Characterization of mosquito larvicidal parasporal inclusions of a Bacillus thuringiensis serovar higo strain

The parasporal inclusion proteins of the type strain of Bacillus thuringiensis serovar higo (H44), that have moderate mosquitocidal activity, were characterized. The purified parasporal inclusions, spherical in shape, were examined for activity against the two mosquito species, Culex pipiens molestus and Anopheles stephensi and the moth-fly, Telmatoscopus albipunctatus . The LC₅₀ values of the inclusion for the two mosquitoes were 3·41 and 0·15 μg ml⁻¹, respectively. No mortality was shown for T. albipunctatus larvae by the inclusions at concentrations up to 1 mg ml⁻¹. Solubilized parasporal inclusions exhibited no haemolytic activity against sheep erythrocytes. Parasporal inclusions consisted of eight proteins with molecular masses of 98, 91, 71, 63, 59, 50, 44 and 27 kDa. Of these, the 50 and 44 kDa proteins were the major components. Analysis with immunoblotting revealed that, among several inclusion proteins of B. thuringiensis serovar israelensis, only two proteins of 130 kDa and 110 kDa reacted weakly with antibodies against higo proteins. N-terminal amino acid sequences of the 98, 91, and 71 kDa proteins showed 85–100% identity to those of the two established Cry protein classes, Cry4A and Cry10A.     

On-Chip Immunoelectrophoresis of Extracellular Vesicles Released from Human Breast Cancer Cells

Extracellular vesicles (EVs) including exosomes and microvesicles have attracted considerable attention in the fields of cell biology and medicine. For a better understanding of EVs and further exploration of their applications, the development of analytical methods for biological nanovesicles has been required. In particular, considering the heterogeneity of EVs, methods capable of measuring individual vesicles are desired. Here, we report that on-chip immunoelectrophoresis can provide a useful method for the differential protein expression profiling of individual EVs. Electrophoresis experiments were performed on EVs collected from the culture supernatant of MDA-MB-231 human breast cancer cells using a measurement platform comprising a microcapillary electrophoresis chip and a laser dark-field microimaging system. The zeta potential distribution of EVs that reacted with an anti-human CD63 (exosome and microvesicle marker) antibody showed a marked positive shift as compared with that for the normal immunoglobulin G (IgG) isotype control. Thus, on-chip immunoelectrophoresis could sensitively detect the over-expression of CD63 glycoproteins on EVs. Moreover, to explore the applicability of on-chip immunoelectrophoresis to cancer diagnosis, EVs collected from the blood of a mouse tumor model were analyzed by this method. By comparing the zeta potential distributions of EVs after their immunochemical reaction with normal IgG, and the anti-human CD63 and anti-human CD44 (cancer stem cell marker) antibodies, EVs of tumor origin circulating in blood were differentially detected in the real sample. The result indicates that the present method is potentially applicable to liquid biopsy, a promising approach to the low-invasive diagnosis of cancer.     

Protein disulfide isomerase-P5, down-regulated in the final stage of boar epididymal sperm maturation,catalyzes disulfide formation to inhibit protein function in oxidative refolding of reduced denatured lysozyme

In mammalian spermiogenesis, sperm mature during epididymal transit to get fertility. The pig sharing many physiological similarities with humans is considered a promising animal model in medicine. We examined the expression profiles of proteins from boar epididymal caput, corpus, and cauda sperm by two-dimensional gel electrophoresis and peptide mass fingerprinting. Our results indicated that protein disulfide isomerase-P5 (PDI-P5) human homolog was down-regulated from the epididymal corpus to cauda sperm, in contrast to the constant expression of protein disulfide isomerase A3 (PDIA3) human homolog. To examine the functions of PDIA3 and PDI-P5, we cloned and sequenced cDNAs of pig PDIA3 and PDI-P5 protein precursors. Each recombinant pig mature PDIA3 and PDI-P5 expressed in Escherichia coli showed thiol-dependent disulfide reductase activities in insulin turbidity assay. Although PDIA3 showed chaperone activity to promote oxidative refolding of reduced denatured lysozyme, PDI-P5 exhibited anti-chaperone activity to inhibit oxidative refolding of lysozyme at an equimolar ratio. SDS-PAGE and Western blotting analysis suggested that disulfide cross-linked and non-productively folded lysozyme was responsible for the anti-chaperone activity of PDI-P5. These results provide a molecular basis and insights into the physiological roles of PDIA3 and PDI-P5 in sperm maturation and fertilization.     

