SV40 T antigen binds directly to the large subunit of purified DNA polymerase alpha.

Purified SV40 large T antigen and purified DNA polymerase alpha-primase form a complex detectable by ELISA and by a modified immunoblotting technique. The interaction is specific for the large catalytic subunit of polymerase alpha. The amino terminal 83 amino acids of T antigen are both necessary and sufficient for binding to the polymerase. However, antibody epitopes located in the carboxy terminal ATPase domain of T antigen are masked in the polymerase-T antigen complex, and complex formation is inhibited by an antibody directed against the carboxy terminus of T antigen, suggesting that this region of T antigen, though not required for binding, is in close proximity to the bound polymerase. The affinity of human DNA polymerase alpha for T antigen is approximately 10-fold greater than that of polymerase alpha from calf thymus, consistent with the interpretation that polymerase alpha is at least in part responsible for the primate-specific replication of SV40 DNA in vivo and in vitro. The results suggest that specific protein-protein interaction between DNA polymerase alpha and T antigen plays an important role in viral DNA replication.     

Long-term survival and concomitant gene expression of ribozyme-transduced CD4+ T-lymphocytes in HIV-infected patients

BACKGROUND: An anti-HIV-1 tat ribozyme, termed Rz2, has been shown to inhibit HIV-1 infection/replication and to decrease HIV-1-induced pathogenicity in T-lymphocyte cell lines and normal peripheral blood T-lymphocytes. We report here the results of a phase I gene transfer clinical trial using Rz2. METHODS: Apheresis was used to obtain a peripheral blood cell population from each of four HIV-negative donors. After enrichment for CD4+ T-lymphocytes, ex vivo expansion and genetic manipulation (approximately equal aliquots of the cells were transduced with the ribozyme-containing (RRz2) and the control (LNL6) retroviral vector), these cells were infused into the corresponding HIV-1-positive twin recipient. Marking was assessed over an initial 24-week period and in total over an approximate 4-year period. RESULTS: The gene transfer procedure was shown to be safe, and technically feasible. Both RRz2- and LNL6-gene-containing peripheral blood mononuclear cells (PBMC) were detected at all time points examined to 4 years. There was concomitant gene construct expression in the absence of the need for ex vivo peripheral blood cell stimulation and there was no evidence of immune elimination of the neoR T-lymphocytes nor of silencing of the Moloney murine leukemia virus long terminal repeat. CONCLUSIONS: The proof of principle results reported here demonstrate safety and feasibility of this type of gene transfer approach. While not specifically tested, T-lymphocytes containing an anti-HIV gene construct may impact on HIV-1 viral load and CD4+ T-lymphocyte count, potentially representing a new therapeutic modality for HIV-1 infection.     

The single Drosophila ZO-1 protein Polychaetoid regulates embryonic morphogenesis in coordination with Canoe/afadin and Enabled

Adherens and tight junctions play key roles in assembling epithelia and maintaining barriers. In cell culture zonula occludens (ZO)-family proteins are important for assembly/maturation of both tight and adherens junctions (AJs). Genetic studies suggest that ZO proteins are important during normal development, but interpretation of mouse and fly studies is limited by genetic redundancy and/or a lack of null alleles. We generated null alleles of the single Drosophila ZO protein Polychaetoid (Pyd). Most embryos lacking Pyd die with striking defects in morphogenesis of embryonic epithelia including the epidermis, segmental grooves, and tracheal system. Pyd loss does not dramatically affect AJ protein localization or initial localization of actin and myosin during dorsal closure. However, Pyd loss does affect several cell behaviors that drive dorsal closure. The defects, which include segmental grooves that fail to retract, a disrupted leading edge actin cable, and reduced zippering as leading edges meet, closely resemble defects in canoe zygotic null mutants and in embryos lacking the actin regulator Enabled (Ena), suggesting that these proteins act together. Canoe (Cno) and Pyd are required for proper Ena localization during dorsal closure, and strong genetic interactions suggest that Cno, Pyd, and Ena act together in regulating or anchoring the actin cytoskeleton during dorsal closure.     

