Molecular cloning and characterization of genes encoding FK506-binding proteins (FKBPs) in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The family of FK506-binding proteins (FKBPs) consists of several members, which show peptidyl prolyl cis–trans isomerase (PPIase) activity. PPIases facilitate the conversion of peptidyl prolyl bonds from cis to trans conformation, a rate-limiting step in protein folding. In the present study, we carried out cloning of cDNAs encoding three different wheat FKBPs viz., TaFKBP20-1, TaFKBP16-1 and TaFKBP15-1. In silico analysis suggested their likely localization to nucleus, cytosol and endoplasmic reticulum, respectively. Biochemical analyses demonstrated that none of the three purified FKBP proteins possesses detectable PPIase activity. Several putative interacting partners of TaFKBP20-1, TaFKBP16-1 and TaFKBP15-1were identified using online software tools. The results of this study provide further evidence that PPIase activity in plant FKBPs is not conserved, and these proteins may be playing important roles in the cell through interaction with target proteins.     

<Emphasis Type="Italic">ZmFKBP20-1</Emphasis> improves the drought and salt tolerance of transformed Arabidopsis

FK506-binding proteins (FKBPs), which belong to the peptidyl-prolyl cis/trans isomerase superfamily, are involved in plant response to abiotic stresses. A number of FKBP family genes have been isolated in plants, but little has been reported of FKBP genes in maize. In this study, a drought-induced FKBP gene, ZmFKBP20-1, was isolated from maize and was characterized for its role in stress responses using gene expression, protein subcellular localization, transformation in Arabidopsis, expression patterns of the stress-responsive genes, and physiological parameter analysis. During drought and salt stresses, ZmFKBP20-1 transgenic Arabidopsis plants exhibited enhanced tolerance, which was concomitant with the altered expression of stress/ABA-responsive genes, such as COR15a, COR47, ERD10, RD22, KIN1, ABI1, and ABI2. The resistance characteristics of ZmFKBP20-1 overexpression were associated with a significant increase in survival rate. These results suggested that ZmFKBP20-1 plays a positive role in drought and salt stress responses in Arabidopsis and provided new insights into the mechanisms of FKBP in response to abiotic stresses in plants.     

Inhibition and Substrate Specificity Properties of FKBP22 from a Psychrotrophic Bacterium, <Emphasis Type="Italic">Shewanella</Emphasis> sp. SIB1

SIB1 FKBP22 is a peptidyl prolyl cis–trans isomerase (PPIase) member from a psychrotrophic bacterium, Shewanella sp. SIB1, consisting of N- and C-domains responsible for dimerization and catalytic PPIase activity, respectively. This protein was assumed to be involved in cold adaptation of SIB1 cells through its dual activity of PPIase activity and chaperone like-function. Nevertheless, the catalytic inhibition by FK506 and its substrate specificity remain unknown. Besides, ability of SIB1 FKBP22 to inhibit phosphatase activity of calcinuerin is also interesting to be studied since it may reflect wider cellular functions of SIB1 FKBP22. In this study, we found that wild type (WT) SIB1 FKBP22 bound to FK506 with IC₅₀ of 77.55 nM. This value is comparable to that of monomeric mutants (NNC-FKBP22, C-domain+ and V37R/L41R mutants), yet significantly higher than that of active site mutant (R142A). In addition, WT SIB1 FKBP22 and monomeric variants were found to prefer hydrophobic residues preceding proline. Meanwhile, R142A mutant has wider preferences on bulkier hydrophobic residues due to increasing hydrophobicity and binding pocket space. Surprisingly, in the absence of FK506, SIB1 FKBP22 and its variants inhibited, with the exception of N-domain, calcineurin phosphatase activity, albeit low. The inhibition of SIB1 FKBP22 by FK506 is dramatically increased in the presence of FK506. Altogether, we proposed that local structure at substrate binding pocket of C-domain plays crucial role for the binding of FK506 and peptide substrate preferences. In addition, C-domain is essential for inhibition, while dimerization state is important for optimum inhibition through efficient binding to calcineurin.     

