Effects of the termination of LC30 imidacloprid stress on the multigeneration adaptive strategies of Aphis glycines population.

Aphis glycines Matsumura (Hemiptera: Aphididae) is a major soybean pest that often poses a serious threat to soybean production. Imidacloprid is one of the commonly used insecticides to control the soybean aphid. To investigate the effect of termination of imidacloprid stress on the adaptive strategies of soybean aphid populations, we studied the growth, development, and related metabolism changes when the stress was terminated after 24 generations of imidacloprid stress on A. glycines. The results show that the A. glycines population accelerated its recovery and expanded its population size across generations. The longevity of the adults of the recovering population in the F12, F18, and F24 generations, respectively, was 1.11, 1.15, and 1.11 times longer than the control, while the fecundity was 10.38%, 11.74%, and 11.61% higher than that of the control. The net reproductive rate (R 0) of the recovering population was always significantly higher than that of the control in the F1 to F24 generations. In addition, metabolisms related to the regulation of cell proliferation and oocyte meiosis were significantly upregulated in the recovering population. Even when the imidacloprid pressure disappeared, intergenerational stimuli still affected the adaptive strategies of soybean aphid populations. This effect was manifested as inhibiting the growth and development of the soybean aphid in the early generations and improving the fecundity of the soybean aphid in the later generations. Adaptive soybean aphid populations would surge in the absence of imidacloprid pressure. This study provides an important reference for exploring the adaptability of the A. glycines population under termination of stress from low lethal concentrations of imidacloprid across generations. It also provides important data for monitoring the population dynamics of A. glycines in the field and analyzing the degree of pharmacodynamic stress.

Effects of multigenerational imidacloprid and thiamethoxam stress on metabolism and physiology of Aphis glycines Matsumura (Hemiptera: Aphididae).

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), a primary pest of soybean, poses a severe threat to soybean production. In this study, the 4th instar nymphs were exposed to the LC50 and LC30 of imidacloprid and thiamethoxam from F0 to F4 generations to evaluate the activities of peroxidase, pyruvate kinase, and trehalase using microassay. We found that peroxidase and pyruvate kinase activities in soybean aphids increased rapidly, first to peak and then decreased slowly generation by generation under imidacloprid and thiamethoxam stress. In contrast, the trehalase activity was significantly decreased in F1 to F5 generations when treated with the LC50 and LC30 and imidacloprid and thiamethoxam compared to control. In addition, the Enzyme-Linked Immunosorbent Assay (ELISA) was used to monitor the changes in molting and juvenile hormone expressions of the soybean aphids in each generation (F1-F5). The expression of juvenile hormone in soybean aphids was increased significantly in each generation under continuous stress of imidacloprid and thiamethoxam LC50 imidacloprid and LC50 thiamethoxam inhibited the expression of molting hormones in soybean aphids of each generation. LC30 imidacloprid or LC30 thiamethoxam significantly stimulated the expression of molting hormone in the 1st and 2nd instar nymphs in each generation. In this paper, the differences in antioxidant regulation, energy metabolism intensity, and hormone expression of multi-generation soybean aphids were monitored under continuous stress of imidacloprid and thiamethoxam. Our results revealed the effects of continuous insecticide stress on the main endogenous substances. Further, they clarified the regulation rules of resistance in soybean aphids, providing a reference for efficient control with imidacloprid and thiamethoxam.

The effects of acetamiprid multigeneration stress on metabolism and physiology of Aphis glycines Matsumura (Hemiptera: Aphididae).

Aphis glycines Matsumura (Hemiptera: Aphididae) is a major soybean pest that often poses a serious threat to soybean production. In this study, we checked the effects of acetamiprid on redox, energy metabolism, and hormone expression in A. glycines. The LC50 and LC30 of acetamiprid were used to treat the fourth instar nymphs in each generation from F0 to F4 to measure the activity of peroxidase, pyruvate kinase, and trehalase using a microassays approach. The peroxidase activity was significantly higher than control when treated with the LC30 of acetamiprid in F2-F5 generations. The activity of pyruvate kinase was significantly higher, while trehalase activity was substantially lower than control in each generation. Besides, we monitored molting and juvenile hormone expression in soybean aphids using enzyme-linked immunosorbent assay. The juvenile hormone titer of third instar nymphs was significantly higher in the treatment group (F1, F2, F4, and F5), while no effects were noted in the F3 generation. Taken together, the activity of peroxidase and pyruvate kinase in soybean aphid first increased to the peak and then decreased, while the trehalase activity continuously decreased in all generations following exposure to acetamiprid. The juvenile hormone titer was significantly higher, while the molting hormone titer was significantly lower in LC50 -treated aphids than in control. Moreover, the LC30 of acetamiprid increased the molting hormone expression in soybean aphids. These findings indicated a baseline for the effective use of acetamiprid in controlling soybean aphids.

