A -1 ribosomal frameshift in a double-stranded RNA virus of yeast forms a gag-pol fusion protein.

The L-A double-stranded RNA (dsRNA) virus of Saccharomyces cerevisiae has two open reading frames (ORFs). ORF1 encodes the 80-kDa major coat protein (gag). ORF2, which is expressed only as a 180-kDa fusion protein with ORF1, encodes a single-stranded RNA-binding domain and has the consensus sequence for RNA-dependent RNA polymerases of (+)-strand and double-stranded RNA viruses (pol). We show that the 180-kDa protein is formed by -1 ribosomal frame-shifting by a mechanism indistinguishable from that of retro-viruses. Analysis of the "slippery site" suggests that a low probability of unpairing of the aminoacyl-tRNA from the 0-frame codon at the ribosomal A site reduces the efficiency of frameshifting more than the reluctance of a given tRNA to have its wobble base mispaired. Frameshifting of L-A requires a pseudoknot structure just downstream of the shift site. The efficiency of the L-A frameshift site is 1.8%, similar to the observed molar ratio in viral particles of the 180-kDa fusion protein to the major coat protein.     

RNA sequence of astrovirus: distinctive genomic organization and a putative retrovirus-like ribosomal frameshifting signal that directs the viral replicase synthesis.

The genomic RNA of human astrovirus was sequenced and found to contain 6797 nt organized into three open reading frames (1a, 1b, and 2). A potential ribosomal frameshift site identified in the overlap region of open reading frames 1a and 1b consists of a "shifty" heptanucleotide and an RNA stem-loop structure that closely resemble those at the gag-pro junction of some retroviruses. This translation frame-shift may result in the suppression of in-frame amber termination at the end of open reading frame 1a and the synthesis of a nonstructural, fusion polyprotein that contains the putative protease and RNA-dependent RNA polymerase. Comparative sequence analysis indicated that the protease and polymerase of astrovirus are only distantly related to the respective enzymes of other positive-strand RNA viruses. The astrovirus polyprotein lacks the RNA helicase domain typical of other positive-strand RNA viruses of similar genome size. The genomic organization and expression strategy of astrovirus, with the protease and the polymerase brought together by predicted frameshift, most closely resembled those of plant leuteoviruses. Specific features of the sequence and genomic organization support the classification of astroviruses as an additional family of positive-strand RNA viruses, designated Astroviridae.     

Molecular structure of a double helix that has non-Watson—Crick type base pairing formed by 2-substituted poly(A) and poly(U)

X-ray fiber diffraction studies of duplexes formed by 2-substituted poly(A) and poly(U) provide evidence for the existence of a double helical structure of poly(A)·poly(U) held together by Hoogsteen-type base pairing with parallel chain polarity.     

Minor histocompatibility antigens HA-1-, -2-, and -4-, and HY-specific cytotoxic T-cell clones inhibit human hematopoietic progenitor cell growth by a mechanism that is dependent on direct cell-cell contact

HLA-identical bone marrow transplantation (BMT) may be complicated by graft-versus-host disease or graft rejection. Both complications are thought to be initiated by recognition of minor histocompatibility (mH) antigens by HLA-restricted mH-antigen-specific T lymphocytes. Using HLA- A2-restricted mH antigens HA-1-, -2-, and -4-, and HY-specific cytotoxic T lymphocyte (CTL) clones, we studied the recognition by these CTL clones of interleukin-2 (IL-2)-stimulated T cells (IL-2 blasts), BM mononuclear cells (BMMNCs), and hematopoietic progenitor cells (HPCs). We showed that, when IL-2 blasts from the BM donors who were investigated were recognized by the HA-1-, -2-, and -4-, and HY- specific CTL clones, their BMMNCs and HPCs were recognized as well by these CTL clones, resulting in antigen-specific growth inhibition of erythrocyte burst-forming units (BFU-E), colony-forming units- granulocyte (CFU-G), and CFU-macrophage (CFU-M). the HA-2-specific CTL clone, however, inhibited BFU-E and CFU-G growth from four donors to a lesser extent than from two other donors. We further investigated whether inhibitory cytokines released into the culture medium by the antigen-specific stimulated CTLs or by stimulated BMMNCs were responsible for suppression of HPC growth or whether this effect was caused by direct cell-cell contact between CTLs and HPCs. HPC growth inhibition was only observed after preincubation of BMMNCs and CTLs together for 4 hours before plating the cells in semisolid HPC culture medium. When no cell-cell contact was permitted before plating, neither antigen-stimulated CTL nor antigen-nonstimulated CTLs provoked HPC growth inhibition. Culturing BMMNCs in the presence of supernatants harvested after incubation of BMMNCs and CTL clones together for 4 or 72 hours did also not result in HPC growth inhibition. Both suppression of HPC growth and lysis of IL-2 blasts and BMMNCs in the 51Cr-release assay appeared to be dependent on direct cell-cell contact between target cells and CTLs and were not caused by the release of inhibitory cytokines into the culture medium by antigen-specific stimulated CTLs or by stimulated BMMNCs. Our results show that mH-antigen-specific CTLs can inhibit HPC growth by a direct cytolytic effect and may therefore be responsible for BM graft rejection after HLA-identical BMT.     