Various <Emphasis Type="Italic">Wolbachia</Emphasis> genotypes differently influence host <Emphasis Type="Italic">Drosophila</Emphasis> dopamine metabolism and survival under heat stress conditions

Background

One of the most widespread prokaryotic symbionts of invertebrates is the intracellular bacteria of Wolbachia genus which can be found in about 50% of insect species. Wolbachia causes both parasitic and mutualistic effects on its host that include manipulating the host reproductive systems in order to increase their transmission through the female germline, and increasing the host fitness. One of the mechanisms, promoting adaptation in biological organisms, is a non-specific neuroendocrine stress reaction. In insects, this reaction includes catecholamines, dopamine, serotonin and octopamine, which act as neurotransmitters, neuromodulators and neurohormones. The level of dopamine metabolism correlates with heat stress resistance in Drosophila adults.

Results

To examine Wolbachia effect on Drosophila survival under heat stress and dopamine metabolism we used five strains carrying the nuclear background of interbred Bi90 strain and cytoplasmic backgrounds with different genotype variants of Wolbachia (produced by 20 backcrosses of Bi90 males with appropriate source of Wolbachia). Non-infected Bi90 strain (treated with tetracycline for 3 generations) was used as a control group. We demonstrated that two of five investigated Wolbachia variants promote changes in Drosophila heat stress resistance and activity of enzymes that produce and degrade dopamine, alkaline phosphatase and dopamine-dependent arylalkylamine N-acetyltransferase. What is especially interesting, wMelCS genotype of Wolbachia increases stress resistance and the intensity of dopamine metabolism, whereas wMelPop strain decreases them. wMel, wMel2 and wMel4 genotypes of Wolbachia do not show any effect on the survival under heat stress or dopamine metabolism. L-DOPA treatment, known to increase the dopamine content in Drosophila, levels the difference in survival under heat stress between all studied groups.

Conclusions

The genotype of symbiont determines the effect that the symbiont has on the stress resistance of the host insect.

     

The genomic underpinnings of apoptosis in the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

Background

Apoptosis is regulated in an orderly fashion by a series of genes, and has a crucial role in important physiological processes such as growth development, immunological response and so on. Recently, substantial studies have been undertaken on apoptosis in model animals including humans, fruit flies, and the nematode. However, the lack of genomic data for silkworms limits their usefulness in apoptosis studies, despite the advantages of silkworm as a representative of Lepidoptera and an effective model system. Herein we have identified apoptosis-related genes in the silkworm Bombyx mori and compared them to those from insects, mammals, and nematodes.

Results

From the newly assembled genome databases, a genome-wide analysis of apoptosis-related genes in Bombyx mori was performed using both nucleotide and protein Blast searches. Fifty-two apoptosis-related candidate genes were identified, including five caspase family members, two tumor necrosis factor (TNF) superfamily members, one Bcl-2 family member, four baculovirus IAP (inhibitor of apoptosis) repeat (BIR) domain family members and 1 RHG (Reaper, Hid, Grim, and Sickle; Drosophila cell death activators) family member. Moreover, we identified a new caspase family member, BmCaspase-New, two splice variants of BmDronc, and Bm3585, a mammalian TNF superfamily member homolog. Twenty-three of these apoptosis-related genes were cloned and sequenced using cDNA templates isolated from BmE-SWU1 cells. Sequence analyses revealed that these genes could have key roles in apoptosis.

Conclusions

Bombyx mori possesses potential apoptosis-related genes. We hypothesized that the classic intrinsic and extrinsic apoptotic pathways potentially are active in Bombyx mori. These results lay the foundation for further apoptosis-related study in Bombyx mori.

     

Identification of a novel <Emphasis Type="Italic">Drosophila</Emphasis> gene, <Emphasis Type="Italic">beltless</Emphasis>, using injectable embryonic and adult RNA interference (RNAi)

Background

RNA interference (RNAi) is a process triggered by a double-stranded RNA that leads to targeted down-regulation/silencing of gene expression and can be used for functional genomics; i.e. loss-of-function studies. Here we report on the use of RNAi in the identification of a developmentally important novel Drosophila (fruit fly) gene (corresponding to a putative gene CG5652/GM06434), that we named beltless based on an embryonic loss-of-function phenotype.