Effects of Acetamiprid at Low and Median Lethal Concentrations on the Development and Reproduction of the Soybean Aphid Aphis glycines.

The soybean aphid Aphis glycines Matsumura (Hemiptera: Aphididae) is a major pest of soybean and poses a serious threat to soybean production. Studies on the effect of acetamiprid on the life table parameters of A. glycines, provide important information for the effective management of this pest. We found that exposure to acetamiprid at LC50 significantly extended the mean generation time, adult pre-reproductive period, and total pre-reproduction period compared with the control, whereas exposure to acetamiprid at LC30 significantly shortened these periods. Exposure to acetamiprid at both LC30 and LC50 significantly decreased the fecundity of the female adult, net reproductive rate, intrinsic rate of increase, and finite rate of increase compared with the control. The probability of attaining the adult stage was 0.51, 0.38, and 0.86 for a newly born nymph from the LC30 acetamiprid treatment group, LC50 acetamiprid treatment group, and control group, respectively. Acetamiprid at both LC50 and LC30 exerted stress effects on A. glycines, with the LC50 treatment significantly decreased the growth rate compared with the LC30 treatment. The present study provides reference data that could facilitate the exploration of the effects of acetamiprid on A. glycines in the field.

Effects of imidacloprid and thiamethoxam on the development and reproduction of the soybean aphid Aphis glycines.

The soybean aphid Aphis glycines Matsumura (Hemiptera: Aphididae) is a primary pest of soybeans and poses a serious threat to soybean production. Our studies were conducted to understand the effects of different concentrations of insecticides (imidacloprid and thiamethoxam) on A. glycines and provided critical information for its effective management. Here, we found that the mean generation time and adult and total pre-nymphiposition periods of the LC50 imidacloprid- and thiamethoxam-treatment groups were significantly longer than those of the control group, although the adult pre-nymphiposition period in LC30 imidacloprid and thiamethoxam treatment groups was significantly shorter than that of the control group. Additionally, the mean fecundity per female adult, net reproductive rate, intrinsic rate of increase, and finite rate of increase of the LC30 imidacloprid-treatment group were significantly lower than those of the control group and higher than those of the LC50 imidacloprid-treatment group (P < 0.05). Moreover, both insecticides exerted stress effects on A. glycines, and specimens treated with the two insecticides at the LC50 showed a significant decrease in their growth rates relative to those treated with the insecticides at LC30. These results provide a reference for exploring the effects of imidacloprid and thiamethoxam on A. glycines population dynamics in the field and offer insight to agricultural producers on the potential of low-lethal concentrations of insecticides to stimulate insect reproduction during insecticide application.

Effect of rotenone-induced stress on physiologically active substances in adult Aphis glycines.

The aim of this study was to determine the effect of rotenone stress on Aphis glycines Matsumura (Hemiptera: Aphididae) populations in different habitats of Northeast China. The changes in kinase expression activity of endogenous substances (proteins, total sugars, trehalose, cholesterol, and free amino acids), detoxifying enzymes (cytochrome P450 and glutathione S-transferase), and metabolic enzymes (proteases and phosphofructokinases) in specimens from three populations were compared before and after stress with rotenone at median lethal concentration (LC50) and their response mechanisms were analyzed. Following a 24 h treatment with rotenone, the average LC50 rotenone values in A. glycines specimens from field populations A and B, and a laboratory population were 4.39, 4.61, and 4.03 mg/L, respectively. The degree of changes in the kinase expression activity of endogenous substances also differed, which indicated a difference in the response of A. glycines specimens from varying habitats to LC50 rotenone stress. The content of endogenous substances, detoxifying enzymes, and metabolic enzymes, except for that of free amino acids, changed significantly in all populations treated with rotenone at LC50 compared with that in the control (P < 0.05). The decrease in protein and trehalose content, and the obstruction of cholesterol transportation owing to decreased feeding in stressed individuals were the causes of A. glycines death after rotenone treatment. Aphis glycines resistance to rotenone may be related to cytochrome P450 expression.

