Cloning, functional characterization, and mode of action of a novel insecticidal pore-forming toxin, sphaericolysin, produced by Bacillus sphaericus

An insecticidal protein produced by Bacillus sphaericus A3-2 was purified to elucidate its structure and mode of action. The active principle purified from the culture broth of A3-2 was a protein with a molecular mass of 53 kDa that rapidly intoxicated German cockroaches (Blattela germanica) at a dose of about 100 ng when injected. The insecticidal protein sphaericolysin possessed the undecapeptide motif of cholesterol-dependent cytolysins and had a unique N-terminal sequence. The recombinant protein expressed in Escherichia coli was equally as potent as the native protein. Sphaericolysin-induced hemolysis resulted from the protein's pore-forming action. This activity as well as the insecticidal activity was markedly reduced by a Y159A mutation. Also, coapplication of sphaericolysin with cholesterol abolished the insecticidal action, suggesting that cholesterol binding plays an important role in insecticidal activity. Sphaericolysin-lysed neurons dissociated from the thoracic ganglia of the German cockroaches. In addition, sphaericolysin's activity in ganglia was suppressed by the Y159A mutation. The sphaericolysin-induced damage to the cockroach ganglia was greater than the damage to the ganglia of common cutworms (Spodoptera litura), which accounts, at least in part, for the higher sensitivity to sphaericolysin displayed by the cockroaches than that displayed by cutworms.     

Effects of resource heterogeneity on nitrogen translocation within clonal fragments of Sasa palmata: an isotopic (15N) assessment

BACKGROUND AND AIMS: Clonal fragments of the rhizomatous dwarf bamboo Sasa palmata, which widely predominates in temperate regions of Japan, were grown under heterogeneous resource conditions such as gap understories or nutrient-patchy grassland. Clonal fragments develop multiple ramets with long rhizomes and appear to be physiologically integrated by the translocation of assimilates. The glasshouse experiment reported here was designed to clarify the mechanisms of physiological integration of nitrogen more precisely. METHODS: To assess how resource conditions influence the amount of nitrogen translocation, and which organ acts as the strongest sink, two experiments were conducted that traced movement of 15N label between interconnected pairs of ramets to compare homogeneous and heterogeneous light and soil nitrogen conditions. KEY RESULTS: The amount of 15N translocated to leaves was between 9% and 11% greater in high-N and high-light ramets in the heterogeneous compared with homogeneous treatments. Under heterogeneous soil nitrogen conditions, translocation increased from individual ramets in resource-rich patches to ramets in resource-poor patches, while the reverse was true under heterogeneous light environments, reflecting differences in the positions of leaves that act as the strongest sinks. Neither the mass increments nor the total mass of clonal fragments was significantly affected by heterogeneity of either light or nutrients, possibly because the experimental period was too short for differences to manifest themselves. CONCLUSIONS: This study clearly demonstrated that nitrogen is readily translocated between ramets, particularly under heterogeneous resource conditions. The translocation patterns were governed by functional 'division of labour' mechanisms that resulted in net nitrogen movement from understory sites to gaps, thereby enhancing the carbon acquisition of the whole fragment. Thus, physiological integration may provide benefits for S. palmata when it is growing under heterogeneous conditions in which there are deficits of certain environmental resources.     

Structure–cytotoxic activity relationship of sesquilignan,morinol A

The cytotoxic activities of sesquilignans, (7S,8S,7′R,8′R)- and (7R,8R,7′S,8′S)-morinol A and (7S,8S,7′S,8′S)- and (7R,8R,7′R,8′R)-morinol B were compared, showing no significant difference between stereoisomers (IC₅₀ = 24–35 μM). As a next stage, the effect of substituents at 7, 7′, and 7″-aromatic ring on the activity was evaluated to find out the higher activity of (7S,8S,7′R,8′R)-7,7′,7″-phenyl derivative 18 (IC₅₀ = 6–7 μM). In the research on the structure–activity relationship of 7″-position of (7S,8S,7′R,8′R)-7,7′,7″-phenyl derivative 18, the most potent compounds were 7,7′,7″-phenyl derivative 18 (IC₅₀ = 6 μM) against HeLa cells. Against HL-60 cells, 7″-(4-nitrophenyl)-7,7′-phenyl derivative 33 and 7″-hexyl-7,7′-phenyl derivative 37 (IC₅₀ = 5 μM) showed highest activity. We discovered the compounds showed four to sevenfold potent activity than that of natural (7S,8S,7′R,8′R)-morinol A. It was also confirmed that the 7′-benzylic hydroxy group have an important role for exhibiting activity, on the other hand, the resonance system of cinnamyl structure is not crucial for the potent activity.     