A new case of combined factor V and factor VIII deficiency further suggests that the LMAN1 M1T mutation is a frequent cause in Italian patients.

Combined factor V and factor VIII deficiency (F5F8D) is an extremely rare worldwide congenital hemorrhagic disorder that is more prevalent in the Mediterranean area. We report the clinical presentations and the identification of a LMAN1 mutation in a 3-year-old Italian boy who was diagnosed with F5F8D. The mutation identified (M1T) has already been found in several Italian patients. Since the LMAN1 M1T mutation has been identified in most patients with F5F8D, we suggest that the search for this mutation should be the first step in the molecular characterization of patients from an Italian ethnic background.     

HIV-1 clade A infection and viral control: an immunological perspective on a case of underquantification

Although a vast majority of HIV-1-positive patients in the UK are infected with clade B virus, a large number of newly diagnosed cases of heterosexually transmitted HIV-1 are acquired abroad, in countries where non-B clade HIV-1 predominates. Since the development of the viral load assay in 1988, assessment of HIV-1 plasma viraemia has become an integral part of HIV clinical care; however, the contemporary viral load assay was developed and optimized for clade B HIV-1. Here we report the underquantification of viraemia in an individual infected with clade A virus, and the consequent initial classification of the patient as an HIV controller (HIC). Immunological investigations of interferon (IFN)-γ and lymphoproliferative responses to HIV-1 clade B antigens and peptides, in parallel with mitogenic stimulation, were performed. Subsequent comparison with responses observed within clade B-infected HIC led to viral sequencing, confirmation of infecting clade and recommendation of antiretroviral therapy initiation. We emphasize the growing need for awareness of possible limitations of the commonly used viral load assays, which cannot be relied upon unreservedly in a clinical setting. Furthermore, this case highlights the increasing need for more detailed investigation into both viral genetics and fitness when defining patients as HIC.     

人参皂苷Rg1对Aβ25-35诱导的星形胶质细胞核因子κB活化及炎症反应的影响

目的 探讨人参皂苷Rg1对β淀粉样蛋白25-35（Aβ25-35）诱导的SD大鼠星形胶质细胞核因子（NF）κB的活化及其对炎性因子IL-6的影响.方法 用人参皂苷Rg1预处理星形胶质细胞24 h（终浓度分别为0、2、4、8、16 μmol/L）,然后再用Aβ25-35和人参皂苷Rg1对星形胶质细胞共同孵育24 h,四甲基偶氮唑盐（MTT）检测各组的细胞活性;用激光扫描共聚焦显微镜（LSCM）检测分析各组的细胞浆与细胞核内NF-κB的激活程度;用ELISA的方法检测IL-6的蛋白含量.结果 人参皂苷Rg1各组的细胞活性均明显高于模型组（P＜0.05）,并且能下调NF-κB的激活水平,减少IL-6的分泌（P＜0.05）,该作用以2、4 μmol/L为显著.结论 人参皂苷Rg1对Aβ25-35诱导星形胶质细胞模型的神经保护作用可能是通过阻断NF-κB的活化,减弱炎症反应,从而提高了细胞的生存率,且此作用以低浓度为好.     

A recombinant immunotoxin that is active on prostate cancer cells and that is composed of the Fv region of monoclonal antibody PR1 and a truncated form of Pseudomonas exotoxin.

Monoclonal antibody PR1 binds to the surface of normal prostate cells and to adenocarcinomas of the prostate. The cDNAs coding for the heavy and light chain variable regions of monoclonal antibody PR1 were cloned by PCR techniques. A recombinant toxin was then constructed that has the heavy chain variable region of monoclonal antibody PR1 connected to the light chain variable region by a flexible peptide linker to create a single-chain Fv; the Fv in turn is fused to a truncated form of Pseudomonas exotoxin. The resulting recombinant immunotoxin PR1(Fv)-PE38KDEL was produced in Escherichia coli and accumulated in inclusion bodies. After denaturation and renaturation, active monomeric molecules with a molecular mass of approximately 65 kDa were purified to homogeneity. PR1(Fv)-PE38KDEL binds specifically to cells containing the PR1 antigen and is very cytotoxic toward a subset of LNCaP cells that express the PR1 antigen on their surface.     