RitR is an archetype for a novel family of redox sensors in the streptococci that has evolved from two-component response regulators and is required for pneumococcal colonization.

To survive diverse host environments, the human pathogen Streptococcus pneumoniae must prevent its self-produced, extremely high levels of peroxide from reacting with intracellular iron. However, the regulatory mechanism(s) by which the pneumococcus accomplishes this balance remains largely enigmatic, as this pathogen and other related streptococci lack all known redox-sensing transcription factors. Here we describe a two-component-derived response regulator, RitR, as the archetype for a novel family of redox sensors in a subset of streptococcal species. We show that RitR works to both repress iron transport and enable nasopharyngeal colonization through a mechanism that exploits a single cysteine (Cys128) redox switch located within its linker domain. Biochemical experiments and phylogenetics reveal that RitR has diverged from the canonical two-component virulence regulator CovR to instead dimerize and bind DNA only upon Cys128 oxidation in air-rich environments. Atomic structures show that Cys128 oxidation initiates a "helical unravelling" of the RitR linker region, suggesting a mechanism by which the DNA-binding domain is then released to interact with its cognate regulatory DNA. Expanded computational studies indicate this mechanism could be shared by many microbial species outside the streptococcus genus.

Streptomyces wadayamensis MppP is a PLP-Dependent Oxidase, Not an Oxygenase.

The PLP-dependent l-arginine hydroxylase/deaminase MppP from Streptomyces wadayamensis (SwMppP) is involved in the biosynthesis of l-enduracididine, a nonproteinogenic amino acid found in several nonribosomally produced peptide antibiotics. SwMppP uses only PLP and molecular oxygen to catalyze a 4-electron oxidation of l-arginine to form a mixture of 2-oxo-4(S)-hydroxy-5-guanidinovaleric acid and 2-oxo-5-guanidinovaleric acid. Steady-state kinetics analysis in the presence and absence of catalase shows that one molecule of peroxide is formed for every molecule of dioxygen consumed in the reaction. Moreover, for each molecule of 2-oxo-4(S)-hydroxy-5-guanidinovaleric acid produced, two molecules of dioxygen are consumed, suggesting that both the 4-hydroxy and 2-keto groups are derived from water. This was confirmed by running the reactions using either [18]O2 or H2[18]O and analyzing the products by ESI-MS. Incorporation of [18]O was only observed when the reaction was performed in H2[18]O. Crystal structures of SwMppP with l-arginine, 2-oxo-4(S)-hydroxy-5-guanidinovaleric acid, or 2-oxo-5-guanidinovaleric acid bound were determined at resolutions of 2.2, 1.9. and 1.8 Å, respectively. The structural data show that the N-terminal portion of the protein is disordered unless substrate or product is bound in the active site, in which case it forms a well-ordered helix that covers the catalytic center. This observation suggested that the N-terminal helix may have a role in substrate binding and/or catalysis. Our structural and kinetic characterizations of N-terminal variants show that the N-terminus is critical for catalysis. In light of this new information, we have refined our previously proposed mechanism of the SwMppP-catalyzed oxidation of l-arginine.

Swit_4259, an acetoacetate decarboxylase-like enzyme from Sphingomonas wittichii RW1.

The Gram-negative bacterium Sphingomonas wittichii RW1 is notable for its ability to metabolize a variety of aromatic hydrocarbons. Not surprisingly, the S. wittichii genome contains a number of putative aromatic hydrocarbon-degrading gene clusters. One of these includes an enzyme of unknown function, Swit_4259, which belongs to the acetoacetate decarboxylase-like superfamily (ADCSF). Here, it is reported that Swit_4259 is a small (28.8 kDa) tetrameric ADCSF enzyme that, unlike the prototypical members of the superfamily, does not have acetoacetate decarboxylase activity. Structural characterization shows that the tertiary structure of Swit_4259 is nearly identical to that of the true decarboxylases, but there are important differences in the fine structure of the Swit_4259 active site that lead to a divergence in function. In addition, it is shown that while it is a poor substrate, Swit_4259 can catalyze the hydration of 2-oxo-hex-3-enedioate to yield 2-oxo-4-hydroxyhexanedioate. It is also demonstrated that Swit_4259 has pyruvate aldolase-dehydratase activity, a feature that is common to all of the family V ADCSF enzymes studied to date. The enzymatic activity, together with the genomic context, suggests that Swit_4259 may be a hydratase with a role in the metabolism of an as-yet-unknown hydrocarbon. These data have implications for engineering bioremediation pathways to degrade specific pollutants, as well as structure-function relationships within the ADCSF in general.

