Potential secondary structure at translation-initiation sites.

Since translational start codons also occur internally, more-complex features within mRNA must determine initiation. We compare the potential secondary structure of 123 prokaryotic mRNA start regions to that of regions coding for internal methionines. The latter display an unexpectedly-uniform, almost-periodic pattern of pairing potential. In contrast, sequences 5' to start codons have little self-pairing, and do not pair extensively with the proximal coding region. Pairing potential surrounding start codons was found to be less than half of that found near internal AUGs. In groups of random sequences where the distribution of nucleotides at each position, or of trinucleotides at each in-frame codon position, matched the observed natural distribution, there was no periodicity in the pairing potential of the internal sequences. Randomized internal sequences had less pairing: the ratio of pairing intensity between internals and starts was reduced from 2.0 to 1.6 by randomization. We propose that the transition from the relatively-unstructured start domains to the highly-structured internal sequences may be an important determinant of translational start-site recognition.     

Estrogen-induced lysosomal proteases secreted by breast cancer cells: A role in carcinogenesis?

In an attempt to understand the mechanism by which estrogens stimulate cell proliferation and mammary carcinogenesis, metastatic human breast cancer cell lines (MCF7, ZR75-1) were found to secrete a 52,000 dalton (52K) protein under estrogen stimulation. Following its purification to homogeneity, the 52K protein was identified as a secreted procathepsin-D-like aspartyl protease bearing mannose-6-phosphate signals. This precursor displays an in vitro autocrine mitogenic activity on estrogen-deprived MCF7 cells and is able to degrade basement membrane and proteoglycans following its autoactivation. The total protease (52K + 48K and 34K) was detected and assayed by monoclonal antibodies and was found to be highly concentrated in proliferative and cystic mastopathies. In breast cancer, its cytosolic concentration appears to be correlated more to tumor invasiveness than to hormone responsiveness. The mRNA of the 52K protease accumulates rapidly following estradiol treatment, as was shown by Northern blot analysis with cloned cDNA. The 52K cathepsin-D-like protease is the first example of a lysosomal protease induced by estrogens in cancer cells. Results obtained using different approaches suggest that two cysteinyl cathepsins are also related to cell transformation and invasiveness. It has been proposed that cathepsin-B is involved in breast cancer and metastatic melanoma, and its regulation by estrogen has been shown in the rat uterus. Cathepsin-L corresponds to the major excreted protein (MEP) whose synthesis and secretion are markedly increased by transformation of NIH 3T3 cells with Ki ras and are regulated by several growth factors. In addition to secreted autocrine growth factors and to other proteases (plasminogen activator, collagenase), lysosomal cathepsins may therefore play an important role in the process of tumor growth and invasion as long as their precursor is secreted abundantly.     

Site selection by the tRNA splicing endonuclease of Xenopus laevis

To investigate the mechanism by which the purified Xenopus tRNA splicing endonuclease recognizes its splice sites, we utilized yeast pre-tRNA(3Leu) and pre-tRNA(Phe) variants constructed by in vitro mutagenesis. We found that the endonuclease interacts with conserved features of the mature tRNA domain. In particular, U8 and C56 may be examples of contact points between protein and RNA. Given that there are no conserved sequences at the splice junctions, the specificity of cutting at both splice sites is determined by the length of the anticodon stem. Although in general, the sequence of the intron is unimportant for splicing, there are some structural requirements.     

Distinct role for CD8 T cells toward cutaneous tumors and visceral metastases

The growth of immunogenic tumors in immunocompetent individuals is one of the oldest conundrums in tumor immunology. Although the ability of mouse CD8+ T cells to control transplanted tumors is well documented, little is known about their impact on autochthonous tumors. To gain insight into the role of CD8+ T cells during the course of cancer development, we produced a novel model of spontaneous melanoma. The metallothionein (MT)-ret/AAD mouse is transgenic for the RET oncogene and the chimeric MHC molecule AAD (alpha1-alpha2 domains of HLA-A2 linked to alpha3 domain of H2-Dd). This model recapitulates the natural history of human melanoma, and expression of the AAD molecule makes it suitable for analyzing CD8+ T cell responses directed against peptide Ags that have been previously identified in HLA-A2+ melanoma patients. We found that, as tumors grow, mice develop a broad melanoma-specific CD8+ T cell response. Occurrence of cutaneous nodules is not affected by CD8+ T cell depletion, showing that although CD8+ T cells are functional, they have no effect on established cutaneous tumors. However, depleted mice die from visceral disease much earlier than controls, showing that CD8+ T cells control metastasis spreading and disease progression. Antigenic modulation is observed in visceral metastases, suggesting that visceral nodules may be subject to immunoediting. Our data demonstrate that growth of melanoma in the MT-ret/AAD model involves several tolerance mechanisms sequentially. They also reveal a different role for CD8+ T cells toward early stage of cutaneous tumors and late visceral metastatic stage of the disease.     

