Insecticidal Activity of the Protein Encoded by the cryV Gene of Bacillus thuringiensis kurstaki INA-02

A new host specificity was discovered with the insecticidal protein encoded by the cryV gene. The cryV gene was cloned from the Bacillus thuringiensis kurstaki INA-02 strain, which was selected among a number of B. thuringiensis isolates because of its high activity against Spodoptera litura. Analyses by polymerase chain reaction (PCR) revealed that INA-02 contained the cryIA(a) and cryV genes. Since no Spodoptera activity was observed with B. thuringiensis sotto, which contained only cryIA(a), insecticidal activity of the protein encoded by the cryV gene was investigated with several insect species including S. litura. For bioassay, the cryV gene was highly expressed in an acrystalliferous B. thuringiensis strain, BT51. The CryV protein from BT51 was assayed against larvae of three lepidopteran species, Bombyx mori, S. litura, and Plutella xylostella. The protein was highly active against S. litura and P. xylostella, suggestive that the protein contributes to the unique activity of INA-02.     

Production of styrene oxide from styrene by a recombinant Escherichia coli with enhanced AcrAB-TolC efflux pump level in an aqueous-organic solvent two-phase system

ABSTRACT

The AcrAB-TolC efflux pump is involved in the organic solvent tolerance of Escherichia coli. Most E. coli strains are highly sensitive to organic solvents such as n-hexane and cyclohexane. Here, a recombinant E. coli transformed with an expression plasmid containing acrAB and tolC became tolerant to n-hexane and cyclohexane. The levels of AcrA, AcrB, and TolC in the recombinant increased by 3- to 5-fold compared to those in the control strain without the plasmid for acrAB or tolC. To investigate the usability of the recombinant as a biocatalyst in an aqueous-organic solvent two-phase system, we further introduced xylMA xylene monooxygenase genes from Pseudomonas putida mt-2 into the recombinant and examined the production of styrene oxide from styrene. The resulting recombinant produced 1.8 mg and 1.0 mg styrene oxide mL^?1 of medium in a medium overlaid with a 25% volume of n-hexane and cyclohexane containing 10% (wt vol^?1) styrene, respectively.     

Psychosocial factors associated with smoking and drinking among Japanese early adolescent boys and girls: Cross-sectional study

Background  

Smoking and drinking alcohol among early adolescents are serious public health concerns, but few studies have been conducted in Japan to assess their prevalence and etiology. A regional survey was conducted in eight schools in two Japanese school districts to identify psychosocial factors associated with smoking and drinking behaviors for boys and girls.     

Analysis of Human T-Cell Antigens by One- and Two-Dimesional Polyacrylamide Gel Electrophoresis

One- and two-dimensional polyacrylamide gel electrophoresis (PAGE) was performed on immunoprecipitates formed between anti-human T-cell xenoantiserum (ATS) and cell-surface glycoproteins of human lymphocytes, that had been radioiodinated by lactoperoxidase and purified on a Lentil lectin-coupled Sepharose 4B column. In some experiments, the cells were ³H-labeled by periodate-tritiated borohydride. ATS that was absorbed with B cells recognized a number of cell-surface antigens expressed preferentially on human thymus and T cells, with molecular weights of 150K (T150), 94K (T94), 72K (T72), and 65K (T65) daltons. Whereas T150 appeared to consist of multiple components of heavily sialylated glycoproteins and to be expressed largely on thymus and T cells, and to a much lesser extent on B cells, the remaining T94, T72, and T65 glycoproteins seemed to be present on thymus and T cells but absent from B cells. Two-dimentional PAGE analysis of these T-cell glycoproteins precipitated by ATS demonstrated that T94 was an acidic glycoprotein with pI of 4, while T72 and T65, the latter being found on thymus and T cells but not on T cell-type leukemic cells, exhibited marked electric charge heterogeneity with pI ranging from 4 to 7. These data clearly suggest that human thymus and T cells possess a complex antigenic make-up on their cell surfaces, comparable to that of mouse T cells with a variety of Ly antigen systems.     

An ultraviolet (UV-A) absorbing biopterin glucoside from the marine planktonic cyanobacterium Oscillatoria sp.

We have investigated the physiological response of marine planktonic cyanobacteria to UV-A (320–390 nm) irradiation. Here, we report the isolation of a UV-A absorbing pigment from a UV-A resistant strain of Oscillatoria. This pigment has been purified, and its structure determined to be biopterin glucoside (BG), a compound chemically related to the pteridine pigments found in butterfly wings. A UV-A sensitive isolate did not produce significant levels of this chromophore. UV-A radiation was very effective in eliciting synthesis of BG. In addition, increased UV-A radiation, increased intracellular levels of BG. These data suggest that BG may protect the cyanobacterium from adverse effects of UV-A radiation. Correspondence to: T. Matsunaga     

Simple enzymatic detection method for urinary sulfated 7α-hydroxy bile acids in normal subjects and in patients with acute hepatitis

