RESUMEN

BACKGROUND: To better understand ischaemia-related molecular alterations, temporal changes in angiogenic Aminopeptidase N (APN/CD13) expression and glucose metabolism were assessed with PET using a rat model of peripheral arterial disease (PAD). METHODS: The mechanical occlusion of the base of the left hindlimb triggered using a tourniquet was applied to establish the ischaemia/reperfusion injury model in Fischer-344 rats. 2-[18F]FDG and [68Ga]Ga-NOTA-c(NGR) PET imaging performed 1, 3, 5, 7, and 10 days post-ischaemia induction was followed by Western blotting and immunohistochemical staining for APN/CD13 in ischaemic and control muscle tissue extracts. RESULTS: Due to a cellular adaptation to hypoxia, a gradual increase in [68Ga]Ga-NOTA-c(NGR) and 2-[18F]FDG uptake was observed from post-intervention day 1 to 7 in the ischaemic hindlimbs, which was followed by a drop on day 10. Conforming pronounced angiogenic recovery, the NGR accretion of the ischaemic extremities differed significantly from the controls 5, 7, and 10 days after ischaemia induction (p ≤ 0.05), which correlated with the Western blot and immunohistochemical results. No remarkable radioactivity was depicted between the normally perfused hindlimbs of either the ischaemic or the control groups. CONCLUSIONS: The PET-based longitudinal assessment of angiogenesis-associated APN/CD13 expression and glucose metabolism during ischaemia may continue to broaden our knowledge on the pathophysiology of PAD.

RESUMEN

Extracellular vesicles (EVs), which are found in almost all cells and human body fluids, are currently being studied as a source of pathophysiological information. Previously, we demonstrated that at least two types of EVs can be isolated from human whole saliva (WS) using enzymatic activity of dipeptidyl peptidase IV (DPP IV) as a marker for differentiating the EV subsets. In the present study, EV fractions, termed EV-I 20 k-ppt and EV-II 100 k-ppt, were prepared by a combination of size-exclusion chromatography of improved condition and sequential centrifugation. The EV-I 20 k-ppt fraction contained medium/large EVs with a diameter of 100-1,000 nm, including aminopeptidase N (APN), mucin 1, ezrin, and Annexin A1. EV-II 100 k-ppt contained small EVs with a diameter of 20-70 nm, with DPP IV and CD9, programmed cell death 6-interacting protein, and tumor susceptibility gene 101 as characteristic proteins. Proteomic analyses also revealed distinctive repertoires of constituent proteins. Immunoprecipitation of several membrane proteins of the EVs with respective antibodies suggested their differential local membrane environment between the two types of salivary vesicles. Thus, we identified two distinctive types of EVs, one is APN/MUC1- rich EVs (EV-I, large/medium EVs) and the other is DPP IV/CD9-rich EVs (EV-II, small EVs). Furthermore, analysis of the binding of the EVs to coronavirus spike proteins showed that EV-II 100 k-ppt, but not EV-I 20 k-ppt, significantly bound to the spike protein of Middle East respiratory syndrome coronavirus (MERS-CoV). Finally, we developed a simple method to prepare two distinctive EVs from only 1 mL of human WS using sequential immunoprecipitation. Elucidating the features and functions of these two types of salivary EVs may help us understand their pathophysiological roles in the oral cavity and gastrointestinal tract.

RESUMEN

Swine enteric coronaviruses (SECoVs), including porcine deltacoronavirus (PDCoV), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), and swine acute diarrhea syndrome coronavirus (SADS-CoV), have caused high mortality in piglets and, therefore, pose serious threats to the pork industry. Coronaviruses exhibit a trend of interspecies transmission, and understanding the host range of SECoVs is crucial for improving our ability to predict and control future epidemics. Here, the replication of PDCoV, TGEV, and PEDV in cells from different host species was compared by measuring viral genomic RNA transcription and protein synthesis. We demonstrated that PDCoV had a higher efficiency in infecting human lung adenocarcinoma cells (A549), Madin-Darby bovine kidney cells (MDBK), Madin-Darby canine kidney cells (MDCK), and chicken embryonic fibroblast cells (DF-1) than PEDV and TGEV. Moreover, trypsin can enhance the infectivity of PDCoV to MDCK cells that are nonsusceptible to TGEV. Additionally, structural analyses of the receptor ectodomain indicate that PDCoV S1 engages Aminopeptidase N (APN) via domain II, which is highly conserved among animal species of different vertebrates. Our findings provide a basis for understanding the interspecies transmission potential of these three porcine coronaviruses.