Sex Differences in Flexibility-Arterial Stiffness Relationship and Its Application for Diagnosis of Arterial Stiffening: A Cross-Sectional Observational Study

Purpose

Arterial stiffness might be related to trunk flexibility in middle-aged and older participants, but it is also affected by age, sex, and blood pressure. This cross-sectional observational study investigated whether trunk flexibility is related to arterial stiffness after considering the major confounding factors of age, sex, and blood pressure. We further investigated whether a simple diagnostic test of flexibility could be helpful to screen for increased arterial stiffening.

Methods

According to age and sex, we assigned 1150 adults (male, n = 536; female, n = 614; age, 18–89 y) to groups with either high- or poor-flexibility based on the sit-and-reach test. Arterial stiffness was assessed by cardio-ankle vascular index.

Results

In all categories of men and in older women, arterial stiffness was higher in poor-flexibility than in high-flexibility (P<0.05). This difference remained significant after normalizing arterial stiffness for confounding factors such as blood pressure, but it was not found among young and middle-aged women. Stepwise multiple-regression analysis also supported the notion of the sex differences in flexibility-arterial stiffness relationship. Receiver operating characteristic curve analysis revealed that cut-off values for sit-and-reach among men and women were 33.2 (area under the curve [AUC], 0.711; 95% confidence interval [CI], 0.666–0.756; sensitivity, 61.7%; specificity, 69.7%) and 39.2 (AUC, 0.639; 95% CI, 0.592–0.686; sensitivity, 61.1%; specificity, 62.0%) cm, respectively.

Conclusion

Our results indicate that flexibility-arterial stiffness relationship is not affected by BP, which is a major confounding factor. In addition, sex differences are observed in this relationship; poor trunk flexibility increases arterial stiffness in young, middle-aged, and older men, whereas the relationship in women is found only in the elderly. Also, the sit-and-reach test can offer a simple method of predicting arterial stiffness at home or elsewhere.     

Genetic variation in Miscanthus × giganteus and the importance of estimating genetic distance thresholds for differentiating clones

Miscanthus × giganteus (Mxg) is an important bioenergy feedstock crop, however, genetic diversity among legacy cultivars may be severely constrained. Only one introduction from Japan to Denmark of this sterile, triploid, vegetatively propagated crop was recorded in the 1930s. We sought to determine if the Mxg cultivars in North America were all synonyms, and if they were derived from the European introduction. We used 64 nuclear and five chloroplast simple sequence repeat (SSR) markers to estimate genetic similarity for 27 Mxg accessions from North America, and compared them with six accessions from Europe, including the species’ type‐specimen. A subset of accessions was also evaluated by restriction‐site associated DNA sequencing (RAD‐seq). In addition, we assessed the potential of new crosses to increase Mxg genetic diversity by comparing eight new triploid Mxg progeny grown from seed, along with samples of the parental species M. sacchariflorus and M. sinensis. Estimates of genotyping error rates were essential for distinguishing between experimental error and true genotypic differences among accessions. Given differences in estimated error rates and costs per marker for SSRs and RAD‐seq, the former is currently more cost‐effective for determining if two accessions are genetically identical. We concluded that all of the Mxg legacy cultivars were derived via vegetative propagation from a single genet. In contrast with the Mxg legacy cultivars, genetic similarity to the type‐specimen of eight new triploid Mxg progeny ranged from 0.46 to 0.56. Though genetic diversity among the Mxg legacy cultivars is critically low, new crosses can provide much‐needed variation to growers.     

Evidence for conversion of human salivary alpha-amylase family A to family B by an enzyme action.