Results

Beltless mRNA is expressed in all developmental stages except in 0–6 h embryos. In situ RT-PCR localized beltless mRNA in the ventral cord and brain of late stage embryos and in the nervous system, ovaries, and the accessory glands of adult flies. RNAi was induced by injection of short (22 bp) beltless double-stranded RNAs into embryos or into adult flies. Embryonic RNAi altered cuticular phenotypes ranging from partially-formed to missing denticle belts (thus beltless) of the abdominal segments A2–A4. Embryonic beltless RNAi was lethal. Adult RNAi resulted in the shrinkage of the ovaries by half and reduced the number of eggs laid. We also examined Df(1)RK4 flies in which deletion removes 16 genes, including beltless. In some embryos, we observed cuticular abnormalities similar to our findings with beltless RNAi. After differentiating Df(1)RK4 embryos into those with visible denticle belts and those missing denticle belts, we assayed the presence of beltless mRNA; no beltless mRNA was detectable in embryos with missing denticle belts.

Conclusions

We have identified a developmentally important novel Drosophila gene, beltless, which has been characterized in loss-of-function studies using RNA interference. The putative beltless protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF) NRF-6 gene, as well as with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains. Future studies should elucidate the role and mechanism of action of beltless during Drosophila development and in adults, including in the adult nervous system.

     

Metabolic profiling of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> metamorphosis: a new insight into the central metabolic pathways

Introduction

Metamorphosis is a complicated process in which cell proliferation, differentiation, and death are orchestrated to form the mature structures of insects. In Drosophila, this process is controlled by ecdysone, a steroid hormone responsible for tissue remodeling and organogenesis that gives rise to the adult fly.

Objective

By using a metabolomics approach, this study aimed to elucidate global changes in the central metabolic pathways in Drosophila throughout metamorphosis and then further examine the effects of temperature and origin on metabolic profiles.

Methods

Targeted and non-targeted metabolic profiling of time-course samples from Drosophila were constructed to cover a wide range of cellular metabolites during metamorphosis.

Results

This was the first wide-scale metabolomics study of Drosophila metamorphosis focusing on central metabolism. The abundance of detected metabolites changed drastically and correlated strongly with the development of Drosophila pupae. In non-stress conditions, temperature affected the developmental time, but the metabolic state at a certain stage of metamorphosis remained stable. Between D. melanogaster Canton S and Oregon R, similar metabolic profiles throughout metamorphosis was observed. However, there were still differences in purine and pyrimidine metabolism at an early stage in the pupal period, which was matched by differences in ecdysteroid levels.

Conclusion

This study supported the strength of metabolomics in the field of developmental biology. The results provided a general view on the metabolic profile of Drosophila during metamorphosis, which provides basic 3 knowledge for future metabolomics studies using Drosophila.

     

Resistance Mechanism in a Terbinafine-Resistant Strain of <Emphasis Type="Italic">Microsporum canis</Emphasis>

To clarify the terbinafine (TRF) resistance mechanism in a TRF-resistant strain of Microsporum canis, the expression of the pleiotropic drug resistance (PDR1), multidrug resistance (MDR1), MDR2 and MDR4 genes were investigated by real-time quantitative PCR (RT-qPCR) analysis, given the known interaction of the corresponding proteins with antifungals and with the efflux blocker FK506. The expression of the PDR1, MDR1, MDR2 and MDR4 genes was 2–4 times higher in the TRF-resistant strain grown in the presence of 0.14 µg/mL of TRF than in TRF-susceptible strains cultured in the absence of TRF. The TRF-resistant strain exhibited MICs of > 32 µg/mL for TRF alone; this resistance was attenuated to an MIC of 8 µg/mL in the presence of FK506, indicating that the TRF inhibitory concentration index value was < 0.75. The additive effect of the efflux blocker FK506 on TRF resistance was detected in the TRF-resistant strain. These results indicated that the TRF resistance in this strain reflects overexpression of genes encoding ABC transporter proteins.     