Incidence and survival of non-O157 verocytotoxigenic Escherichia coli in soil

Aims: This study estimated the incidence of non‐O157 verocytotoxigenic Escherichia coli (VTEC) in farm pasture soils and investigated the survival of non‐O157 VTEC in clay and sandy loam soils. Methods and Results: Twenty farms were tested over a 12‐month period by sample enrichment in tryptone soya broth plus vancomycin, followed by PCR screening for the presence of vt1 and vt2 genes. Of the 600 soil samples, 162 (27%), across all farms, were found to contain vt1 and/or vt2 genes. The enrichment cultures from the 162 PCR‐positive samples were plated onto Chromocult tryptone bile X‐glucuronide agar (TBX), presumptive VTEC colonies recovered, confirmed as VTEC by PCR and serotyped. Samples of the two predominant soil types in Ireland (clay and sandy) were homogenized, characterized in terms of pH, boron, cobalt, copper, potassium, magnesium, manganese, phosphorus, zinc and organic matter content, inoculated with washed suspensions of eight non‐O157:H7 soil isolates and six bovine faecal isolates and stored at 10°C for up to 201 days. Inoculum survival rates were determined at regular intervals by recovering and plating soil samples on TBX. All inoculated non‐O157 serotypes had highest D‐values in the sandy loam soil with D‐values ranging from 50·26 to 75·60 days. The corresponding range in clay loam soils was 31·60–48·25 days. Conclusions: This study shows that non‐O157 VTEC occur widely and frequently in pasture soils and can persist in such environments for several months, with considerable opportunity for recycling through farm environments, and cattle, with clear potential for subsequent transmission into the human food chain. Significance and Impact of the Study: This is the first such study of non‐O157 VTEC in farm soils and found that these VTEC are frequent and persistent contaminants in farm soils. In light of recent epidemiological data, non‐O157 VTEC should be seen as an emerging risk to be controlled within the food chain.     

Evaluation of organic insecticides for management of spotted‐wing drosophila (Drosophila suzukii) in berry crops

Spotted‐wing drosophila, Drosophila suzukii (Matsumura), is an invasive pest affecting fruit production in many regions of the world. Insecticides are the primary tactic for controlling D. suzukii in organic as well as conventional production systems. Organic growers have a greater challenge because fewer insecticides are approved for use in organic agriculture. The most effective organically approved product is spinosad, but alternatives are needed because of label restrictions limiting the number of applications per year, toxicity to beneficial arthropods and the risk of developing resistance. We evaluated several organically approved insecticides against D. suzukii in laboratory assays and field trials conducted on organic blueberry and raspberry farms. Spinosad was consistently the most effective insecticide, but a few other insecticides such as azadirachtin + pyrethrins, Chromobacterium subtsugae and sabadilla alkaloids showed moderate activity. None of the treatments had long residual activity. Mortality started to decline by 3 days after treatment, and by 5 days after application, the treatments were not different from the controls. These products may be useful in rotation programmes, necessary for reducing reliance on spinosad and mitigating resistance. Cultural and biological control approaches are needed in fruit production for D. suzukii management, but insecticides will likely continue to be the dominant management tactic while these other approaches are being optimized and adopted.     

A Laminin G-EGF-Laminin G module in Neurexin IV is essential for the apico-lateral localization of Contactin and organization of septate junctions

Septate junctions (SJs) display a unique ultrastructural morphology with ladder-like electron densities that are conserved through evolution. Genetic and molecular analyses have identified a highly conserved core complex of SJ proteins consisting of three cell adhesion molecules Neurexin IV, Contactin, and Neuroglian, which interact with the cytoskeletal FERM domain protein Coracle. How these individual proteins interact to form the septal arrays that create the paracellular barrier is poorly understood. Here, we show that point mutations that map to specific domains of neurexin IV lead to formation of fewer septae and disorganization of SJs. Consistent with these observations, our in vivo domain deletion analyses identified the first Laminin G-EGF-Laminin G module in the extracellular region of Neurexin IV as necessary for the localization of and association with Contactin. Neurexin IV protein that is devoid of its cytoplasmic region is able to create septae, but fails to form a full complement of SJs. These data provide the first in vivo evidence that specific domains in Neurexin IV are required for protein-protein interactions and organization of SJs. Given the molecular conservation of SJ proteins across species, our studies may provide insights into how vertebrate axo-glial SJs are organized in myelinated axons.     