2-Haloacrylate Hydratase,a New Class of Flavoenzyme That Catalyzes the Addition of Water to the Substrate for Dehalogenation

Enzymes catalyzing the conversion of organohalogen compounds are useful in the chemical industry and environmental technology. Here we report the occurrence of a new reduced flavin adenine dinucleotide (FAD) (FADH₂)-dependent enzyme that catalyzes the removal of a halogen atom from an unsaturated aliphatic organohalogen compound by the addition of a water molecule to the substrate. A soil bacterium, Pseudomonas sp. strain YL, inducibly produced a protein named Caa67_YL when the cells were grown on 2-chloroacrylate (2-CAA). The caa67_YL gene encoded a protein of 547 amino acid residues (M_r of 59,301), which shared weak but significant sequence similarity with various flavoenzymes and contained a nucleotide-binding motif. We found that 2-CAA is converted into pyruvate when the reaction was carried out with purified Caa67_YL in the presence of FAD and a reducing agent [NAD(P)H or sodium dithionite] under anaerobic conditions. The reducing agent was not stoichiometrically consumed during this reaction, suggesting that FADH₂ is conserved by regeneration in the catalytic cycle. When the reaction was carried out in the presence of H₂¹⁸O, [¹⁸O]pyruvate was produced. These results indicate that Caa67_YL catalyzes the hydration of 2-CAA to form 2-chloro-2-hydroxypropionate, which is chemically unstable and probably spontaneously dechlorinated to form pyruvate. 2-Bromoacrylate, but not other 2-CAA analogs such as acrylate and methacrylate, served as the substrate of Caa67_YL. Thus, we named this new enzyme 2-haloacrylate hydratase. The enzyme is very unusual in that it requires the reduced form of FAD for hydration, which involves no net change in the redox state of the coenzyme or substrate.Dehalogenases catalyze the removal of halogen atoms from organohalogen compounds. These enzymes have been attracting a great deal of attention partly because of their possible applications to the chemical industry and environmental technology. Several dehalogenases have been discovered and characterized (6, 11, 14, 17, 22). Some of them act on unsaturated aliphatic organohalogen compounds in which a halogen atom is bound to an sp²-hybridized carbon atom. Examples include various corrinoid/iron-sulfur cluster-containing reductive dehalogenases (1, 7), cis- and trans-3-chloroacrylic acid dehalogenases (4, 19), and LinF (maleylacetate reductase), which acts on 2-chloromaleylacetate (5).In order to gain more insight into the enzymatic dehalogenation of unsaturated aliphatic organohalogen compounds, we searched for microorganisms that dissimilate 2-chloroacrylate (2-CAA) as a sole source of carbon and energy (8). 2-CAA is a bacterial metabolite of 2-chloroallyl alcohol, an intermediate or by-product in the industrial synthesis of herbicides (26). Rats treated orally with the herbicides sulfallate, diallate, and triallate excrete urinary 2-CAA (16). Various halogenated acrylic acids are produced by a red alga (27). We obtained three 2-CAA-utilizing bacteria as a result of screening (8). For one of these bacteria, Burkholderia sp. strain WS, we previously discovered a new NADPH-dependent enzyme, 2-haloacrylate reductase (12, 13). Although this enzyme does not directly remove a halogen atom from the substrate, it is supposed to participate in the metabolism of 2-CAA by catalyzing the conversion of 2-CAA into l-2-chloropropionate, which is subsequently dehalogenated by l-2-haloacid dehalogenase.Another bacterium that we obtained, Pseudomonas sp. strain YL, also dissimilates 2-CAA. However, the metabolic fate of 2-CAA in this bacterium remains unclear. In the present study, we analyzed proteins from 2-CAA- and lactate-grown cells of Pseudomonas sp. YL by two-dimensional polyacrylamide gel electrophoresis (PAGE) and identified a 2-CAA-inducible protein. We found that the protein catalyzes the dehalogenation of 2-CAA by the addition of a water molecule to the substrate, representing a new family of dehalogenases that act on unsaturated aliphatic organohalogen compounds. Remarkably, the enzyme requires reduced flavin adenine dinucleotide (FAD) (FADH₂) for its activity, although the reaction does not involve a net change in the redox state of the coenzyme or substrate. Here we describe the occurrence and characteristics of this unusual flavoenzyme.     

Excessive ingestion of long-chain polyunsaturated fatty acids during developmental stage causes strain- and sex-dependent eye abnormalities in mice

The eyes are riched in long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic acid [ARA; 20:4 (n−6)] and docosahexaenoic acid [DHA; 22:6 (n−3)]. Despite their abundance in the eyes, ARA and DHA cannot be sufficiently synthesized de novo in mammals. During gestation, eye development is exceptionally rapid, and substantial amounts of LC-PUFAs are needed to ensure proper eye development. Here, we studied the influences of dietary LC-PUFAs in dams (C57BL/6 and C3H/He) on the eye morphogenesis and organogenesis of their pups. Intriguingly, fetuses and newborn mice from C57BL/6 dams fed an LC-PUFA (particularly ARA)-enriched diet displayed a much higher incidence of eye abnormalities such as microphthalmia (small eye) and corneal opacity than those from dams fed an LC-PUFA-poor diet. The effects of LC-PUFAs on eye anomalies were evident only in the female pups of C57BL/6 inbred mice, not in those of C3H/He mice or male C57BL/6 mice. These results demonstrate a gene-by-environment (GxE) interaction in eye development in mice. Furthermore, our molecular analysis suggested the potential roles of Pitx3 and Pax6 in the above interaction involving ARA.     

