Metachromatic Agar-Diffusion Methods for Detecting Staphylococcal Nuclease Activity

Based on the metachromatic property of Toluidine Blue O, three, convenient agar-diffusion methods have been developed that enable detection of the nuclease of Staphylococcus aureus at concentrations as low as 0.005 mug/ml in agar and broth cultures. The interactions of agar and deoxyribonucleic acid with Toluidine Blue O are discussed.     

Metachromatic Agar-Diffusion Microslide Technique for Detecting Staphylococcal Nuclease in Foods

The metachromatic agar-diffusion (MAD) microslide technique was shown to detect nanogram quantities of staphylococcal thermonuclease in various foods without prior extraction, purification, or concentration.     

Convenient Assay for Staphylococcal Nuclease by the Metachromatic Well-Agar-Diffusion Technique

The metachromatic agar-diffusion (MAD) microslide technique was adapted for quantitative assay for staphylococcal thermonuclease in heterogeneous systems, such as milk and broth. When an enzyme-containing solution was placed in a well cut in the agar, a bright pink halo was obtained. The diameter of the pink zone of hydrolysis was related to time and temperature of incubation and to nuclease concentration. Concentrations of nuclease as low as 0.005 mug/ml and as high as 2.0 mug/ml were conveniently determined after 3 hr at 37 C.     

Staphylococcal Nuclease in Udder Secretions of Cows with Acute Mastitis

High concentrations of nuclease produced by Staphylococcus aureus were demonstrated within a few hours by direct examination of udder secretions from cows with a severe staphylococcal mastitis. A positive correlation between the nuclease concentration and the severity of the mastitis was found. The cows with high nuclease concentrations were generally young individuals and/or in the first 2 weeks of the lactation period. Most had a low titre of antibodies against staphylococcal nuclease. Two-thirds of the cows in which high nuclease concentrations were demonstrated were culled or died because of the mastitis attack. The role which nuclease plays for staphylococcal virulence is discussed, and it is concluded that nuclease contributes to the pathogenicity of S. aureus.     

金黄色葡萄球菌核酸酶A基因在大肠杆菌中的高效表达

这篇文章报道了金黄色葡萄球菌核酸酶A的克隆和在温控启动子P_RP_L调控下在E．Coli中的高效表达。SDS—PAGE分析表明,核酸酶A的含量可占E．Coli细胞总蛋白含量的60%。经有效的增溶和复性处理后,重组酶具有与天然酶相同的活力;N-末端氨基酸分析的结果指出,fMet在转译后被加工去除;对重组SNaseA在构象上的同一性也通过Phenyl—Su—perose疏水柱层析进行了分析。     

Beta Hemolysin: a Persistent Impurity in Preparations of Staphylococcal Nuclease and Enterotoxin

Purified staphylococcal nuclease and enterotoxin B from several sources contained beta hemolysin whose physicochemical resemblances to the other two proteins make its elimination difficult.     

Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation

While early structural models of helix-bundle integral membrane proteins posited that the transmembrane α-helices [transmembrane helices (TMHs)] were orientated more or less perpendicular to the membrane plane, there is now ample evidence from high-resolution structures that many TMHs have significant tilt angles relative to the membrane. Here, we address the question whether the tilt is an intrinsic property of the TMH in question or if it is imparted on the TMH during folding of the protein. Using a glycosylation mapping technique, we show that four highly tilted helices found in multi-spanning membrane proteins all have much shorter membrane-embedded segments when inserted by themselves into the membrane than seen in the high-resolution structures. This suggests that tilting can be induced by tertiary packing interactions within the protein, subsequent to the initial membrane-insertion step.     

Specific Insect-Virus Interactions Are Responsible for Variation in Competency of Different Thrips tabaci Isolines to Transmit Different Tomato Spotted Wilt Virus Isolates

Local adaptation between sympatric host and parasite populations driven by vector genetics appears to be a factor that influences dynamics of disease epidemics and evolution of insect-vectored viruses. Although T. tabaci is the primary vector of Tomato spotted wilt virus (TSWV) in some areas of the world, it is not an important vector of this economically important plant virus in many areas where it occurs. Previous studies suggest that genetic variation of thrips populations, virus isolates, or both are important factors underlying the localized importance of this species as a vector of TSWV. This study was undertaken to quantify variation in transmissibility of TSWV isolates by T. tabaci, in the ability of T. tabaci to transmit isolates of TSWV, and to examine the possibility that genetic interactions and local adaptation contribute to the localized nature of this species as a vector of TSWV. Isofemale lines of Thrips tabaci from multiple locations were tested for their ability to transmit multiple TSWV isolates collected at the same and different locations as the thrips. Results revealed that the probability of an isofemale line transmitting TSWV varied among virus isolates, and the probability of an isolate being transmitted varied among isofemale lines. These results indicate that the interaction of T. tabaci and TSWV isolate genetic determinants underlie successful transmission of TSWV by T. tabaci. Further analysis revealed sympatric vector-virus pairing resulted in higher transmission than allopatric pairing, which suggests that local adaptation is occurring between T. tabaci and TSWV isolates.     