Periplasmic Cytophaga hutchinsonii Endoglucanases Are Required for Use of Crystalline Cellulose as the Sole Source of Carbon and Energy.

UNLABELLED: The soil bacterium Cytophaga hutchinsonii actively digests crystalline cellulose by a poorly understood mechanism. Genome analyses identified nine genes predicted to encode endoglucanases with roles in this process. No predicted cellobiohydrolases, which are usually involved in the utilization of crystalline cellulose, were identified. Chromosomal deletions were performed in eight of the endoglucanase-encoding genes: cel5A, cel5B, cel5C, cel9A, cel9B, cel9C, cel9E, and cel9F Each mutant retained the ability to digest crystalline cellulose, although the deletion of cel9C caused a modest decrease in cellulose utilization. Strains with multiple deletions were constructed to identify the critical cellulases. Cells of a mutant lacking both cel5B and cel9C were completely deficient in growth on cellulose. Cell fractionation and biochemical analyses indicate that Cel5B and Cel9C are periplasmic nonprocessive endoglucanases. The requirement of periplasmic endoglucanases for cellulose utilization suggests that cellodextrins are transported across the outer membrane during this process. Bioinformatic analyses predict that Cel5A, Cel9A, Cel9B, Cel9D, and Cel9E are secreted across the outer membrane by the type IX secretion system, which has been linked to cellulose utilization. These secreted endoglucanases may perform the initial digestion within amorphous regions on the cellulose fibers, releasing oligomers that are transported into the periplasm for further digestion by Cel5B and Cel9C. The results suggest that both cell surface and periplasmic endoglucanases are required for the growth of C. hutchinsonii on cellulose and that novel cell surface proteins may solubilize and transport cellodextrins across the outer membrane. IMPORTANCE: The bacterium Cytophaga hutchinsonii digests crystalline cellulose by an unknown mechanism. It lacks processive cellobiohydrolases that are often involved in cellulose digestion. Critical cellulolytic enzymes were identified by genetic analyses. Intracellular (periplasmic) nonprocessive endoglucanases performed an important role in cellulose utilization. The results suggest a model involving partial digestion at the cell surface, solubilization and uptake of cellodextrins across the outer membrane by an unknown mechanism, and further digestion within the periplasm. The ability to sequester cellodextrins and digest them intracellularly may limit losses of soluble cellobiose to other organisms. C. hutchinsonii uses an unusual approach to digest cellulose and is a potential source of novel proteins to increase the efficiency of conversion of cellulose into soluble sugars and biofuels.

Heterologous expression of carcinoembryonic antigen in Lactococcus lactis via LcsB-mediated surface displaying system for oral vaccine development.

BACKGROUND/PURPOSE: Carcinoembryonic antigen (CEA) is an attractive target for immunotherapy because it is expressed minimally in normal tissue, but is overexpressed in a wide variety of malignant epithelial tissues. Lactic acid bacteria (LABs), widely used in food processes, are attractive candidates for oral vaccination. Thus, we examined whether LABs could be used as a live vaccine vector to deliver CEA antigen. METHODS: CEA was cloned into an Escherichia coli/Lactococcus lactis shuttle vector pSEC:LEISS under the control of a nisin promoter. For displaying the CEA on the cell surface of the L. lactis strain, the anchor motif LcsB from the S-layer protein of Lactobacillus crispatus was fused with CEA. Intracellular and cell surface expression of the CEA-LcsB fusion was confirmed by western blot analysis. RESULTS: Significantly higher levels of CEA-specific secretory immunoglobulin A in the sera of mice were observed upon oral administration of strain cultures containing the CEA-LcsB fused protein. In addition, the CEA-LcsB antigen group showed a higher spleen index compared to the CEA antigen alone or negative control, demonstrating that surface-displayed CEA antigen could induce a higher immune response. CONCLUSION: These results provided the first evidence for displaying CEA antigen on the cell surfaces of LABs as oral vaccines against cancer or infectious diseases.