RESUMEN

Since angiogenesis/neoangiogenesis has a major role in tumor development, progression and metastatic spread, the establishment of angiogenesis-targeting imaging and therapeutic vectors is of utmost significance. Aminopeptidase N (APN/CD13) is a pivotal biomarker of angiogenic processes abundantly expressed on the cell surface of active vascular endothelial and various neoplastic cells, constituting a valuable target for cancer diagnostics and therapy. Since the asparagine-glycine-arginine (NGR) sequence has been shown to colocalize with APN/CD13, the research interest in NGR-peptide-mediated vascular targeting is steadily growing. Earlier preclinical experiments have already demonstrated the imaging and therapeutic feasibility of NGR-based probes labeled with different positron emission tomography (PET) and single-photon emission computed tomography (SPECT) radionuclides, including Gallium-68 (68Ga), Copper-64 (64Cu), Technetium-99m (99mTc), Lutetium-177 (177Lu), Rhenium-188 (188Re) or Bismuth-213 (213Bi). To improve the tumor binding affinity and the retention time of single-receptor targeting peptides, NGR motifs containing heterodimers have been introduced to identify multi-receptor overexpressing malignancies. Preclinical studies with various tumor-bearing experimental animals provide useful tools for the investigation of the in vivo imaging behavior of NGR-based heterobivalent ligands. Herein, we review the reported preclinical achievements on NGR heterodimers that could be highly relevant for the development of further target-specific multivalent compounds in diagnostic and therapeutic settings.

RESUMEN

Angiogenesis plays a crucial role in tumour progression and metastatic spread; therefore, the development of specific vectors targeting angiogenesis has attracted the attention of several researchers. Since angiogenesis-associated aminopeptidase N (APN/CD13) is highly expressed on the surface of activated endothelial cells of new blood vessels and a wide range of tumour cells, it holds great promise for imaging and therapy in the field of cancer medicine. The selective binding capability of asparagine-glycine-arginine (NGR) motif containing molecules to APN/CD13 makes radiolabelled NGR peptides promising radiopharmaceuticals for the non-invasive, real-time imaging of APN/CD13 overexpressing malignancies at the molecular level. Preclinical small animal model systems are major keystones for the evaluation of the in vivo imaging behaviour of radiolabelled NGR derivatives. Based on existing literature data, several positron emission tomography (PET) and single-photon emission computed tomography (SPECT) radioisotopes have been applied so far for the labelling of tumour vasculature homing NGR sequences such as Gallium-68 (68Ga), Copper-64 (64Cu), Technetium-99m (99mTc), Lutetium-177 (177Lu), Rhenium-188 (188Re), or Bismuth-213 (213Bi). Herein, a comprehensive overview is provided of the recent preclinical experiences with radiolabelled imaging probes targeting angiogenesis.

Asunto(s)