Gene- and Protein-Delivered Zinc Finger–Staphylococcal Nuclease Hybrid for Inhibition of DNA Replication of Human Papillomavirus

Previously, we reported that artificial zinc-finger proteins (AZPs) inhibited virus DNA replication in planta and in mammalian cells by blocking binding of a viral replication protein to its replication origin. However, the replication mechanisms of viruses of interest need to be disentangled for the application. To develop more widely applicable methods for antiviral therapy, we explored the feasibility of inhibition of HPV-18 replication as a model system by cleaving its viral genome. To this end, we fused the staphylococcal nuclease cleaving DNA as a monomer to an AZP that binds to the viral genome. The resulting hybrid nuclease (designated AZP–SNase) cleaved its target DNA plasmid efficiently and sequence-specifically in vitro. Then, we confirmed that transfection with a plasmid expressing AZP–SNase inhibited HPV-18 DNA replication in transient replication assays using mammalian cells. Linker-mediated PCR analysis revealed that the AZP–SNase cleaved an HPV-18 ori plasmid around its binding site. Finally, we demonstrated that the protein-delivered AZP–SNase inhibited HPV-18 DNA replication as well and did not show any significant cytotoxicity. Thus, both gene- and protein-delivered hybrid nucleases efficiently inhibited HPV-18 DNA replication, leading to development of a more universal antiviral therapy for human DNA viruses.     

Phase Transitions in a Logistic Metapopulation Model with Nonlocal Interactions

The presence of one or more species at some spatial locations but not others is a central matter in ecology. This phenomenon is related to ecological pattern formation. Nonlocal interactions can be considered as one of the mechanisms causing such a phenomenon. We propose a single-species, continuous time metapopulation model taking nonlocal interactions into account. Discrete probability kernels are used to model these interactions in a patchy environment. A linear stability analysis of the model shows that solutions to this equation exhibit pattern formation if the dispersal rate of the species is sufficiently small and the discrete interaction kernel satisfies certain conditions. We numerically observe that traveling and stationary wave-type patterns arise near critical dispersal rate. We use weakly nonlinear analysis to better understand the behavior of formed patterns. We show that observed patterns arise through both supercritical and subcritical bifurcations from spatially homogeneous steady state. Moreover, we observe that as the dispersal rate decreases, amplitude of the patterns increases. For discontinuous transitions to instability, we also show that there exists a threshold for the amplitude of the initial condition, above which pattern formation is observed.     

Different Reading Frames Are Responsible for Is1-Dependent Deletions and Recombination

Two classes of plasmids in addition to the parent become apparent when plasmids that contain direct repeats of IS1. One class of plasmids has deleted sequences from the end of IS1 to nonrandom sites within the plasmid. The appearance of these plasmids in the population requires intact insA and insB reading frames, but not insC. The other class of plasmids has undergone an exchange within the direct repeats of IS1 on the plasmid. Their appearance requires InsC but neither InsA nor InsB. The two reactions may represent two distinguishable steps in IS1 transposition. The InsC-catalyzed exchange is independent of RecA and resembles hologous recombination since the frequency of recombinants arising from exchanges in different regions of IS1 appears to be roughly proportional to the size of the region. InsC can also catalyze an exchange between direct repeats of non-IS1 DNA.     

Structural Analysis and Deletion Mutagenesis Define Regions of QUIVER/SLEEPLESS that Are Responsible for Interactions with Shaker-Type Potassium Channels and Nicotinic Acetylcholine Receptors

Ly6 proteins are endogenous prototoxins found in most animals. They show striking structural and functional parallels to snake α-neurotoxins, including regulation of ion channels and cholinergic signaling. However, the structural contributions of Ly6 proteins to regulation of effector molecules is poorly understood. This question is particularly relevant to the Ly6 protein QUIVER/SLEEPLESS (QVR/SSS), which has previously been shown to suppress excitability and synaptic transmission by upregulating potassium (K) channels and downregulating nicotinic acetylcholine receptors (nAChRs) in wake-promoting neurons to facilitate sleep in Drosophila. Using deletion mutagenesis, co-immunoprecipitations, ion flux assays, surface labeling and confocal microscopy, we demonstrate that only loop 2 is required for many of the previously described properties of SSS in transfected cells, including interactions with K channels and nAChRs. Collectively our data suggest that QVR/SSS, and by extension perhaps other Ly6 proteins, target effector molecules using limited protein motifs. Mapping these motifs may be useful in rational design of drugs that mimic or suppress Ly6-effector interactions to modulate nervous system function.     

Histidines Are Responsible for Zinc Potentiation of the Current in KDC1 Carrot Channels

Unlike all plant inward-rectifying potassium channels, the carrot channel KDC1 has two histidine pairs (H161,H162) in the S3–S4 and (H224,H225) in the S5–S6 linkers. When coinjected with KAT1 in Xenopus oocytes, KDC1 participates in the formation of heteromultimeric KDC1:KAT1 channels and the ionic current is potentiated by extracellular Zn²⁺. To investigate the potential interactions between KDC1 and zinc, a KDC1-KAT1 dimer was constructed. The dimeric and heteromeric channels displayed similar characteristics and the same sensitivity to zinc and other metals; this result suggests that zinc binding is mediated by residues in a single channel subunit. The KDC1:KAT1 currents were also potentiated by external Pb²⁺ and Cd²⁺ and inhibited by Ni²⁺. To investigate further the role of KDC1-histidines, these amino acids were mutated into alanines. The single mutations H225A, H161A, and H162A did not affect the response of the heteromeric channels to zinc. Conversely, the single mutant H224A and the double mutants (H224A,H225A) and (H161A,H162A) abolished zinc potentiation, but not that induced by Pb²⁺ or Cd²⁺. These results suggest that Zn²⁺ potentiation cannot be ascribed to simple electrostatic interactions between zinc and channel residues and that histidine 224 is crucial for zinc but not for lead potentiation of the current.