Streptomyces wadayamensis MppP Is a Pyridoxal 5'-Phosphate-Dependent L-Arginine α-Deaminase, γ-Hydroxylase in the Enduracididine Biosynthetic Pathway.

L-Enduracididine (L-End) is a nonproteinogenic amino acid found in a number of bioactive peptides, including the antibiotics teixobactin, enduracidin, and mannopeptimycin. The potent activity of these compounds against antibiotic-resistant pathogens like MRSA and their novel mode of action have garnered considerable interest for the development of these peptides into clinically relevant antibiotics. This goal has been hampered, at least in part, by the fact that L-End is difficult to synthesize and not currently commercially available. We have begun to elucidate the biosynthetic pathway of this unusual building block. In mannopeptimycin-producing strains, like Streptomyces wadayamensis, L-End is produced from L-Arg by the action of three enzymes: MppP, MppQ, and MppR. Herein, we report the structural and functional characterization of MppP. This pyridoxal 5'-phosphate (PLP)-dependent enzyme was predicted to be a fold type I aminotransferase on the basis of sequence analysis. We show that MppP is actually the first example of a PLP-dependent hydroxylase that catalyzes a reaction of L-Arg with dioxygen to yield a mixture of 2-oxo-4-hydroxy-5-guanidinovaleric acid and 2-oxo-5-guanidinovaleric acid in a 1.7:1 ratio. The structure of MppP with PLP bound to the catalytic lysine residue (Lys221) shows that, while the tertiary structure is very similar to those of the well-studied aminotransferases, there are differences in the arrangement of active site residues around the cofactor that likely account for the unusual activity of this enzyme. The structure of MppP with the substrate analogue D-Arg bound shows how the enzyme binds its substrate and indicates why D-Arg is not a substrate. On the basis of this work and previous work with MppR, we propose a plausible biosynthetic scheme for L-End.

Antitumor Activity of 3-Indolylmethanamines 31B and PS121912.

AIM: To investigate the in vivo effects of 3-indolylmethanamines 31B and PS121912 in treating ovarian cancer and leukemia, respectively. MATERIALS AND METHODS: Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and western blotting were applied to demonstrate the induction of apoptosis. Xenografted mice were investigated to show the antitumor effects of 3-indolylmethanamines. (13)C-Nuclear magnetic resource (NMR) and western blotting were used to demonstrate inhibition of glucose metabolism. RESULTS: 31B inhibited ovarian cancer cell proliferation and activated caspase-3, cleaved poly (ADP-ribose) polymerase 1 (PARP1), and phosphorylated mitogen-activated protein kinases (MAPK), JUN N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38. 31B reduced ovarian cancer xenograft tumor growth and PS121912 inhibited the growth of HL-60-derived xenografts without any sign of toxicity. Compound 31B inhibited de novo glycolysis and lipogenesis mediated by the reduction of fatty acid synthase and lactate dehydrogenase-A expression. CONCLUSION: 3-Indolylmethanamines represent a new class of antitumor agents. We have shown for the first time the in vivo anticancer effects of 3-indolylmethanamines 31B and PS121912.

The aspartate-less receiver (ALR) domains: distribution, structure and function.

Two-component signaling systems are ubiquitous in bacteria, Archaea and plants and play important roles in sensing and responding to environmental stimuli. To propagate a signaling response the typical system employs a sensory histidine kinase that phosphorylates a Receiver (REC) domain on a conserved aspartate (Asp) residue. Although it is known that some REC domains are missing this Asp residue, it remains unclear as to how many of these divergent REC domains exist, what their functional roles are and how they are regulated in the absence of the conserved Asp. Here we have compiled all deposited REC domains missing their phosphorylatable Asp residue, renamed here as the Aspartate-Less Receiver (ALR) domains. Our data show that ALRs are surprisingly common and are enriched for when attached to more rare effector outputs. Analysis of our informatics and the available ALR atomic structures, combined with structural, biochemical and genetic data of the ALR archetype RitR from Streptococcus pneumoniae presented here suggest that ALRs have reorganized their active pockets to instead take on a constitutive regulatory role or accommodate input signals other than Asp phosphorylation, while largely retaining the canonical post-phosphorylation mechanisms and dimeric interface. This work defines ALRs as an atypical REC subclass and provides insights into shared mechanisms of activation between ALR and REC domains.