RESUMEN

Angiogenesis-related cell-surface molecules, including integrins, aminopeptidase N, vascular endothelial growth factor, and gastrin-releasing peptide receptor (GRPR), play a crucial role in tumour formation. Radiolabelled imaging probes targeting angiogenic biomarkers serve as valuable vectors in tumour identification. Nowadays, there is a growing interest in novel radionuclides other than gallium-68 (68Ga) or copper-64 (64Cu) to establish selective radiotracers for the imaging of tumour-associated neo-angiogenesis. Given its ideal decay characteristics (Eß+average: 632 KeV) and a half-life (T1/2 = 3.97 h) that is well matched to the pharmacokinetic profile of small molecules targeting angiogenesis, scandium-44 (44Sc) has gained meaningful attention as a promising radiometal for positron emission tomography (PET) imaging. More recently, intensive research has been centered around the investigation of 44Sc-labelled angiogenesis-directed radiopharmaceuticals. Previous studies dealt with the evaluation of 44Sc-appended avb3 integrin-affine Arg-Gly-Asp (RGD) tripeptides, GRPR-selective aminobenzoyl-bombesin analogue (AMBA), and hypoxia-associated nitroimidazole derivatives in the identification of various cancers using experimental tumour models. Given the tumour-related hypoxia- and angiogenesis-targeting capability of these PET probes, 44Sc seems to be a strong competitor of the currently used positron emitters in radiotracer development. In this review, we summarize the preliminary preclinical achievements with 44Sc-labelled angiogenesis-specific molecular probes.

Asunto(s)

RESUMEN

Bufadienolides are steroids that inhibit Na+/K+-ATPase; recent evidence shows that bufalin inhibits the activity of porcine aminopeptidase N (pAPN). We evaluated the selectivity of some bufadienolides on metallo-aminopeptidases. Among the enzymes of the M1 and M17 families, pAPN and porcine aminopeptidase A (pAPA) were the only targets of some bufadienolides. ѱ-bufarenogin, telocinobufagin, marinobufagin, bufalin, cinobufagin, and bufogenin inhibited the activity of pAPN in a dose-dependent manner in the range of 10-7-10-6 M. The inhibition mechanism was classical reversible noncompetitive for telocinobufagin, bufalin and cinobufagin. Bufogenin had the lowest Ki value and a non-competitive behavior. pAPA activity was inhibited by ѱ-bufarenogin, cinobufagin, and bufogenin, with a classical competitive type of inhibition. The models of enzyme-inhibitor complexes agreed with the non-competitive type of inhibition of pAPN by telocinobufagin, bufalin, cinobufagin, and bufogenin. Since APN is a target in cancer therapy, we tested the effect of bufadienolides on the MeWo APN+ human melanoma cell line; they induced cell death, but we obtained scant evidence that inhibition of APN contributed to their effect. Thus, APN is a selective target of some bufadienolides, and we suggest that inhibition of APN activity by bufadienolides is not a major contributor to their antiproliferative properties in MeWo cells.

Asunto(s)

RESUMEN

Members of deltacoronavirus (DCoV) have mostly been identified in diverse avian species as natural reservoirs, though the porcine DCoV (PDCoV) is a major swine enteropathogenic virus with global spread. The important role of aminopeptidase N (APN) orthologues from various mammalian and avian species in PDCoV cellular entry and interspecies transmission has been revealed recently. In this study, comparative analysis indicated that three avian DCoVs, bulbul DCoV HKU11, munia DCoV HKU13, and sparrow DCoV HKU17 (Chinese strain), and PDCoV in the subgenera Buldecovirus are grouped together at whole-genome levels; however, the spike (S) glycoprotein and its S1 subunit of HKU17 are more closely related to night heron DCoV HKU19 in Herdecovirus. Nevertheless, the S1 protein of HKU11, HKU13, or HKU17 bound to or interacted with chicken APN (chAPN) or porcine APN (pAPN) by flow cytometry analysis of cell surface expression of APN and by coimmunoprecipitation in APN-overexpressing cells. Expression of chAPN or pAPN allowed entry of pseudotyped lentiviruses with the S proteins from HKU11, HKU13 and HKU17 into nonsusceptible cells and natural avian and porcine cells, which could be inhibited by the antibody against APN or anti-PDCoV-S1. APN knockdown by siRNA or knockout by CRISPR/Cas9 in chicken or swine cell lines significantly or almost completely blocked infection of these pseudoviruses. Hence, we demonstrate that HKU11, HKU13, and HKU17 with divergent S genes likely engage chAPN or pAPN to enter the cells, suggesting a potential interspecies transmission from wild birds to poultry and from birds to mammals by certain avian DCoVs. IMPORTANCE The receptor usage of avian deltacoronaviruses (DCoVs) has not been investigated thus far, though porcine deltacoronavirus (PDCoV) has been shown to utilize aminopeptidase N (APN) as a cell receptor. We report here that chicken or porcine APN also mediates cellular entry by three avian DCoV (HKU11, HKU13, and HKU17) spike pseudoviruses, and the S1 subunit of three avian DCoVs binds to APN in vitro and in the surface of avian and porcine cells. The results fill the gaps in knowledge about the avian DCoV receptor and elucidate important insights for the monitoring and prevention of potential interspecies transmission of certain avian DCoVs. In view of the diversity of DCoVs, whether this coronavirus genus will cause novel virus to emerge in other mammals from birds, are worthy of further surveillance and investigation.