Urinary sulfated primary bile acids, 7α-hydroxy bile acids, are detected by an enzymatic method using 7α-hydroxysteroid dehydrogenase (EC 1.1.1.-, 7α-HSD) after chromatographic fractionation on Sephadex G-25. Urinary sulfated or glucuronated bile acids are hydrolyzed by β-glucuronidase/sulfatase (EC 3.2.1.31/EC 3.1.6.1) from Helix pomatia and then released 7α-hydroxy bile acids are detected with 7α-HSD in the presence of β-NAD⁺, diaphorase (EC 1.6.99.2, from Clostridium kluyveri) and 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride. The absorbance of formazan formed during the enzymic reaction is measured at 500 nm. Excretion values of 7α-hydroxy bile acids in normal subjects and in patients with acute hepatitis were compared. This enzymatic detection method for the excretion pattern of urinary 7α-hydroxy bile acids may be useful for clinical diagnosis.     

Expression of high activity of ornithine decarboxylase and occurrence of unusual chromophobic cells in anterior pituitary gland of a novel growth-retarded strain of mice, grm/grm

Extremely high activity of ornithine decarboxylase (ODC) was detected in the pituitary gland of growth- retarded mice, grm/grm at 2 months after birth. The elevated enzyme activity gradually decreased to the control level in 14 months after birth. In the pituitary gland of the growth-retarded mice, unusual chromophobic cells were also present from the early stages after birth. The chromophobic cells showed conspicuous proliferations and resulted in a distinct hyperplasia of the tissue after 4 months after birth. These findings suggest that OCD is correlated to the progressive transformation of pituitary cells into the chromophobic cells.     

Loss of symbiont infectivity following thermal stress can be a factor limiting recovery from bleaching in cnidarians

Increases in seawater temperature can cause coral bleaching through loss of symbiotic algae (dinoflagellates of the family Symbiodiniaceae). Corals can recover from bleaching by recruiting algae into host cells from the residual symbiont population or from the external environment. However, the high coral mortality that often follows mass-bleaching events suggests that recovery is often limited in the wild. Here, we examine the effect of pre-exposure to heat stress on the capacity of symbiotic algae to infect cnidarian hosts using the Aiptasia (sea-anemone)-Symbiodiniaceae model system. We found that the symbiont strain Breviolum sp. CS-164 (ITS2 type B1), both free-living and in symbiosis, loses the capacity to infect the host following exposure to heat stress. This loss of infectivity is reversible, however, a longer exposure to heat stress increases the time taken for reversal. Under the same experimental conditions, the loss of infectivity was not observed in another strain Breviolum psygmophilum CCMP2459 (ITS2 type B2). Our results suggest that recovery from bleaching can be limited by the loss of symbiont infectivity following exposure to heat stress.Subject terms: Microbial ecology, Biodiversity

