Signal transduction of somatostatin in human B lymphoblasts

Somatostatin (SST) and somatostatin receptors (SSTR) are widely distributed in lymphoid tissues. Here, we report on the stimulatory effects of SST in Epstein-Barr virus-immortalized B lymphoblasts. By RT-PCR, we demonstrated the exclusive expression of the somatostatin receptor isoform 2A (SSTR2A) in B lymphoblasts. Addition of SST rapidly increased the cytosolic free calcium concentration [Ca(2+)](i) maximally by about 200 nM, with an EC(50) of 1.3 nM, and stimulated the formation of inositol phosphates. Furthermore, SST increased binding of guanosine 5'-O-(3-thiotriphosphate) by 50% above basal. These effects were partly inhibited by pertussis toxin (PTX), which indicates the involvement of PTX-sensitive G proteins. We provide further evidence that Galpha(16,) a PTX-insensitive G protein confined to lymphohematopoietic cells, is involved in the otherwise unusual coupling of SSTR2A to phospholipase C activation. In addition, SST activated extracellular regulated kinases and induced a 3.5-fold stimulation of DNA synthesis and a 4.4-fold stimulation of B lymphoblast proliferation, which was accompanied by an enhanced immunoglobulin formation. Thus SST exerts a growth factor-like activity on human B lymphoblasts.     

A novel sequence framework (bla(TEM-1G)) encoding the parental TEM-1 beta-lactamase

A novel parental bla(TEM) gene (bla(TEM-1G)), encoding a TEM-1 beta-lactamase (pI of 5.4) produced by the uropathogenic Escherichia coli strain FMV194 was isolated from a dog. We report PCR-restriction fragment length polymorphism analysis and nucleotide sequencing of this gene. The bla(TEM-1G) sequence was identical to the bla(TEM-1C) gene framework in the coding and promoter (P3) regions, except for a silent G(604)-->T mutation in the coding region. Molecular phylogenetic analysis of parental bla(TEM) genes indicated two distinct groups, one comprising bla(TEM-1F) and bla(TEM-2). The other group comprises bla(TEM-1C) which is the probable ancestor of bla(TEM-1A), bla(TEM-1D) and bla(TEM-1G). The bla(TEM-1G) gene has the same framework as a gene encoding an inhibitor-resistant TEM beta-lactamase produced by an E. coli strain of human origin. Thus, parental bla(TEM) genes encoding beta-lactamases in E. coli strains isolated from different host species, in this case human and canine, may be phylogenetically very close.     

Structural and Sequence Evolution of U17 Small Nucleolar RNA (snoRNA) and Its Phylogenetic Congruence in Chelonians

Vertebrate U17 RNA is an intron-encoded H/CA box containing snoRNA, which has been intensively studied in the last decade, though its precise role in ribosome biogenesis is not yet clear. A consensus secondary structure for the U17 RNA molecule has been derived from the comparative sequence and structural evolution analysis of U17 snoRNA among vertebrates. Its phylogenetic congruence above class level has been tested and preliminary data on chelonians suggest that also in this order, U17 snoRNA evolved congruently with phylogeny. We herein extend our analysis to other components of this reptile group. According to the sequence data that have also emerged from chelonians, the U17 RNA molecule can be divided into two main domains: the 5-variable domain, which presents the sequence motifs capable of base-pairing with the 18S rRNA target and spanning STEM1, -2, and -3, and the 3-conserved domain, consisting of STEM4. In vertebrates, the latter RNA region shows a high conservation both in secondary structure and in the presence of the three sequence motifs 5-AUUCCUA-3, 5-U(G/U)ACU-3, and 5-AACCC-3. We tested the phylogenetic congruence of U17 evolution with chelonian relationships: Our results are significantly similar to those emerging from mtDNA and morphological systematics. Some discrepancies (e.g., the position of Platysternon) need to be addressed in greater depth.     

Receptor usage and fetal expression of ovine endogenous betaretroviruses: implications for coevolution of endogenous and exogenous retroviruses

Betaretroviruses of sheep include two exogenous viruses, Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV), and a group of endogenous viruses known as enJSRVs. The exogenous JSRV and ENTV are the etiological agents of ovine pulmonary adenocarcinoma (OPA) and enzootic nasal tumor (ENT), respectively. Sheep affected by OPA or ENT do not show an appreciable antibody response to JSRV or ENTV. Consequently, it is conceivable that enJSRV expression in the fetal lamb tolerizes sheep to the related exogenous viruses. In this study, possible mechanisms of interference between the sheep exogenous and endogenous betaretroviruses were investigated. In situ hybridization detected enJSRV RNAs in lymphoid cells associated with the lamina propria of the small intestine and in the thymus of sheep fetuses. Low-level expression of enJSRVs was also detected in the lungs. In addition, expression of enJSRVs was found to block entry of the exogenous JSRV, presumably via mechanisms of receptor interference. Indeed, enJSRVs, like JSRV and ENTV, were found to utilize hyaluronidase-2 as a cellular receptor.     

Comparison of fluorescence and resonance light scattering for highly sensitive microarray detection of bacterial pathogens

Microarrays have emerged as potential tools for bacterial detection and identification. Given their high parallelism, they might represent a breakthrough in current diagnostic methods, provided they can be coupled to simplified labeling protocols and detected with adequate sensitivities. We describe here a technique to directly label total bacterial RNA, thus avoiding the multiple steps and possible biases associated with enzymatic amplification (e.g. PCR). We have then compared the performances of one white-light source and two laser-based fluorescence scanners for detection reliability and sensitivity. Our study reveals that nanoparticle-labeled bacterial RNA generates reproducible resonance light scattering signals that are at least 50 times more intense than state-of-the-art confocal-based fluorescence signals.     