Asunto(s)

RESUMEN

Determination of the mechanisms of interspecies transmission is of great significance for the prevention of epidemic diseases caused by emerging coronaviruses (CoVs). Recently, porcine deltacoronavirus (PDCoV) was shown to exhibit broad host cell range mediated by surface expression of aminopeptidase N (APN), and humans have been reported to be at risk of PDCoV infection. In the present study, we first demonstrated overexpression of APN orthologues from various species, including mice and felines, in the APN-deficient swine small intestine epithelial cells permitted PDCoV infection, confirming that APN broadly facilitates PDCoV cellular entry and perhaps subsequent interspecies transmission. PDCoV was able to limitedly infect mice in vivo, distributing mainly in enteric and lymphoid tissues, suggesting that mice may serve as a susceptible reservoir of PDCoV. Furthermore, elements (two glycosylation sites and four aromatic amino acids) on the surface of domain B (S1B) of the PDCoV spike glycoprotein S1 subunit were identified to be critical for cellular surface binding of APN orthologues. However, both domain A (S1A) and domain B (S1B) were able to elicit potent neutralizing antibodies against PDCoV infection. The antibodies against S1A inhibited the hemagglutination activity of PDCoV using erythrocytes from various species, which might account for the neutralizing capacity of S1A antibodies partially through a blockage of sialic acid binding. The study reveals the tremendous potential of PDCoV for interspecies transmission and the role of two major PDCoV S1 domains in receptor binding and neutralization, providing a theoretical basis for development of intervention strategies. IMPORTANCE Coronaviruses exhibit a tendency for recombination and mutation, which enables them to quickly adapt to various novel hosts. Previously, orthologues of aminopeptidase N (APN) from mammalian and avian species were found to be associated with porcine deltacoronavirus (PDCoV) cellular entry in vitro. Here, we provide in vivo evidence that mice are susceptible to PDCoV limited infection. We also show that two major domains (S1A and S1B) of the PDCoV spike glycoprotein involved in APN receptor binding can elicit neutralizing antibodies, identifying two glycosylation sites and four aromatic amino acids on the surface of the S1B domain critical for APN binding and demonstrating that the neutralization activity of S1A antibodies is partially attributed to blockage of sugar binding activity. Our findings further implicate PDCoV's great potential for interspecies transmission, and the data of receptor binding and neutralization may provide a basis for development of future intervention strategies.

Asunto(s)

RESUMEN

Circulating tumor cells (CTCs) have been considered as a potential biomarker for evaluation of cancer metastasis and prognosis, especially in hepatocellular carcinoma (HCC). However, the isolation and detection of rare CTCs in HCC patients face enormous challenges due to omittance and nonspecific binding. We previously designed a small molecular NIR fluoresent agent, named MLP, which had high affinity with a tumor cell-overexpressed enzyme, aminopeptidase N (APN). Based on that, in this work we introduced a novel strategy via coassembling the antiepithelial cell adhesion molecule (EpCAM) antibody and MLPinto theFe3O4 magnetic nanobeads (MB-MLP-EpCAM) to isolate and identify HCC-CTCs coinstantaneously. MB-MLP-EpCAM significantly improved the CTC-capture efficiency (>85%) without sacrificing cell viability (>90%). Most importantly, the advantages of precise dual-targetability, high resolution of fluorescence imaging, and prominent selectivity make our nanoplatform have great potential to achieve in vivo real-time identification and monitoring of CTCs clinically.

