) with chronic obstructive pulmonary diseases (COPD) with haemo gas analysis values PO₂<76 mmHg. The subjects underwent transcutaneous blood gas monitoring, 16 minutes during therapy (I.P.P.V. or aerosol) and 14 minutes of recovery. ANOVA analysis of variance was used for statistical analysis. During I.P.P.V. tcm PO₂ increase from the base value (60.6±9 mmHg) already after 2 minutes until the 16th minute (69.7±9); 2 minutes after the end of I.P.P.V. tcm PO₂ falls below the basic one (57.6±7) and then exceeds the basic value until the 14th minute (63.5±10) (p=ns). The base tcm PCO₂ (41.2±7) decreases reaching the maximum decrement at the 16th minute of therapy (32±6.6); at the end of I.P.P.V. it increases without exceeding the base value (39.5±7) (p<0.01). During aerosol therapy, tcm PO₂ increases from the basic one (62.4±10) (maximum after 16 minutes 70±9.5), then it decreases (64±11) and increases againg reaching the maximum growth after 14 minutes of recovery (65.7±11) (p=ns). The tcm PCO₂ values show a decrement below the base value (42.7±4.5) reaching its maximum at the 16th minute of therapy (38.8±7); at the end of aerosol the tem PCO₂ increases (42.5±4.6) (p=ns). The tcm PO₂ variations (I.P.P.V. vs aerosol) did not show any significant statistical difference. At the end of the therapies the tcm PO₂ falls a little more after I.P.P.V. than after aerosol (57.6±7.3 vs 64±11, p=ns); the tcm PCO₂ base values are similar during the two therapies (41.2±7 vs 42.7±4.5, p=ns), but the tcm PCO₂ decrements reach a significant statistical difference after 8–10–12–16 minutes (p<0.05); during the recovery time after 2 minutes (34±6.5 vs 40±6.3, p<0.05) and this difference disappeared at the last recording. We calculate the tcm PO₂ increment and the tcm PCO₂ decrement from the base value. The tcm PO₂ increment is higher during I.P.P.V. than during aerosol therapy. The tcm PCO₂ decrement shows how the tcm PCO₂ falls during I.P.P.V. and how these values remain below the base one until the end of the rest time. Data reported here demonstrate that mechanical physiokinesi therapy improves COPD ventilation so that the respiratory pattern could be ameliorated and preserved.     

Exploring the homogeneity of terrestrial subterranean communities at a local spatial scale

1. Although caves are generally perceived as isolated habitats, at the local scale, they are often interconnected via a network of fissures in the bedrock. Accordingly, caves in close proximity are expected to host the same, or very similar, animal communities. 2. We explored the extent to which subterranean arthropod communities are homogenous at a local spatial scale of less than 1 km², along with which cave-specific environmental features result in a departure from the expected homogeneous pattern. We approached this question by studying richness and turnover in terrestrial invertebrate communities of 27 caves in a small karst massif in the Western Italian Alps. 3. Specialised subterranean species were homogeneously distributed among caves and were not influenced by seasonality. The only factor driving their distribution was the distance from the cave entrance, with deeper caves yielding a greater diversity of species. 4. Significant spatio-temporal turnover in species not specialised to subterranean life was observed. In summer, there was a significant homogenisation of the community and a more even distribution of species among sites; in winter, these species were missing or found exclusively at greater depths, where environmental conditions were more stable. Furthermore, caves at lower elevations yielded, on average, a greater diversity and abundance of these species. 5. This study demonstrated that the theoretical expectation of no turnover in community composition in caves in close proximity is not always met. Turnover can be mostly attributed to seasonal patterns and sampling depth; thus, our findings have implications for planning sampling and monitoring activities in caves.