Baculovirus replication factor LEF-1 is a DNA primase

The baculovirus replication factors LEF-1 and LEF-2 of the Autographa californica multinucleocapsid nucleopolyhedrovirus were overexpressed as fusions containing a hemagglutinin (HA) epitope and a HIS(6) tag using recombinant baculoviruses. LEF-1 was purified to near homogeneity and found to have primase activity in an indirect assay employing Escherichia coli DNA polymerase I (Klenow enzyme) and poly(dT) template. The LEF-1 primase products were also directly characterized by electrophoresis in 20% polyacrylamide-8 M urea gels and agarose gels. Primer synthesis was time dependent, and products of several hundred nucleotides or more were observed from the M13 single-stranded DNA (ssDNA) template. The LEF-1 primase was absolutely dependent on divalent cations (Mg(2+)), and optimal activity was supported by 10 mM MgCl(2). An alkaline pH (8.8 to 9.4) was optimal, whereas monovalent salt (KCl) was inhibitory. Mutation of an invariant aspartic acid in a putative primase domain caused LEF-1 activity to be abolished. Upon ultracentrifugation in glycerol gradients, LEF-1 was found to have a sedimentation coefficient of 3S that is consistent with its being present as a monomer. Elution profiles of LEF-1 and LEF-2 from ssDNA-cellulose and DEAE resin suggested that LEF-2 may bind to both DNA and LEF-1.     

Characterization of a baculovirus alkaline nuclease

All baculovirus genomes sequenced to date encode a homolog of an alkaline nuclease that has been characterized in the Herpesviridae. In this report we describe the characterization of the alkaline nuclease (AN) homolog of the Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) (open reading frame 133). His-tagged AN constructs were expressed in recombinant baculoviruses and affinity purified, and then their enzymatic activity was characterized. AN was found to degrade linear DNA at alkaline pH, preferred Mg(2+) over Mn(2+), had optimal activity at 35 degrees C, and did not appear to have a salt requirement. To rule out contamination by the endogenous baculovirus gene product or a cellular enzyme, point mutations were introduced into a highly conserved domain of the gene. These mutations were found to markedly reduce or eliminate most of the activity of the affinity-purified enzyme. An antibody generated against the protein was used to analyze its expression by Western blot analysis. AN was found to be expressed at low levels by 12 h postinfection, with maximal expression at 24 h postinfection. Attempts to generate a virus with this gene inactivated were unsuccessful, suggesting that AN may be encoded by an essential gene.     

The baculovirus single-stranded DNA binding protein, LEF-3, forms a homotrimer in solution.

LEF-3 is one of six proteins from Autographa californica multinucleocapsid polyhedrosis virus required for transient DNA replication and has the properties of a single-stranded DNA binding protein. In this report we demonstrate that LEF-3 interacts with itself in both yeast two-hybrid assays and glutathione S-transferase fusion affinity assays. LEF-3 deletion clones which were unable to interact with full-length LEF-3 also failed to support transient DNA replication, suggesting that this interaction is required for the proper function of LEF-3. LEF-3 was purified to homogeneity and characterized by analytical ultracentrifugation and native polyacrylamide gel electrophoresis. These studies revealed that LEF-3 was present as a 132-kDa complex, indicating that its native conformation is that of a homotrimer. This result was confirmed by cross-linking with glutaraldehyde followed by matrix-assisted laser desorption/ionization mass spectrometry.     

Production of polyhedrin monoclonal antibodies for distinguishing two Orgyia pseudotsugata baculoviruses.

Monoclonal antibodies were produced to polyhedrins from Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) and single-capsid nuclear polyhedrosis virus (OpSNPV). Although the polyhedrins are closely related, antibodies were selected which allowed differentiation between the two viruses. In an indirect enzyme-linked immunosorbent assay, purified OpMNPV and OpSNPV polyhedrins could be detected by specific monoclonal antibodies at concentrations as low as 2 and 5 ng/ml, respectively. The antibodies were also capable of identifying their homologous polyhedrin in extracts of infected insects. These antibodies would be useful for monitoring production of the viral insecticide, TM Biocontrol-1, which by license must contain only OpMNPV, and to confirm that insect mortality after aerial spraying with this insecticide is attributable to OpMNPV infection.     