Asunto(s)

RESUMEN

Aminopeptidase N is an important metalloenzyme from the M1 zinc metallopeptidase family, which is present in numerous apicomplexan parasites, including Plasmodium, Eimeria, and Cryptosporidium. Aminopeptidase N is a potential drug target, and hence, its properties have been widely investigated. In the current study, the cellular localization and enzyme characteristics of Toxoplasma gondii aminopeptidase N3 (TgAPN3) were evaluated in vitro. Cellular localization analysis revealed that TgAPN3 and GRA protein were co-located in the organelle and parasitophorous vacuole of T. gondii. The secretion assay showed that TgAPN3 could be co-secreted from the tachyzoites with GRA protein. A functional recombinant Toxoplasma aminopeptidase N3 (rTgAPN3) was produced in Escherichia coli. The enzyme activity was first determined using a fluorogenic H-Ala-MCA substrate. Some activity of rTgAPN3 was observed between pH 3.0 and 8.0, with a peak at pH 7.0. The activity was significantly enhanced in the presence of Co2+ ions. Substrate specificity of rTgAPN3 was then evaluated. The enzyme showed a preference for substrates containing N-terminal Ala residues, followed by Tyr and Cys. The rTgAPN3 activity was significantly inhibited by bestatin and phebestatin. In general, TgAPN3 was a structurally conserved member of the M1 family, although it also displayed unique biochemical characteristics. These results lay the foundation for a functional study of TgAPN3 and constitute its putative identification as a drug target.

Asunto(s)

RESUMEN

The development of tumor-specific imaging nanoprobes with the potential to improve the accuracy of cancer diagnosis has become an area of intense research. Aminopeptidase N (CD13) predominantly expresses on the surface of ovarian tumor cells and can be specifically recognized by Asn-Gly-Arg (NGR) peptide. The applicability of CD13 as a target for specific ovarian tumor imaging, however, remains unexploited so far. In this study, Cy5.5-labeled, NGR-conjugated iron oxide nanoparticles (Cy5.5-NGR-Fe3O4 NPs) were prepared as an ovarian tumor specific bimodal imaging nanoprobe. It is demonstrated that the conjugation of NGR targeting moiety leads to a higher cellular uptake toward ES-2 cells, the human ovarian carcinoma cells that highly express CD13. Moreover, magnetic resonance imaging of ovarian tumor xenograft reveals that the Fe3O4-Cy5.5-NGR NPs results in a significant T2* signal reduction in the tumor. Meanwhile, near infrared fluorescence imaging indicates a higher accumulation of Fe3O4-Cy5.5-NGR NPs in the tumor xenograft. Therefore, CD13 could be applied as a novel and efficient target for constructing ovarian tumor specific nanoprobes with improved accuracy for ovarian tumor diagnosis.

Asunto(s)

RESUMEN

A monkey cell line Vero (ATCC CCL-81) is commonly used for porcine epidemic diarrhea virus (PEDV) propagation in vitro. However, it is still controversial whether the porcine aminopeptidase N (pAPN) counterpart on Vero cells (Vero-APN) confers PEDV entry. We found that endogenous expression of Vero-APN was undetectable in the mRNA and the protein levels in Vero cells. We cloned the partial Vero-APN gene (3340-bp) containing exons 1 to 9 from cellular DNA and subsequently generated two APN-knockout Vero cell lines by CRISPR/Cas9 approach. PEDV infection of two APN-knockout Vero cells had the same efficiency as the Vero cells with or without neuraminidase treatment. A Vero cells stably expressing pAPN did not increase PEDV production. SiRNA-knockdown of pAPN in porcine jejunum epithelial cells had no effects on PEDV infection. The results suggest that there exists an additional cellular receptor on Vero or porcine jejunal cells independent of APN for PEDV entry.

