Mechanism of in vitro expansion of long DNA repeats: effect of temperature, repeat length, repeat sequence, and DNA polymerases.

Studies of sequence repeat expansions from duplexes consisting of DNA repeat sequences greater than three bases are currently lacking. These studies are needed in order to gain a better understanding of DNA expansions in general and as a first step in understanding expansions of longer sequence repeats that have been implicated in human diseases. We have undertaken an in vitro study of tetranucleotide, hexanucleotide, and octanucleotide repeat expansions from short DNA duplexes using Taq DNA polymerase. Expansions of hexanucleotide repeats were also studied with the Klenow fragment of DNA polymerase I and with T4 DNA polymerase. Studies with Taq DNA polymerase show that expansions occur more readily as the length of the repeat sequence decreases but are generally more efficient at reaction temperatures closer to the melting point of the starting duplex. A mechanism for the observed expansions with Taq DNA polymerase is proposed that does not invoke strand slippage or DNA structure. Studies at 37 degrees C with Klenow pol I and T4 DNA polymerase indicate that the template-switching and/or strand-displacement activities of the polymerases used can play a major role in the apparent in vitro expansions of short repetitive DNA duplexes.     

Separation of proteases from yellowfin tuna spleen by ultrafiltration

Separation of protease, trypsin and chymotrypsin from yellowfin tuna spleen extract by ultrafiltration (UF) using regenerated cellulose membranes with molecular weight cut off (MWCO) 30 and 100 kDa was studied. The 100 kDa membrane had a higher transmission of enzymes than that of the 30 kDa membrane. The enzyme transmission varied from 0.01 to 0.18 and from 0.6 to 0.8 for the 30 kDa membrane and 100 kDa membrane, respectively. The protein transmission was about 0.8 for both membranes. Increasing cross-flow rate and transmembrane pressure (TMP) increased permeate flux. The limiting fluxes at cross-flow rate 120, 240 and 360 L/h for the 30 kDa membrane were 17.3, 43.9 and 54.7 L/m2h, respectively and the limiting fluxes at the same flow rate for 100 kDa membrane were 34.1, 51.1 and 68.4 L/m2h, respectively. The separation of these proteases was achieved using the 30 kDa membrane. The purities of proteases were increased more than ten times at TMP 1.5 bar and cross-flow rate 360 L/h by diafiltration using 30 kDa membrane.     

The influence of pH on the G-quadruplex binding selectivity of perylene derivatives

Three new perylene derivatives with branched ionizable side chains were synthesized, and their G-quadruplex binding specificities were compared by spectroscopic and electrophoretic analysis with two well-studied G-quadruplex ligands: PIPER and TmPyP4. The value of pH and consequent charge formation and self-aggregation of these perylene derivatives influences not only the type of G-quadruplex formation, but also the G-quadruplex binding selectivity.     

Role of different habitats for the functional response of Crytorrhinus lividipennis (Hemiptera: Miridae) on Nilaparvata lugens (Hemiptera: Delphacidae)

The functional response of a predator, Cyrtorrhinus lividipennis, on the brown planthopper, Nilaparvata lugens, was investigated in two different experimental habitats: a piece of tiller in a petri dish (5.5?cm in diameter) and a rice plant in a net cage (5.5?cm diameter?×?43?cm height) for 24?h at room temperature. In the petri dish experiment, 2nd–5th instar nymphs, adult male, and female C. lividipennis were introduced in separate experiments to eggs of N. lugens at densities of five, 10, 20, 30, and 40 eggs per piece of rice. In the rice plant experiment, each C. lividipennis was introduced to a cage with a rice plant containing N. lugens eggs. After 24?h, the number of dead eggs and remaining eggs were counted and data fitted to three functional response models. Among the three types of functional responses, Type II best described the predator response to host densities in N. lugens in both experimental habitats, according to the logistic regression analysis value. The results showed that C. lividipennis was a more effective predator in the rice plant experiment compared to the disc experiment. Additionally, the searching efficiency and handling time parameters were different in the two different experimental habitats. This may cause errors when applying the functional response to biological control and predator–prey models. Different habitats and other environmental conditions from the experiment and natural rice field have to be considered.     

Cassane diterpenoids from the stem of Caesalpinia pulcherrima

Cassane diterpenoids: pulcherrin A, pulcherrin B, pulcherrin C, neocaesalpin P, neocaesalpin Q and neocaesalpin R, together with eight known compounds: isovouacapenol C, 6β-cinnamoyl-7β-hydroxy-vouacapen-5α-ol, pulcherrimin E, pulcherrimin C, α-cadinol, 7-hydroxycadalene, teucladiol and bonducellin were isolated from the stem of Caesalpinia pulcherrima. The chemical structures were elucidated by analysis of their spectroscopic data.     

DNA modification by 4-aza-3-ene-1,6-diynes: DNA cleavage, pH-dependent cytosine-specific interactions, and cancer cell cytotoxicity

The (Z)-hex-1,5-diyne-3-ene reactive core common to the enediyne antitumor antibiotics undergoes a Bergman cyclization after proper activation to afford reactive diradical intermediates that are responsible for initiating DNA cleavage. Direct modification of the enediyne core has been proposed as a method to permit cancer cell-specific triggering of the diradical-generating cyclization. For example, 3-aza-3-ene-1,5-diynes undergo an aza-Bergman cyclization to afford the fleeting 2,5-didehydropyridine diradicals. While protonation of these aza-enediynes can afford products of diradical trapping, the hydrolytic instability of the 3-aza-3-ene-1,5-diyne moiety prevents its use in pH-triggered DNA cleaving anticancer agents. Recently, more hydrolytically stable systems incorporating the 4-aza-3-ene-1,6-diyne moiety were developed. We report here studies of the 4-aza-3-ene-1,6-diyne-containing benzimidazolium salt AZB002 [1-methyl-2-(phenylethynyl)-3-(3-phenylprop-2-ynyl)-3H-benzimidazolium tetrafluoroborate] and two structurally related heterocycles that lack the aza-enediyne functionality, AZB016 [1,3-dimethyl-2-(phenylethynyl)-3H-benzimidazolium triflate] and AZB004 [3-methyl-2-(phenylethynyl)benzothiazolium triflate]. The interaction of these compounds with supercoiled DNA, a double-stranded DNA fragment, and a short DNA duplex oligonucleotide was investigated. There are three distinct DNA interactions exhibited by AZB002: a frank strand scission leading to the relaxation of supercoiled DNA and formation of at least two different DNA adducts, one of which leads to cytosine-specific cleavage after piperidine/heat treatment. In contrast, analogues lacking the aza-enediyne functionality either fail to interact with DNA (AZB016) or cleave DNA at guanine residues, presumably through alkylation of the N-7 position (AZB004). We also investigated the cytotoxicity of AZB002 and the related heterocyclic compounds AZB004 and AZB016 and find that only the DNA interactive compounds AZB002 and AZB004 display significant cytotoxicity. In particular, AZB002 is cytotoxic against a wide range of cancer cell lines.     

Response of Steinernema carpocapsae Infective Juveniles to the Plasma of Three Insect Species

Exsheathed infective juveniles of Steinernema carpocapsae All strain were attracted to the plasma of three species of insects in agar plate bioassays. Plasma of Pieris rapae crucivora, Spodoptera litura, and Agrotis segetum attracted 88.6%, 80.4%, and 64.4%, respectively, of Steinernema carpocapsae juveniles added to plates. Autoclaved plasma of S. litura larvae attracted more juveniles than saline controls, but less than nonautoclaved plasma. The active agent passed through a 14,000 MW dialysis membrane.