Expression profiles of the heat shock protein 70 gene in response to heat stress in Agrotis c-nigrum (Lepidoptera: Noctuidae).

Heat shock proteins (HSPs) are molecular chaperones, and their overexpression enhances the survivability and stress tolerance of the cell. To understand the characteristics of HSP70 in Agrotis c-nigrum Linnaeus larvae, the coding sequence of this protein was cloned, and the effect of heat stress on transcription and protein properties was assessed. The obtained cDNA sequence of HSP70 was 2,213 bp, which contained an ORF of 1,965 bp and encoded 654 amino acid residues. Isolated HSP70 cDNA demonstrated more than 80% identity with the sequences of other known insect HSP70s. Next, HSP70 was expressed in Escherichia coli BL21 (DE3) cells and identified using SDS-PAGE and western blotting analyses. In addition, anti-HSP70-specific antisera were prepared using a recombinant HSP70 protein, and the results showed that this antisera was very specific to AcHSP70. Real-time quantitative polymerase chain reaction detected the relative transcription of the HSP70 gene in larvae and the transcription of A. c-nigrum in response to high temperatures. Induction of HSP70 was up-regulated to peak expression at 36°C.

IDENTIFICATION OF VDR ANTAGONISTS AMONG NUCLEAR RECEPTOR LIGANDS USING VIRTUAL SCREENING.

Herein, we described the development of two virtual screens to identify new vitamin D receptor (VDR) antagonists among nuclear receptor (NR) ligands. Therefore, a database of 14330 nuclear receptor ligands and their NR affinities was assembled using the online available "Binding Database". Two different virtual screens were carried out in conjunction with a reported VDR crystal structure applying a stringent and less stringent pharmacophore model to filter docked NR ligand conformations. The pharmacophore models were based on the spatial orientation of the hydroxyl functionalities of VDR's natural ligands 1,25(OH2)D3 and 25(OH2)D3. The first virtual screen identified 32 NR ligands with a calculate free energy of VDR binding of more than -6.0 kJ/mol. All but nordihydroguaiaretic acid (NDGA) are VDR ligands, which inhibited the interaction between VDR and coactivator peptide SRC2-3 with an IC50 value of 15.8 µM. The second screen identified 162 NR ligands with a calculate free energy of VDR binding of more than -6.0 kJ/mol. More than half of these ligands were developed to bind VDR followed by ERα/ß ligands (26%), TRα/ß ligands (7%) and LxRα/ß ligands (7%). The binding between VDR and ERα ligand H6036 as well as TRα/ß ligand triiodothyronine and a homoserine analog thereof was confirmed by fluorescence polarization.

Phenotypic plasticity of HSP70s gene expression during diapause: signs of evolutionary responses to cold stress among Soybean Pod Borer populations (Leguminivora glycinivorella) in Northeast of China.

The soybean pod borer (Leguminivora glycinivorella Matsumura) successfully survives the winter because of its high expression of 70-kDa heat shock proteins (HSP70s) during its overwintering diapause. The amount of HSP70s is different under different environmental stresses. In this study, inducible heat shock protein 70 and its constitutive heat shock cognate 70 were cloned by RT-PCR and RACE. These genes were named Lg-hsp70 and Lg-hsc70, respectively. Gene transcription and protein expression after cold stress treatment (5°C to -5°C) were analyzed by western blotting and by qRT-PCR for four populations that were sampled in the northeast region of China, including Shenyang, Gongzhuling, Harbin and Heihe, when the soybean pod borer was in diapause. As the cold shock temperature decreased, the levels of Lg-HSP70s were significantly up-regulated. The amount of cold-induced Lg-HSP70s was highest in the southernmost population (Shenyang, 41°50'N) and lowest in the northernmost population (Heihe, 50°22'N). These results support the hypothesis that the soybean pod borer in the northeast region of China displays phenotypic plasticity, and the accumulation of Lg-HSP70s is a strategy for overcoming environmental stress. These results also suggest that the induction of HSP70 synthesis, which is a complex physiological adaptation, can evolve quickly and inherit stability.

Anticancer activity of VDR-coregulator inhibitor PS121912.