Abfallverwertung nach dem Vorbild der Natur: Versuche zur mikrobiellen Kompostierung

The substance circles represent a perfect recycling just as required by sophisticated, environment‐oriented waste management concepts and the substance management act. Environmental education at schools also focuses an the topic of recycling, and attempts — via imparting the necessary technical knowledge — to establish the readiness for the separation of useful matter by sorts (e.g.biodegradable waste) among students and pupils. The access to the topic of biodegradable waste composting is supported by praxis‐oriented experiments. The article depicts the types of experiments by which the metabolism activity of microorganisms can be proved (“Selbsterhitzungsexperiment” = “self‐heat‐up experiment”) and the microbial disintegration of biogenic materials can be made visible respectively (“Diarähmchentest” = “slide‐frame‐test”). Thus, these simple and inexpensive student experiments are being compared to experiments from research praxis. On the basis of transparencies from biodegradable substances, “Latex” products (condoms)and peels of citric fruits (natural conservation substances), the question of whether or not such materials belong into the trash‐can for biodegradable waste can be answered in a praxis‐oriented way.     

The novel immunosuppressive protein kinase C inhibitor sotrastaurin has no pro-viral effects on the replication cycle of hepatitis B or C virus

The pan-protein kinase C (PKC) inhibitor sotrastaurin (AEB071) is a novel immunosuppressant currently in phase II trials for immunosuppression after solid organ transplantation. Besides T-cell activation, PKC affects numerous cellular processes that are potentially important for the replication of hepatitis B virus (HBV) and hepatitis C virus (HCV), major blood-borne pathogens prevalent in solid organ transplant recipients. This study uses state of the art virological assays to assess the direct, non-immune mediated effects of sotrastaurin on HBV and HCV. Most importantly, sotrastaurin had no pro-viral effect on either HBV or HCV. In the presence of high concentrations of sotrastaurin, well above those used clinically and close to levels where cytotoxic effects become detectable, there was a reduction of HCV and HBV replication. This reduction is very likely due to cytotoxic and/or anti-proliferative effects rather than direct anti-viral activity of the drug. Replication cycle stages other than genome replication such as viral cell entry and spread of HCV infection directly between adjacent cells was clearly unaffected by sotrastaurin. These data support the evaluation of sotrastaurin in HBV and/or HCV infected transplant recipients.     

Evolution of biological interaction networks: from models to real data

We are beginning to uncover common mechanisms leading to the evolution of biological networks. The driving force behind these advances is the increasing availability of comparative data in several species.     

Calcium Intake and Serum Concentration in Relation to Risk of Cardiovascular Death in NHANES III

Background

Evidence for an association between calcium intake and risk of cardiovascular death remains controversial. By assessing dietary intake, use of supplements, and serum levels of calcium, we aimed to disentangle this link in the third National Health and Nutrition Examination Survey (NHANES III).

Methods

Mortality linkage of NHANES III to death certificate data for those aged 17 years or older (n = 20,024) was used to estimate risk of overall cardiovascular death as well as death from ischemic heart disease (IHD), acute myocardial infarction (AMI), heart failure (HF), and cerebrovascular disease (CD) with multivariate Cox proportional hazards regression analysis.

Results

About 10.0% of the population died of cardiovascular disease and the majority (5.4%) died of IHD. There was increased risk of overall CVD death for those in the bottom 5% of serum calcium compared to those in the mid 90% (HR: 1.51 (95% CI: 1.03–2.22)). For women there was a statistically significant increased risk of IHD death for those with serum calcium levels in the top 5% compared to those in the mid 90% (HR: 1.72 (95%CI: 1.13–2.61)), whereas in men, low serum calcium was related to increased IHD mortality (HR: 2.32 (95% CI 1.14–3.01), P_interaction: 0.306). No clear association with CVD death was observed for dietary or supplemental calcium intake.

Conclusions

Calcium as assessed by serum concentrations is involved in cardiovascular health, though differential effects by sex may exist. No clear evidence was found for an association between dietary or supplementary intake of calcium and cardiovascular death.