The ecological risks of transgenic plants.

Biotechnologies have been utilized "ante litteram" for thousands of years to produce food and drink and genetic engineering techniques have been widely applied to produce many compounds for human use, from insulin to other medicines. The debate on genetically modified (GM) organisms broke out all over the world only when GM crops were released into the field. Plant ecologists, microbiologists and population geneticists carried out experiments aimed at evaluating the environmental impact of GM crops. The most significant findings concern: the spread of transgenes through GM pollen diffusion and its environmental impact after hybridisation with closely related wild species or subspecies; horizontal gene transfer from transgenic plants to soil microbes; the impact of insecticide proteins released into the soil by transformed plants on non-target microbial soil communities. Recent developments in genetic engineering produced a technology, dubbed "Terminator", which protects patented genes introduced in transgenic plants by killing the seeds in the second generation. This genetic construct, which interferes so heavily with fundamental life processes, is considered dangerous and should be ex-ante evaluated taking into account the data on "unexpected events", as here discussed, instead of relying on the "safe until proven otherwise" claim. Awareness that scientists, biotechnologists and genetic engineers cannot answer the fundamental question "how likely is that transgenes will be transferred from cultivated plants into the natural environment?" should foster long-term studies on the ecological risks and benefits of transgenic crops.     

Human cutaneous melanoma expresses a significant phosphate-dependent glutaminase activity: a comparison with the surrounding skin of the same patient

The protein content and the activity and type of phosphate-dependent glutaminase were determined in freshly pigmented lesions obtained from human melanoma and adjacent skin. Significant phosphate-dependent glutaminase activity was found in both the melanoma and non-pigmented adjacent skin areas. A comparison between the pigmented and adjacent skin areas suggests the occurrence of gradual metabolic changes that result in an increased protein content in the centre of the neoplasia. The presence of a kidney-type glutaminase (K(m) of 2-5 mm) indicates a high sensitivity of the melanoma to variations in glutamine plasma levels (0.6 to 1 mm). These data lead us to postulate that glutamine supply is an important factor for melanoma cell proliferation, being a source of nitrogen for DNA and RNA synthesis. The intense neovascularization observed in melanoma ensures the oxygen supply that is required for glutamine oxidation. These findings support the proposition that glutamine is an important fuel for melanoma.     

Selection-driven evolution of emergent dengue virus

In the last four decades the incidence of dengue fever has increased 30-fold worldwide, and over half the world's population is now threatened with infection from one or more of four co-circulating viral serotypes (DEN-1 through DEN-4). To determine the role of viral molecular evolution in emergent disease dynamics, we sequenced 40% of the genome of 82 DEN-4 isolates collected from Puerto Rico over the 20 years since the onset of endemic dengue on the island. Isolates were derived from years with varying levels of DEN-4 prevalence. Over our sampling period there were marked evolutionary shifts in DEN-4 viral populations circulating in Puerto Rico; viral lineages were temporally clustered and the most common genotype at a particular sampling time often arose from a previously rare lineage. Expressed changes in structural genes did not appear to drive this lineage turnover, even though these regions include primary determinants of viral antigenic properties. Instead, recent dengue evolution can be attributed in part to positive selection on the nonstructural gene 2A (NS2A), whose functions may include replication efficiency and antigenicity. During the latest and most severe DEN-4 epidemic in Puerto Rico, in 1998, viruses were distinguished by three amino acid changes in NS2A that were fixed far faster than expected by drift alone. Our study therefore demonstrates viral genetic turnover within a focal population and the potential importance of adaptive evolution in viral epidemic expansion.     

The Candida albicans Cdr2p ATP-binding cassette (ABC) transporter confers resistance to caspofungin

Multidrug resistance may pose a serious problem to antifungal therapy. The Candida albicans Cdr2p is one of two ATP-binding cassette (ABC) transporters mediating antifungal resistance in vivo through increased drug efflux. Echinocandins such as caspofungin represent the newest class of antifungals that target cell wall synthesis. We show here by agar plate resistance assays that cross-resistant clinical isolates of C. albicans display high minimal inhibitory concentrations (MICs) to caspofungin when compared with a sensitive ATCC reference strain. Northern analysis and immunoblotting indicate that these isolates also show high levels of CDR1 and CDR2 expression. To determine a possible contribution of Cdr1p or Cdr2p to caspofungin resistance, we have functionally expressed Cdr1p and Cdr2p in appropriate recipient strains of the yeast Saccharomyces cerevisiae. Yeast cells expressing Cdr1p or Cdr2p exhibit cross-resistance to established antifungal drugs such as azoles and terbinafine. However, Cdr2p and, to a much lesser extent, Cdr1p confer caspofungin hyper-resistance when expressed in yeast. Likewise, Cdr2p confers caspofungin resistance when constitutively overexpressed in a drug-sensitive C. albicans strain. We therefore propose that Cdr2p may contribute to clinical candin resistance. Finally, our data suggest that cross-resistance phenotypes of clinical isolates are the consequence of distinct mechanisms that may operate simultaneously.