PURPOSE: PS121912 has been developed as selective vitamin D receptor (VDR)-coregulator inhibitor starting from a high throughput screening campaign to identify new agents that modulate VDR without causing hypercalcemia. Initial antiproliferative effects of PS121912 were observed that are characterized herein to enable future in vivo investigation with this molecule. METHODS: Antiproliferation and apoptosis were determined using four different cancer cell lines (DU145, Caco2, HL-60 and SKOV3) in the presence of PS121912, 1,25-(OH)2D3, or a combination of 1,25-(OH)2D3 and PS121912. VDR si-RNA was used to identify the role of VDR during this process. The application of ChIP enabled us to determine the involvement of coregulator recruitment during transcription, which was investigated by RT-PCR with VDR target genes and those affiliated with cell cycle progression. Translational changes of apoptotic proteins were determined with an antibody array. The preclinical characterization of PS121912 includes the determination of metabolic stability and CYP3A4 inhibition. RESULTS: PS121912 induced apoptosis in all four cancer cells, with HL-60 cells being the most sensitive. At sub-micromolar concentrations, PS121912 amplified the growth inhibition of cancer cells caused by 1,25-(OH)2D3 without being antiproliferative by itself. A knockout study with VDR si-RNA confirmed the mediating role of VDR. VDR target genes induced by 1,25-(OH)2D3 were down-regulated with the co-treatment of PS121912. This process was highly dependent on the recruitment of coregulators that in case of CYP24A1 was SRC2. The combination of PS121912 and 1,25-(OH)2D3 reduced the presence of SRC2 and enriched the occupancy of corepressor NCoR at the promoter site. E2F transcription factors 1 and 4 were down-regulated in the presence of PS121912 and 1,25-(OH)2D3 that in turn reduced the transcription levels of cyclin A and D, thus arresting HL-60 cells in the S or G2/M phase. In addition, proteins with hematopoietic functions such as cyclin-dependent kinase 6, histone deacetylase 9 and transforming growth factor beta 2 and 3 were down-regulated as well. Elevated levels of P21 and GADD45, in concert with cyclin D1, also mediated the antiproliferative response of HL-60 in the presence of 1,25-(OH)2D3 and PS121912. Studies at higher concentration of P121912 identified a VDR-independent pathway of antiproliferation that included the enzymatic and transcriptional activation of caspase 3/7. CONCLUSION: Overall, we conclude that PS121912 behaves like a VDR antagonist at low concentrations but interacts with more targets at higher concentrations leading to apoptosis mediated by caspase 3/7 activation. In addition, PS121912 showed an acceptable metabolic stability to enable in vivo cancer studies.

Heterologous protein display on the cell surface of lactic acid bacteria mediated by the s-layer protein.

BACKGROUND: Previous studies have revealed that the C-terminal region of the S-layer protein from Lactobacillus is responsible for the cell wall anchoring, which provide an approach for targeting heterologous proteins to the cell wall of lactic acid bacteria (LAB). In this study, we developed a new surface display system in lactic acid bacteria with the C-terminal region of S-layer protein SlpB of Lactobacillus crispatus K2-4-3 isolated from chicken intestine. RESULTS: Multiple sequence alignment revealed that the C-terminal region (LcsB) of Lb. crispatus K2-4-3 SlpB had a high similarity with the cell wall binding domains SA and CbsA of Lactobacillus acidophilus and Lb. crispatus. To evaluate the potential application as an anchoring protein, the green fluorescent protein (GFP) or beta-galactosidase (Gal) was fused to the N-terminus of the LcsB region, and the fused proteins were successfully produced in Escherichia coli, respectively. After mixing them with the non-genetically modified lactic acid bacteria cells, the fused GFP-LcsB and Gal-LcsB were functionally associated with the cell surface of various lactic acid bacteria tested. In addition, the binding capacity could be improved by SDS pretreatment. Moreover, both of the fused proteins could simultaneously bind to the surface of a single cell. Furthermore, when the fused DNA fragment of gfp:lcsB was inserted into the Lactococcus lactis expression vector pSec:Leiss:Nuc, the GFP could not be secreted into the medium under the control of the nisA promoter. Western blot, in-gel fluorescence assay, immunofluorescence microscopy and SDS sensitivity analysis confirmed that the GFP was successfully expressed onto the cell surface of L. lactis with the aid of the LcsB anchor. CONCLUSION: The LcsB region can be used as a functional scaffold to target the heterologous proteins to the cell surfaces of lactic acid bacteria in vitro and in vivo, and has also the potential for biotechnological application.