Vaccination of a melanoma patient with mature dendritic cells pulsed with MAGE-3 peptides triggers the activity of nonvaccine anti-tumor cells

We previously characterized the CTL response of a melanoma patient who experienced tumor regression following vaccination with an ALVAC virus coding for a MAGE-A3 Ag. Whereas anti-vaccine CTL were rare in the blood and inside metastases of this patient, anti-tumor CTL recognizing other tumor Ags, mainly MAGE-C2, were 100 times more frequent in the blood and considerably enriched in metastases following vaccination. In this study we report the analysis of the CTL response of a second melanoma patient who showed a mixed tumor response after vaccination with dendritic cells pulsed with two MAGE-A3 antigenic peptides presented, respectively, by HLA-A1 and HLA-DP4. Anti-MAGE-3.A1 CD8 and anti-MAGE-3.DP4 CD4 T cells became detectable in the blood after vaccination at a frequency of approximately 10(-5) among the CD8 or CD4 T cells, respectively, and they were slightly enriched in slowly progressing metastases. Additional anti-tumor CTL were present in the blood at a frequency of 2x10(-4) among the CD8 T cells and, among these, an anti-MAGE-C2 CTL clone was detected only following vaccination and was enriched by >1,000-fold in metastases relative to the blood. The striking similarity of these results with our previous observations further supports the hypothesis that the induction of a few anti-vaccine T cells may prime or restimulate additional anti-tumor T cell clones that are mainly responsible for the tumor regression.     

Phase-I study of Innacell γδ™, an autologous cell-therapy product highly enriched in γ9δ2 T lymphocytes, in combination with IL-2, in patients with metastatic renal cell carcinoma

PURPOSE: gamma9delta2 T lymphocytes have been shown to be directly cytotoxic against renal carcinoma cells. Lymphocytes T gammadelta can be selectively expanded in vivo with BrHPP (IPH1101, Phosphostim) and interleukin 2 (IL-2). A phase I Study was conducted in patients with metastatic renal cell carcinoma (mRCC) to determine the maximum-tolerated dose and safety of Innacell gammadeltatrade mark, an autologous cell-therapy product based on gamma9delta2 T lymphocytes, in patients with mRCC. EXPERIMENTAL DESIGN: A 1-h intravenous infusion of gamma9delta2 T lymphocytes was administered alone during treatment cycle 1 and combined with a low dose of subcutaneous interleukin-2 (IL-2, 2 MIU/m(2) from Day 1 to Day 7) in the two subsequent cycles (at 3-week intervals). The dose of gamma9delta2 T lymphocytes was escalated from 1 up to 8 x 10(9) cells. RESULTS: Ten patients underwent a total of 27 treatment cycles. Immunomonitoring data demonstrate that gamma9delta2 T lymphocytes are initially cleared from the blood to reappear at the end of IL-2 administration. Dose-limiting toxicity occurred in one patient at the dose of 8 x 10(9) cells (disseminated intravascular coagulation). Other treatment-related adverse events (AEs) included mainly gastrointestinal disorders and flu-like symptoms (fatigue, pyrexia, rigors). Hypotension and tachycardia also occurred, especially with co-administered IL-2. Six patients showed stabilized disease. Time to progression was 25.7 weeks. CONCLUSION: The data collected in ten patients with mRCC indicate that repeated infusions of Innacell gammadeltatrade mark at different dose levels (up to 8 x 10(9) total cells), either alone or with IL-2 is well tolerated. These results are in favor of the therapeutic value of cell therapy with Innacell gammadeltatrade mark for the treatment of cancers.     

Massive presence of insertion sequences in the genome of SOPE, the primary endosymbiont of the rice weevil Sitophilus oryzae

Bacteria that establish an obligate intracellular relationship with eukaryotic hosts undergo an evolutionary genomic reductive process. Recent studies have shown an increase in the number of mobile elements in the first stage of the adaptive process towards intracellular life, although these elements are absent in ancient endosymbionts. Here, the genome of SOPE, the obligate mutualistic endosymbiont of rice weevils, was used as a model to analyze the initial events that occur after symbiotic integration. During the first phases of the SOPE genome project, four different types of insertion sequence (IS) elements, belonging to well-characterized IS families from gamma-proteobacteria, were identified. In the present study, these elements, which may represent more than 20% of the complete genome, were completely characterized; their relevance as a source of gene inactivation, chromosomal rearrangements, and as participants in the genome reductive process are discussed herein.     

DING proteins; novel members of a prokaryotic phosphate-binding protein superfamily which extends into the eukaryotic kingdom

PstS proteins are the cell-bound phosphate-binding elements of the ubiquitous bacterial ABC phosphate uptake mechanisms. Primary and tertiary structures, characteristic of pstS proteins, are conserved in proteins, which are expressed in secretory operons and induced by phosphate deprivation, in Pseudomonas species. There are two subsets of these proteins; AP proteins, which are alkaline phosphatases, and DING proteins, named for their N-terminal sequence, which are phosphate-binding proteins. Both form elements of a proposed phosphate-scavenging system in pseudomonads. DING proteins have also been isolated from many eukaryotic sources, and are associated with both normal and pathological functions in mammals. Their phosphate-binding function suggests a role in biomineralization, but the ability to bind other ligands may be related to signal transduction in eukaryotes. Though it has been claimed that all such proteins may originate from pseudomonads, many eukaryotic DING proteins have unique features which are incompatible with a bacterial origin.     

Comparative evaluation of Strepto B ID chromogenic medium and Granada media for the detection of Group B streptococcus from vaginal samples of pregnant women

Two types of selective media, the chromogenic medium Strepto B ID® and two non-chromogenic media Strepto B agar® and the Granada® medium, were tested and compared to blood agar plates (BAP) for screening of Group B streptococcus vaginal colonization in pregnant women. All tested media were comparable in terms of sensitivity however, their use in routine laboratories may markedly facilitate the rapid detection of GBS in vaginal samples.