SDS-PAGE showed that human salivary alpha-amylase family A (HSA-A) was converted to family B (HSA-B) in human saliva. This conversion did not occur in the supernatant of saliva which had been centrifuged at 105,000 x g for 60 min. An enzyme which catalyzed the conversion existed in the insoluble fraction of human saliva. The enzyme was solubilized with nonionic or zwitterionic detergents, and showed the maximum activity around pH 6. It was stable between pH 4 and 10, and at a temperature lower than 40 degrees C. The enzyme reduced the molecular weight of HSA-A (62,000) to the same molecular weight (58,000) as that of HSA-B without forming any intermediate. It also changed the PAGE pattern of multiple forms of HSA-A to the same pattern as that of HSA-B. It was not inhibited by protease inhibitors, and it did not destroy the reactivity of HSA-A with anti-human salivary alpha-amylase antiserum. The enzyme diminished the reactivity of HSA-A with concanavalin A. These results indicate that HSA-A was converted to HSA-B through the release of sugar chains by the action of the enzyme in the insoluble fraction of human saliva.     

Tissue architecture in the Caenorhabditis elegans gonad depends on interactions among fibulin-1, type IV collagen and the ADAMTS extracellular protease

Molecules in the extracellular matrix (ECM) regulate cellular behavior in both development and pathology. Fibulin-1 is a conserved ECM protein. The Caenorhabditis elegans ortholog, FBL-1, regulates gonad-arm elongation and expansion by acting antagonistically to GON-1, an ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family protease. The elongation of gonad arms is directed by gonadal distal tip cells (DTCs). Here we report that a dominant mutation in the EMB-9/type IV collagen α1 subunit can compensate for loss of FBL-1 activity in gonadogenesis. A specific amino acid substitution in the noncollagenous 1 (NC1) domain of EMB-9 suppressed the fbl-1 null mutant. FBL-1 was required to maintain wild-type EMB-9 in the basement membrane (BM), whereas mutant EMB-9 was retained in the absence of FBL-1. EMB-9 (either wild type or mutant) localization in the BM enhanced PAT-3/β-integrin expression in DTCs. In addition, overexpression of PAT-3 partially rescued the DTC migration defects in fbl-1 mutants, suggesting that EMB-9 acts in part through PAT-3 to control DTC migration. In contrast to the suppression of fbl-1(tk45), mutant EMB-9 enhanced the gonadal defects of gon-1(e1254), suggesting that it gained a function similar to that of wild-type FBL-1, which promotes DTC migration by inhibiting GON-1. We propose that FBL-1 and GON-1 control EMB-9 accumulation in the BM and promote PAT-3 expression to control DTC migration.     

High accumulation of minerals in the human arteries of lower limb

To elucidate accumulation of minerals in the human arteries, the relative contents (RCs) of minerals in the arteries of the upper and lower limbs were analyzed by inductively coupled plasma atomic emission spectrometry. It was found that the RCs of calcium and phosphorus in the femoral and popliteal arteries of the lower limb increased with aging, whereas those in the axillary and radial arteries of the upper limb did not increase with aging. This result indicates that higher accumulation of calcium and phosphorus occurs in the arteries of the lower limb with aging as compared with that in the arteries of the upper limb, and the prevalence of arteriosclerosis increases in the arteries of the lower limb with aging but not in the arteries of the upper limb.     

Effect of the structure of dietary epoxylignan on its cytotoxic activity: relationship between the structure and the activity of 7,7′-epoxylignan and the introduction of apoptosis by caspase 3/7

We compared the cytotoxic activities of dietary epoxylignans and their stereoisomers and found (?)-verrucosin, which is (7S,7′R,8R,8′R)-7,7′-epoxylignan, to be the most cytotoxic epoxylignan against HeLa cells (IC₅₀ = 6.6 μM). On the other hand, the activity was about a factor of 10 less against HL-60. In this research on the relationship between the structure and cytotoxic activity of (?)-verrucosin 13, the 7-(4-methoxyphenyl)-7′-(3,4-dimethoxyphenyl) derivative 60, for which the activity (IC₅₀ = 2.4 μM) is three times greater than (?)-verrucosin 13, was discovered. The induction of apoptosis by caspase 3/7 was observed upon treatment with the (?)-verrucosin derivative.