The Expression of Dectin-1, Irak1 and Rip2 During the Host Response to <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>

Background

C-type lectin receptors (CLRs), Toll-like receptors (TLRs), and Nod-like receptors (NLRs) have the ability to recognize Aspergillus fumigatus (A. fumigates) and induce innate immune response. Dectin-1 is a well-described CLR, while interleukin-1 receptor-associated kinase 1 (Irak1) and receptor-interacting protein 2 (Rip2) are pivotal adaptor proteins of TLRs and NLRs signaling pathways, respectively.

Objectives

Our primary aim is to elucidate whether Dectin-1 regulates the expression of Irak1 and Rip2, and confirm that CLRs, TLRs, and NLRs pathways act synergistically in response to A. fumigatus infection.

Methods

Pulmonary infection mouse models were established. Myeloid cells were differentiated in cell culture and examined by inverted microscopy, flow cytometry, and scanning electron microscopy. The relative mRNA levels were determined by qRT-PCR. The protein expression levels were determined by immunohistochemistry and Western blot.

Results

The expression of Dectin-1, Irak1, Rip2, and phosphorylation level of nuclear factor (NF)-κB p65 were induced by conidia in immunocompetent mice, while their expression and phosphorylation level were inhibited in immunocompromised mice after the administration of conidia. Conidia increased the expression of Dectin-1, Irak1, and Rip2 in myeloid cells, while Dectin-1 silencing significantly reduced their expression.

Conclusion

Our findings demonstrate that Dectin-1, Irak1, and Rip2 are involved in response to A. fumigatus infection. Dectin-1 modulates the expression of Irak1 and Rip2. Additionally, these three signaling pathways are interconnected, and CLRs pathway plays a dominant role against A. fumigatus invasion.

     

Evidence for a role for the putative <Emphasis Type="Italic">Drosophila</Emphasis> hGRX1 orthologue in copper homeostasis

Glutaredoxins are a family of small molecular weight proteins that have a central role in cellular redox regulation. Human GRX1 (hGRX1) has also been shown to play an integral role in copper homeostasis by regulating the redox activity of the metalated sites of copper chaperones such as ATOX1 and SOD1, and the copper efflux proteins ATP7A and ATP7B. To further elucidate the role of hGRX1 in copper homeostasis, we examined the impact of RNA interference-mediated knockdown of CG6852, a putative Drosophila orthologue of hGRX1. CG6852 shares ~41 % amino acid identity with hGRX1 and key functional domains including the metal-binding CXXC motif are conserved between the two proteins. Knockdown of CG6852 in the adult midline caused a thoracic cleft and reduced scutellum, phenotypes that were exacerbated by additional knockdown of copper uptake transporters Ctr1A and Ctr1B. Knockdown of CG6852 in the adult eye enhanced a copper-deficiency phenotype caused by Ctr1A knockdown while ubiquitous knockdown of CG6852 resulted a mild systemic copper deficiency. Therefore we conclude that CG6852 is a putative orthologue of hGRX1 and may play an important role in Drosophila copper homeostasis.     

Metabolic changes of genetically engineered grapes (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) studied by <Superscript>1</Superscript>H-NMR,metabolite heatmaps and <Emphasis Type="Italic">i</Emphasis>PLS

Introduction

The Deficiens Homologue 9-iaaM (DefH9-iaaM) gene is an ovule-specific auxin-synthesizing gene which is expressed specifically in placenta/ovules and promotes auxin-synthesis. It was introduced into the genome of two grape cultivars Thompson Seedless and Silcora and both transgenic cultivars had an increased number of berries per bunch.

Objectives

This study investigates the down-stream metabolic changes of Silcora and Thompson seedless grape cultivars when genetically modified through the insertion of the DefH9-iaaM gene into their genome.

Methods

The effects of the genetic modification upon the grape metabolome were evaluated through ¹H-NMR and exploratory data analysis. Chemometric tools such as Interval Partial Least Squares regression and metabolite heatmaps were employed for scrutinizing the changes in the transgenic metabolome as compared to the wild type one.