Asunto(s)

RESUMEN

Substantial progress has been described in the study of puromycin and its analogs for antibiotic properties. However, the peptidase inhibitory activity of related analogs has not been explored as extensively. Specifically, inhibiting aminopeptidases for achieving antitumor effect has been sparsely investigated. Herein, we address this challenge by reporting the synthesis of a series of analogs based on the structural template of puromycin. We also present exhaustive biochemical and in vitro analyses in support of our thesis. Analyzing the structure-activity relationship revealed a steric requirement for maximum potency. Effective inhibitors of Puromycin-Sensitive Aminopeptidase (PSA) are disclosed here. These potential therapeutic agents display superior in vitro antitumor potency against two leukemic cell lines, as compared to known inhibitors of aminopeptidases.

Asunto(s)

RESUMEN

Aminopeptidase N (APN) is a member of the highly conserved M1 family of metalloproteases, and is considered to be a valuable target for the treatment of a variety of diseases, e.g., cancer, malaria, and coccidiosis. In this study, we identified an APN gene (TgAPN2) in the Toxoplasma gondii genome, and performed a biochemical characterization of the recombinant TgAPN2 (rTgAPN2) protein. Active rTgAPN2 was first produced and purified in Escherichia coli. The catalytic activity of the enzyme was verified using a specific fluorescent substrate, H-Ala-MCA; the rTgAPN2 was relatively active in the absence of added metal ions. The addition of some metal ions, especially Zn2+, inhibited the activity of the recombinant enzyme. The activity of rTgAPN2 was reduced in the presence of the EDTA chelator in the absence of added metal ions. The optimum pH for enzyme activity was 8.0; the enzyme was active in the 3-10 pH range. The substrate preference of rTgAPN2 was evaluated. The enzyme showed a preference for substrates containing N-terminal Ala and Arg residues. Finally, bestatin and amastatin were shown to inhibit the activity of the enzyme. In conclusion, rTgAPN2 shared general characteristics with the M1 family of aminopeptidases but also had some unique characteristics. This provides a basis for the function of aminopeptidases and the study of drug targets.

Asunto(s)

RESUMEN

RNAi is a powerful tool to target the insect genes involved in host-pest interactions. Key insect genes are the choice for silencing to achieve pest derived resistance where resistance genes are not available in gene pool of host plant. In this study, an attempt was made to determine the effect of dsRNA designed from two genes Cytochrome P450 derivative (CYP6) and Aminopeptidase N (APN) of rice yellow stem borer (YSB) on growth and development of insect. The bioassays involved injection of chemically synthesized 5' FAM labeled 21-nt dsRNA into rice cut stems and allowing the larvae to feed on these stems which resulted in increased mortality and observed growth and development changes in larval length and weight compared with its untreated control at 12-15 days after treatment. These results were further supported by observing the reduction in transcripts expression of these genes in treated larvae. Fluorescence detection in treated larvae also proved that dsRNA was readily taken by larvae when fed on dsRNA treated stems. These results from the present study clearly show that YSB larvae fed on dsRNA designed from Cytochrome P450 and Aminopeptidase N has detrimental effect on larval growth and development. These genes can be deployed to develop YSB resistance in rice using RNAi approach.

RESUMEN

13F-1 is a 5-fluorouracil prodrug containing an Asn-Gly-Arg (NO2) COOCH3 tripeptide. 13F-1 might possess the activity against cancer growth by targeting Aminopeptidase N (APN/CD13). Our goal in this study was to evaluate the inhibitory effect of 13F-1 on the growth of human colonic carcinoma by both in vitro and in vivo studies. Experiments were performed in colonic carcinoma Colo205 cells, which highly express APN/CD13 on cell surface. The inhibition of 13F-1 on cancer cell growth was estimated by the colorimetric and clonogenic assays. The assays of Annexin V-FITC/PI and JC-1 fluorescence probe were employed to determine the apoptotic cells. Further experiment was performed in mice bearing Colo205 xenografts. 13F-1 was injected for three consecutive weeks. The specimens of Colo205 xenografts were removed for TUNEL staining and western blotting analysis. The expressions of APN/CD13 were analyzed by immunofluorescent flow cytometry and western blotting assays. 13F-1 significantly inhibited Colo205 cell proliferation. 13F-1 by injection delayed the expansion of Colo205 xenografts without significant toxicity to mice. The inhibitory effect of 13F-1 might arise from its role in apoptotic induction. Further analysis indicated that 13F-1 strongly inhibited APN/CD13 expression on cancer cell surface. In contrast, 5-FU did not affect APN/CD13 expression. These results indicated the mechanism of 13F-1 action that 13F-1׳s effect was associated with its role in suppression of APN/CD13 expression. Conclusion, 13F-1 could be developed as a promising agent for treatment of cancers with high expression of APN/CD13.