Different activities of the largest subunit of replication protein A cooperate during SV40 DNA replication

Replication protein A (RPA) is a stable heterotrimeric complex consisting of p70, p32 and p14 subunits. The protein plays a crucial role in SV40 minichromosome replication. Peptides of p70 representing interaction sites for the smaller two subunits, DNA as well as the viral initiator protein large T-antigen (Tag) and the cellular DNA polymerase alpha-primase (Pol) all interfered with the replication process indicating the importance of the different p70 activities in this process. Inhibition by the peptide disrupting protein-protein interactions was observed only during the pre-initiation stage prior to primer synthesis, suggesting the formation of a stable initiation complex between RPA, Tag and Pol at the primer end.     

Human natural killer cells analyzed by B73.1, a monoclonal antibody blocking Fc receptor functions. II. Studies of B73.1 antibody-antigen interaction on the lymphocyte membrane

In this paper, we characterize the antigen recognized by the monoclonal antibody B73.1 and the modification occurring at the membrane of the positive cells after interaction with the antibody. The B73.1-defined antigen is a protein of 50,000 to 72,000 daltons that is sensitive to pronase but not to trypsin treatment. B73.1 antibody, and its F(ab')2 fragment, directly block, at high concentrations, the binding of IgG antibody-sensitized erythrocytes to the Fc receptors (FcR) of a subpopulation of lymphocytes and neutrophils. B73.1 antibody dissociates rapidly from the positive cells, but concomitant modulation of both B73.1 antigen and FcR is induced when cells are incubated in the continuous presence of antibody or when B73.1 antibody is cross-linked at the cell membrane with an anti-mouse immunoglobulin antiserum. Reaction of lymphocytes with immune complexes also induces modulation of both FcR and B73.1 antigen, without affecting the expression of other antigens on the positive cells. The possibility that the antigen is internalized and digested by the cell after reaction with the antibody is discussed. B73.1 antibody inhibits antibody-dependent cytotoxicity mediated by lymphocytes (K cells) and neutrophils, whereas it does not affect spontaneous cytotoxicity of NK cells. These results suggest the B73.1-defined antigen might be the FcR or a structure closely related to it on K/NK cells.     

Satellite DNA sequences in the New World primateCebus apella (Platyrrhini,Primates)

Two satellite DNAs, designated CapA and CapB, were isolated from the neotropical primate,Cebus apella. The satellites exhibit nonoverlapping distributions onC. apella chromosomes. CapA is a major component of interstitial regions of constitutive heterochromatin, a very large block of heterochromatin comprising most of the long arm of chromosome 11, and some telomeres. The CapA monomer has a length of about 1500 bp and appears recently to have undergone an amplification episode in theC. apella genome. CapA-like sequences are probably present in members of the family Cebidae (to whichC. apella belongs), but not in members of the family Callitrichidae (marmosets). CapB sequences can be detected at the centromeres of manyC. apella chromosomes, and similar sequences are present in all neotropical primates. The 342 bp CapB monomer shares 60%–64% sequence identity with several alpha satellite sequences of human origin. Because of its structure, sequence, and location, it appears that CapB is the New World primate homolog of Old World primate alpha satellite DNA.     

A simplified method for mapping deletion/substitution mutants of bacteriophage lambda.

A convenient and rapid method for mapping deletion/substitution mutants of phage λ is presented. The method involves: (a) forming heteroduplex DNA molecules between the wild-type and the mutant: (b) digestion of the single-stranded regions with S₁ nuclease; (c) cleavage of a portion of the remaining duplex DNA with EcoRI nuclease or any convenient restriction endonuclease; and (d) separation of the resulting DNA fragments by gel electrophoresis. Using three deletion/substitution mutants with known endpoints, we show that the values obtained by this method deviate, on the average, by ±120 base pairs from the values obtained by electron microscopy.