Results

The results show that the pleiotropic effect on the grape metabolome as a function of the gene modifications is relatively low, although the insertion of the transgene caused a decrement in malic acid and proline and an increment in p-coumaric acid content. In addition, the concentration of malic acid was successfully correlated with the number of inserted copies of transgene in the Silcora cultivar, proving that the increased production of berries, promoted by the inserted gene, is achieved at the expense of a decrement in malic acid concentration.

Conclusion

NMR together with chemometrics is able to identify specific metabolites that were up- or down regulated in the genetically engineered plants allowing highlighting alterations in the down-stream metabolic pathways due to the up-stream genetic modifications.

     

Metabolomics reveals immunomodulation as a possible mechanism for the antibiotic effect of <Emphasis Type="Italic">Persicaria capitata</Emphasis> (Buch.-Ham. ex D. Don) H.Gross

Introduction

In spite of advances in antibiotics, urinary tract infection (UTI) is still among the most common reasons for antibiotic medication worldwide. Persicaria capitata (Buch.-Ham. ex D. Don) H.Gross (P. capitata) is a herbal medicine used by the Miao people in China to treat UTI. However studies of its mechanism are challenging, owing to the complexity of P. capitata with multiple constituents acting on multiple metabolic pathways.

Objective

The objective of this study was to explore the working mechanism of P. capitata on urinary tract infection.

Methods

Relinqing® granule, which is solely made from aqueous extracts of the whole P. capitata plant, was used in this study. Urine metabolomics based on gas chromatography-mass spectroscopy was employed to assess the metabolic changes caused by administration of Relinqing® granule in a UTI mouse model. Female specific-pathogen-free Kunming mice were divided into control group (mock infection, saline treatment), model group (E.coli infection, saline treatment), Relinqing® group (E.coli infection, Relinqing® granule treatment), ciprofloxacin group (E.coli infection, ciprofloxacin treatment), and sham-Relinqing® group (no surgery, Relinqing® granule treatment).

Results

The results showed that after the treatments, urine levels of itaconic acid in Relinqing® group increased by 4.9 fold and 11.3 fold compared with model and ciprofloxacin groups respectively. Itaconic acid is an endogenous antibacterial metabolite produced by macrophages, which also functions as a checkpoint for metabolic reprogramming of macrophage.

Conclusion

Our findings suggest that this herbal medicine can cure urinary tract infection through modulation of immune system.

     

Performance in daily activities,cognitive impairment and perception in multiple sclerosis patients and their caregivers

Background

The relationship between cognitive assessment results in multiple sclerosis (MS) and performance in daily activities (DAs) remains unclear. Our study aimed to evaluate the relationship between cognitive functions (CF) measured by tests, performance in DAs, and the perception of CF in patients and their caregivers (CG) in MS.

Methods

The Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS) battery was used to evaluate cognitive status. We created an ad hoc questionnaire (DaQ) to assess performance in DAs not requiring specific motor skills. We used the Multiple Sclerosis Neuropsychological Questionnaire (MSNQ) to measure each patient self-judgment and caregiver’s perception of CF.

Results

Forty-nine patients and their caregivers were included in the study. Significant correlations were found between the BICAMS and the DaQ (Symbol Digit Modalities Test (SDMT): r?=???0.48, p?<?0.001; California Verbal Learning Test (CVLT): r?=???0.33, p?=?0.01; Brief Visual Memory Test (BVMT-R): r?=???0.42; p?=?0.002); patients self-judgment (SDMT: r?=???0.38, p?=?0.004; CVLT: r?=???0.26, p?=?0.03); caregiver perception of patient’s CF (SDMT: r?=???0.52, p?<?0.001; CVLT: r?=???0.3, p?=?0.01; BVMT-R: r?=???0.42, p?=?0.002). The difference in perception between the patients and their caregivers was related to patient age (p?=?0.001) and severity of cognitive impairment (p?=?0.03).

Conclusions

Cognitive assessment results show a significant correlation with performance in daily activities and with patients and, especially, caregiver perception of cognitive impairment. These data support the importance of a routine evaluation of cognitive function in MS that includes an anamnestic evaluation of patients, and, when possible, consideration of the caregiver’s point of view.