Asunto(s)

RESUMEN

Aminopeptidases N (APNs), the receptors of Bacillus thuringiensis (Bt) toxin in the lepidopteran midgut, are involved in the Bt pathogen infection mechanism. In the present work, we screened 102 APNs from SilkDB, ButterflyBase and MonarchBase; 16 APNs were identified from the silkworm (Bombyx mori) and 24 from the monarch butterfly (Danaus plexippus). Syntenic and phylogenetic tree analysis showed that APN genes have developed multi-family genes before evolutionary divergence of the Lepidoptera. The tissue-expression pattern shows some BmAPNs are specifically or highly expressed in the midgut. Bacillus bombysepticus (Bb) is a specific pathogen of B. mori, leading to acute fuliginosa septicemia of the larva. BmAPNs were modulated by real time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis after Bb or Bt oral infection. There were different patterns of induced expression between Bb and Bt challenges, suggesting that B. mori has different responses to infection by the specific pathogen Bb and the nonspecific pathogen Bt. Research on BmAPNs will help us to better understand the evolutionary conservation and functions in Bb or Bt pathogen interaction with the host and to apply this knowledge in agricultural and forestry pest control.

Asunto(s)

RESUMEN

Bacillus thuringiensis (Bt) crystal proteins (Cry) bind to aminopeptidase N (APN) receptors on insect midgut membrane leading to pore formation and subsequent death. However, evolution of insect resistance to Bt toxins threatens their long-term application. Therefore, search for new targets which could function as Cry toxin receptors is an immediate mandate. In the present study, two full-length APN cDNAs were cloned from Malpighian tubule and salivary gland tissues of the moth, Achaea janata. Both these APNs showed 99% and 32% sequence homology with fat body and midgut APNs respectively. Tissue distribution analysis revealed the presence of two different APN isoforms, one predominant in non-gut visceral tissues while the other exclusively expressed in the midgut. Immunofluorescence and western blot analyses showed cross-reactivity in Malpighian tubule and salivary gland when probed with anti-fat body APN antiserum. These results clearly indicated the presence of non-gut (AjAPN1) and gut-specific (AjAPN4) isoforms in this moth. The expression of both the isoforms steadily increased during the larval development. Hormonal studies indicated regulation of the APN genes by the morphogenetic hormones, 20-hydroxyecdyone and juvenile hormone. Further, in vitro ligand-blotting studies demonstrated binding of Cry toxins to APNs in Malpighian tubule and salivary gland indicating their potential as alternate targets.

Asunto(s)

RESUMEN

Aminopeptidase N (APN) is known to play important roles in tumor angiogenesis, tumor cell invasion, and metastasis. Thus, APN is an attractive biomarker for imaging tumor angiogenesis. Here we report results obtained from biodistribution and single photon emission computed tomography (SPECT) imaging studies of a technetium-99m labeled probestin (a potent APN inhibitor) conjugate containing a tripeptide, Asp-DAP-Cys (DAP=2,3-diaminopropionic acid), chelator and a 8-amino-3,6-dioxaoctanoic acid (PEG2) linker conducted in nude mice xenografted with HT-1080 human fibrosarcoma tumors (APN-positive tumors). These results collectively demonstrate that (99m)Tc-probestin uptake by tumors and other APN expressing tissues in vivo is specific and validate the use of probestin as a vector for targeting APN in vivo.

Asunto(s)