Roles for the N- and C-terminal domains of phytochrome B in interactions between phytochrome B and cryptochrome signaling cascades

Plants fine-tune light responses through interactions betweenphotoreceptors. We have previously reported that the greeningof Arabidopsis thaliana roots is regulated synergistically byphytochromes and cryptochromes. In the present study, we investigatedthe functions of the N- and C-terminal domains of phytochromeB (phyB) in the interactions between phyB and cryptochrome signalingcascades. Transgenic Arabidopsis expressing the phyB N-terminaldomain fused to green fluorescent protein (GFP), ß-glucuronidase(GUS) and the nuclear localization signal (NLS) showed intenseroot greening under blue light, indicating that the C-terminaldomain was dispensable for the synergistic interaction in theinduction of root greening. However, root greening under redlight was substantially reduced in the absence of the C-terminaldomain. This effect was opposite to the previous observationthat removal of the C-terminal domain enhanced the signalingactivity of phyB in the inhibition of hypocotyl elongation.In addition, we found that overexpression of the isolated C-terminaldomain of phyB enhanced the blue light response not only forroot greening but also for the inhibition of hypocotyl elongation.Analysis of this activity on various photoreceptor mutant backgroundsdemonstrated that the isolated C-terminal domain enhanced cryptochromesignaling. In summary, these results demonstrate that differentdomains of phyB can play various roles which are dependent onlight conditions as well as on the specific physiological response.     

Biotransformation of raspberry ketone and zingerone by cultured cells of Phytolacca americana

The biotransformation of raspberry ketone and zingerone were individually investigated using cultured cells of Phytolacca americana. In addition to (2S)-4-(4-hydroxyphenyl)-2-butanol (2%), (2S)-4-(3,4-dihydroxyphenyl)-2-butanol (5%), 4-[4-(beta-d-glucopyranosyloxy)phenyl]-2-butanone (19%), 4-[(3S)-3-hydroxybutyl]phenyl-beta-d-glucopyranoside (23%), and (2S)-4-(4-hydroxyphenyl)but-2-yl-beta-d-glucopyranoside (20%), two biotransformation products, i.e., 2-hydroxy-4-[(3S)-3-hydroxybutyl]phenyl-beta-d-glucopyranoside (12%) and 2-hydroxy-5-[(3S)-3-hydroxybutyl]phenyl-beta-d-glucopyranoside (11%), were isolated from suspension cells after incubation with raspberry ketone for three days. On the other hand, two compounds, i.e., (2S)-4-(4-hydroxy-3-methoxyphenyl)but-2-yl-beta-d-glucopyranoside (17%) and (2S)-2-(beta-d-glucopyranosyloxy)-4-[4-(beta-d-glucopyranosyloxy)-3-methoxyphenyl]butane (16%), together with (2S)-4-(4-hydroxy-3-methoxyphenyl)-2-butanol (15%), 4-[4-(beta-d-glucopyranosyloxy)-3-methoxyphenyl]-2-butanone (21%), and 4-[(3S)-3-hydroxybutyl]-2-methoxyphenyl-beta-d-glucopyranoside (24%) were obtained upon addition of zingerone. Cultured cells of P. americana can reduce, and regioselectively hydroxylate and glucosylate, these food ingredients to their beta-glycosides.     

A protein kinase Cepsilon-anti-apoptotic kinase signaling complex protects human vascular endothelial cells against apoptosis through induction of Bcl-2

Endothelial cell apoptosis is associated with vascular injury and predisposes to atherogenesis. Endothelial cells express anti-apoptotic genes including Bcl-2, Bcl-XL and survivin, which also contribute to angiogenesis and vascular remodeling. We report a central role for protein kinase Cepsilon (PKCepsilon) in the regulation of Bcl-2 expression and cytoprotection of human vascular endothelium against apoptosis. Using myristoylated inhibitory peptides, a predominant role for PKCepsilon in vascular endothelial growth factor-mediated endothelial resistance to apoptosis was revealed. Immunoblotting of endothelial cells infected with an adenovirus expressing a constitutively active form of PKCepsilon (Adv-PKCepsilon-CA) or control Adv-beta-galactosidase demonstrated a 3-fold, PKCepsilon-dependent increase in Bcl-2 expression, with no significant change in Bcl-XL, Bad, Bak, or Bax. The induction of Bcl-2 inhibited apoptosis induced by serum starvation or etoposide, and PKCepsilon activation attenuated etoposide-induced caspase-3 cleavage. The functional role of Bcl-2 was confirmed with Bcl-2 antagonist HA-14-1. Inhibition of phosphoinositide 3-kinase attenuated vascular endothelial growth factor-induced protection against apoptosis, and this was rescued by overexpression of constitutively active PKCepsilon, suggesting PKCepsilon acts downstream of phosphoinositide 3-kinase. Co-immunoprecipitation studies demonstrated a physical interaction between PKCepsilon and Akt, which resulted in formation of a signaling complex, leading to optimal induction of Bcl-2. This study reveals a pivotal role for PKCepsilon in endothelial cell cytoprotection against apoptosis. We demonstrate that PKCepsilon forms a signaling complex and acts co-operatively with Akt to protect human vascular endothelial cells against apoptosis through induction of the anti-apoptotic protein Bcl-2 and inhibition of caspase-3 cleavage.