     

FKBP51, a novel T-cell-specific immunophilin capable of calcineurin inhibition.

The immunosuppressive drugs FK506 and cyclosporin A block T-lymphocyte proliferation by inhibiting calcineurin, a critical signaling molecule for activation. Multiple intracellular receptors (immunophilins) for these drugs that specifically bind either FK506 and rapamycin (FK506-binding proteins [FKBPs]) or cyclosporin A (cyclophilins) have been identified. We report the cloning and characterization of a new 51-kDa member of the FKBP family from murine T cells. The novel immunophilin, FKBP51, is distinct from the previously isolated and sequenced 52-kDa murine FKBP, demonstrating 53% identity overall. Importantly, Western blot (immunoblot) analysis showed that unlike all other FKBPs characterized to date, FKBP51 expression was largely restricted to T cells. Drug binding to recombinant FKBP51 was demonstrated by inhibition of peptidyl prolyl isomerase activity. As judged from peptidyl prolyl isomerase activity, FKBP51 had a slightly higher affinity for rapamycin than for FK520, an FK506 analog. FKBP51, when complexed with FK520, was capable of inhibiting calcineurin phosphatase activity in an in vitro assay system. Inhibition of calcineurin phosphatase activity has been implicated both in the mechanism of immunosuppression and in the observed toxic side effects of FK506 in nonlymphoid cells. Identification of a new FKBP that can mediate calcineurin inhibition and is restricted in its expression to T cells suggests that new immunosuppressive drugs may be identified that, by virtue of their specific interaction with FKBP51, would be targeted in their site of action.     

Development of a novel engineered antibody targeting <Emphasis Type="Italic">Neisseria</Emphasis> species

Objectives

A Neissaria bacterial pilus sugar, bacillosamine, was synthesized and, for the first time, used as a probe to screen a single-chain variable fragment (scFv).

Results

Four Neisseria, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria sicca and Neisseria subflava, and two negative controls, Streptococcus pneumoniae and Escherichia coli, were tested through ELISA, immunostaining and gold nanoparticle immunological assay. All results indicated that the selected scFv is feasible for the specific detection of Neisseria species via the recognition of bacillosamine.

Conclusions

The recombinant scFv could detect Neisseria strains at 10⁶ CFU/ml.

     

Enhanced <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> immune responses caused by heterologous plant genes from <Emphasis Type="Italic">Pinellia ternata</Emphasis>

Background

Pinellia ternata is a Chinese traditional medicinal herb, used to cure diseases including insomnia, eclampsia and cervical carcinoma, for hundreds of years. Non-self-recognition in multicellular organisms can initiate the innate immunity to avoid the invasion of pathogens. A design for pathogen independent, heterosis based, fresh resistance can be generated in F₁ hybrid was proposed.

Results

By library functional screening, we found that P. ternata genes, named as ptHR375 and ptHR941, were identified with the potential to trigger a hypersensitive response in Nicotiana benthamiana. Significant induction of ROS and Callose deposition in N. benthamiana leaves along with activation of pathogenesis-related genes viz.; PR-1a, PR-5, PDF1.2, NPR1, PAL, RBOHB and ERF1 and antioxidant enzymes was observed. After transformation into N. benthamiana, expression of pathogenesis related genes was significantly up-regulated to generate high level of resistance against Phytophthora capsici without affecting the normal seed germination and morphological characters of the transformed N. benthamiana. UPLC-QTOF-MS analysis of ptHR375 transformed N. benthamiana revealed the induction of Oxytetracycline, Cuelure, Allantoin, Diethylstilbestrol and 1,2-Benzisothiazol-3(2H)-one as bioactive compounds. Here we also proved that F₁ hybrids, produced by crossing of the ptHR375 and ptHR941 transformed and non-transformed N. benthamiana, show significant high levels of PR-gene expressions and pathogen resistance.

Conclusions

Heterologous plant genes can activate disease resistance in another plant species and furthermore, by generating F₁ hybrids, fresh pathogen independent plant immunity can be obtained. It is also concluded that ptHR375 and ptHR941 play their role in SA and JA/ET defense pathways to activate the resistance against invading pathogens.