Mice expressing both B7-1 and viral glycoprotein on pancreatic beta cells along with glycoprotein-specific transgenic T cells develop diabetes due to a breakdown of T-lymphocyte unresponsiveness.

T lymphocytes have been implicated in the onset of many autoimmune diseases; however, the mechanisms underlying T-cell activation toward self antigens are poorly understood. To study whether T-lymphocyte costimulation can overcome the immunologic unresponsiveness observed in an in vivo model, we have created transgenic mice expressing the costimulatory mouse molecule B7-1, a ligand for the CD28 receptor, on pancreatic beta cells. We now report that triple-transgenic mice expressing both B7-1 and a viral glycoprotein on their beta cells, along with T cells expressing the viral-glycoprotein-specific transgenic T-cell receptor, all develop insulitis (lymphocytic infiltration of the pancreatic islets) and diabetes. In striking contrast, the T cells in double-transgenic mice expressing the same viral glycoprotein (but no B7) on their pancreatic beta cells and the transgenic T-cell receptor on their T cells, reported earlier, remain indifferent to the glycoprotein-expressing beta cells. In fact, all three transgenes are required to initiate immune-mediated destruction of the beta cells. Mice expressing any of the transgenes alone, or any two in combination, maintain normal islet architecture and never spontaneously develop insulitis or diabetes. Our results show that aberrant B7 expression on peripheral tissues may play an important role in the activation of self-reactive T cells and further suggest that abnormal expression of costimulatory receptors may be involved in various T-cell-mediated autoimmune diseases.     

Comparison between the action of estradiol and that of the antiestrogen U 11-100 A on the induction in the rat uterus of a specific protein (the induced protein).

When measured by an in vitro approach, involving incubation in the presence of labeled leucine, the rate of synthesis of the specific estrogen-induced protein (IP) after in vivo stimulation of the rat uterus by the antiestrogens U 11-100 A (UA; 1-(2-[p-(3,4-dihydro-6-methoxy-2-phenyl-1-naphtyl)-phenoxy]ethyl)pyrrolidine hydrochloride) or CI-628 (alpha-[4-pyrrolidinoethyoxy]phenyl-4-methoxy-d-nitrostilbene) was very low compared to the response measured under the same conditions after in vivo stimulation with 17 beta-estradiol (E2). In the course of investigations aimed at clarifying the role of IP in estrogen action, we have conducted similar experiments, but the labeling step aimed at detecting and measuring IP synthesis was carried out in vivo. We have observed that UA promoted a full IP response which is lost or missed in incubated uteri. Similar results were obtained with CI-628 and tamoxifen. A comparison between IP responses obtained and measured in vivo after E2 and those after UA action revealed that the responses paralleled the number of receptors that are translocated to the nucleus by each compound. Thus, while transient after E2 treatment, the IP (or IP-like) response was maintained for as long as 12 h (as is the nuclear receptor occupancy) when UA was used as inducer. Several explanations for the disappearance of the UA-induced IP response under in vitro conditions are considered.     

Mouse DNA polymerase alpha-primase terminates and reinitiates DNA synthesis 2-14 nucleotides upstream of C2A1-2(C2-3/T2) sequences on a minute virus of mice DNA template.

The distribution of termination and initiation sites in a 5081-nucleotide minute virus of mice DNA template being copied by a highly purified mouse DNA polymerase alpha-DNA primase complex in the presence of GTP has been examined. The 3'-hydroxyl termini (17 in all) were clustered at six sites that were located 2-14 nucleotides upstream of C2A2C2, C2AC3, or C2A2T2 sequences. When either [alpha-32P]- or [gamma-32P]GTP was included in the DNA polymerase reaction mixtures, nascent DNA became radiolabeled. Analysis of the 32P-labeled material following treatment of the DNA with tobacco acid pyrophosphatase, bacterial alkaline phosphatase, or ribonuclease T1 revealed the presence of oligoribonucleotide chains averaging 5-7 nucleotides long and beginning with 5' GTP residues. Eight presumptive DNA primase initiation sites were located opposite C4 or C5 sequences 3-9 nucleotides upstream of one of the three closely related hexanucleotides C2A2C2, C2AC3, and C2A2T2. RNA-DNA junctions were found 3-10 nucleotides downstream of DNA primase initiation sites. The results indicate that hexanucleotides having the general formula C2A1-2(C2-3/T2), herein referred to as psi, are involved in promoting termination of DNA synthesis and/or de novo initiation of RNA-primed DNA chains by DNA polymerase alpha-primase.