Cnidarians including reef-building corals harbor endosymbiotic dinoflagellates of the family Symbiodiniaceae, from which they derive the majority of their energy. Therefore, the breakdown of the symbiotic relationship, a process known as bleaching, can result in the host starving. However, bleaching is not always lethal because symbiont densities can recover [1, 2]. Recovery from bleaching is driven mainly by symbiotic algae that remain within the bleached corals (the residual population) dividing and spreading throughout the colony [3], and also possibly through the recruitment of free-living symbiotic algae from the external environment [4]. In the last few decades, coral cover has drastically decreased in many regions, due to frequent mass coral bleaching events caused by global warming [5], implying that recovery from bleaching is often limited by unknown factors. In the present study, we demonstrate that both free-living and residual symbiont cells lose their capacity to infect cnidarian host cells once they are exposed to high temperature stress, and present this mechanism as a limiting factor for the host’s recovery from bleaching.We first examined the effect of pre-exposure to high temperature on infectivity using aposymbiotic Exaiptasia pallida (or “Aiptasia”) polyps (Supplementary Fig. 1) and cultured strains of Breviolum sp. CS-164 (ITS2 type B1). Symbiotic algae and polyps were separately incubated at either 25 or 32 °C for 3 days. Following this initial treatment, polyps were inoculated with symbiotic algae at 25 °C for 3 days. Infectivity was then determined by counting the number of algae in the tentacles where algal colonization occurs quickly and individual symbiont cells are easily visualized [6]. When both symbiotic algae and hosts were pre-exposed to 25 °C, significant numbers of algae were seen in tentacles (Fig. 1a). In contrast, the number (Fig. 1a) and density (Fig. 1b) of algae in the tentacles were significantly lower when both the algae and the host were exposed simultaneously to 32 °C, and when the algae alone were exposed to 32 °C. Thus, symbiotic algae, but not host polyps, lose their capacity to form a symbiotic relationship once they are exposed to high temperature. Neither cell viability measured by Evans blue staining (Supplementary Figs. 2a, b and 3) nor cell density (Supplementary Fig. 2c) differed between CS-164 cells exposed to 25 and 32 °C, indicating that infectivity was not lost by the lethal damage to cells. We repeated this experiment with another strain, B. psygmophilum CCMP2459 (ITS2 type B2). In contrast to the results with CS-164, high temperature had no effect on infectivity of CCMP2459 (Supplementary Fig. 4a). These results demonstrate that symbiotic algae can lose their capacity to infect host cells following exposure to high temperature and that thermal sensitivity differs between these two algal strains (Fig. 1b and Supplementary Fig. 4a).Open in a separate windowFig. 1Loss of infectivity in Breviolum sp. CS-164 following exposure to elevated temperature.a Fluorescent photographs of Aiptasia polyps 3 days after culturing with symbiont cells in four different treatments (i) neither symbionts nor polyps exposed to high temperature (32 °C) for 3 days, (ii) only symbionts exposed to high temperature, (iii) only polyps exposed to high temperature, (iv) both symbionts and polyps exposed to high temperature. Red dots show chlorophyll fluorescence from algal symbionts. b The density of symbionts in tentacles was measured 3 days after culturing Aiptasia polyps (H) with symbiont cells (S) in four different treatments, as indicated below the panel and outlined in the text. c The density of symbionts was measured 3 days after culturing Aiptasia polyps with symbiotic algae in different treatments. In this experiment, symbiotic algae that had been expelled from Aiptasia polyps cultured at 25 or 32 °C for 3 days were used to infect Aiptasia. b, c Values are log₂ fold changes with respect to the samples without any temperature treatment. Each point represents an independent experiment. ns, not significant (with p > 0.05); **, p < 0.01.We next examined whether the loss of infectivity following pre-exposure to high temperature also occurs when symbiotic algae are in symbiosis with the host rather than free living (Fig. 1c and Supplementary Fig. 4b). We prepared symbiotic Aiptasia polyps with either CCMP2459 or CS-164 by separately inoculating them in aposymbiotic polyps, and then exposed each group to either 25 or 32 °C for 3 days. Algae expelled from the polyps during this treatment were collected and then used to inoculate aposymbiotic Aiptasia at 25 °C. In CS-164, after 3 days of inoculation, symbiont density in Aiptasia became lower with algae collected at 32 °C than 25 °C (Fig. 1c). However, in CCMP2459, there was no difference in the infectivity between algae collected at 25 and 32 °C (Supplementary Fig. 4b). Our results demonstrate that symbiont cells, both free-living and in symbiosis, can lose infectivity following exposure to high temperature and that thermal sensitivity differs between these two algal strains.We then tested whether or not the temperature-induced loss of infectivity was reversible (Fig. 2). Free-living CS-164 cells were pre-exposed to 25 or 32 °C for either 2 or 3 days after which they were allowed to recover for a maximum of 10 days at 25 °C. After these treatments, symbiotic algae were used to inoculate aposymbiotic Aiptasia polyps at 25 °C for 3 days. Cells with pre-exposure to 32 °C for 2 days had lower infectivity but regained the capacity to infect host cells after a 5-day recovery period (Fig. 2). However, after 3 days exposure, infectivity gradually recovered but remained lower than the controls even after 10 days (Fig. 2). Our results demonstrate that the loss of infectivity following temperature stress is reversible in algal cells but a longer exposure to heat stress increases the time taken to reverse the loss of infectivity.Open in a separate windowFig. 2Reversibility of the lost infectivity upon the exposure to elevated temperature in Breviolum sp. CS-164.The density of symbionts in tentacles was measured following treatments as described in the text and shown by the relative to control. Values are log₂ fold changes with respect to the control. The box and line represent the quartiles and median, respectively. Each point represents an independent experiment. *, p < 0.05.In the present study, infectivity was tested by introducing symbiotic algae directly into the host’s body cavity, suggesting that the loss of infectivity seen in our experiments is likely due to a failure of the host to take up the algal cells via phagocytosis or for symbiont cells to persist within host cells. Factors such as symbiont cell size [7] and the symbiont surface glycome [8, 9] are potentially important determinants of symbiont uptake and persistence, though we still know little about this topic (see review [10]). Furthermore, it is unknown how these various discriminatory factors are influenced by thermal stress.In coral larvae and juveniles, the initiation of symbiosis with algae is reduced at high temperatures, suggesting that global warming will complicate the relationship between host and symbiont [11–13]. However, the mechanism for this reduction in the rate of symbiosis establishment is not clear. Our results suggest that the loss of symbiont infectivity is one possible cause of this phenomenon.Recovery of symbiont densities following coral bleaching relies on a supply of symbiont cells either from within the host or from the external environment. Our results show that symbiotic algae, both free-living and symbiotic, lose the capacity to infect the host following exposure to high temperature stress (Fig. 1). This loss of infectivity is reversible but dependent on the duration of the thermal stress (Fig. 2). Thus, following coral bleaching events, especially those induced by thermal anomalies that can last for weeks, symbiont densities within the host are unlikely to recover in time to avoid the host starving due to physiological compromise of the symbionts, rather than the host. Nonetheless, given the differences in sensitivity between two strains tested (Fig. 1b and Supplementary Fig. 4a), if heat tolerant symbionts are available in the environment, this might provide a chance for recovery.