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The conventional inactivated split seasonal influenza vaccine offers low efficacy, particularly in the elderly and against antigenic variants. Here, to improve the efficacy of seasonal vaccination for the elderly population, we tested whether supplementing seasonal bivalent (H1N1 + H3N2) split (S) vaccine with M2 ectodomain repeat and multi-subtype consensus neuraminidase (NA) proteins (N1 NA + N2 NA + flu B NA) on a virus-like particle (NA-M2e) would induce enhanced cross-protection against different influenza viruses in aged mice. Immunization with split vaccine plus NA-M2e (S + NA-M2e) increased vaccine-specific IgG antibodies towards T-helper type 1 responses and hemagglutination inhibition titers. Aged mice with NA-M2e supplemented vaccination were protected against homologous and heterologous viruses at higher efficacies, as evidenced by preventing weight loss, lowering lung viral loads, inducing broadly cross-protective humoral immunity, and IFN-γ + CD4 and CD8 T cell responses than those with seasonal vaccine. Overall, this study supports a new strategy of NA-M2e supplemented vaccination to enhance protection against homologous and antigenically different viruses in the elderly., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this work, we reported a fluorescent probe Fur-SH, a derivative of benzofuranone, which was used to detect H 2 S in living cells and zebrafish. Based on the three structural characteristics of the probe, the effects of different structural modifications on the optical properties of the fluorophore were compared. Then, the fluorophore Fur-OH was synthesized by modifying diethylamino group with benzofuranone as the main skeleton. With 2,4-dinitrofluorobenzene as the recognition group and diethylamino as the electron donor, the push-pull electron effect occurred with nitro group, which led to fluorescence quenching, and an openable fluorescent probe Fur-SH was formed. The probe Fur-SH (λ ex  = 510 nm; λ em  = 570 nm) had the advantages of smaller full width at half maxima, rapid response (5 min) and wide pH window. The quantitative properties of the probe were excellent, reaching saturation at 50 equivalents of substrate. The probe Fur-SH showed high sensitivity to H 2 S, with LOD of 48.9 nM and LOQ of 50 nM. At present, the probe Fur-SH had been applied to fluorescence imaging of MCF-7 cells and zebrafish. By comparing the effects of different structures on the optical properties of fluorophores, this work was expected to be helpful to the development of fluorescent probes in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

We investigated the priming effect of nanoscale zero-valent iron (nZVI) on carbon sink and iron uptake, and the possible mediation by AMF (arbuscular mycorrhizal fungi, Funneliformis mosseae) in semiarid agricultural soils. Maize seed dressings comprised of three nZVI concentrations of 0, 1, 2 g·kg -1 and was tested with and without AMF inoculation under high and low soil moistures, respectively. The ICP-OES observations indicated that both low dose of nZVI (1 g·kg -1 ) and high dose of nZVI (2 g·kg -1 ) significantly increased the iron concentrations in roots (L: 54.5-109.8 %; H: 119.1-245.4 %) and shoots (L: 40.8-78.9 %; H: 81.1-99.4 %). Importantly, the absorption and translocation rate of iron were substantially improved by AMF inoculation under the low-dose nZVI. Yet, the excess nanoparticles as a stress were efficiently relieved by rhizosphere hyphae, and the iron concentration in leaves and stems can maintain as high as about 300 mg·kg -1 while the iron translocation efficiency was reduced. Moreover, next-generation sequencing confirmed that appropriate amount of nZVI clearly improved the rhizosphere colonization of Funneliformis mosseae (p < 0.001) and the development of soil fungal community. Soil observations further showed that the hyphae development and GRSP (glomalin-related soil protein) secretion were significantly promoted (p < 0.05), with the increased R 0.25 (< 0.25 mm) by 35.97-41.16 %. As a return, AMF and host plant turned to input more organic matter into soils for microbial growth and Fe uptake, and such interactions became more pronounced under drought stress. In contrast, high dose of nZVI (2 g·kg -1 ) tended to agglomerate on the surface of hyphae and spores, causing severe deformation and inactivation of AMF symbionts. Therefore, the priming effects of nZVI on carbon sequestration and Fe uptake in agricultural soils were positively mediated by AMF via the feedback loop of the plant-soil-microbe system for enhanced adaptation to global climate change., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Universal influenza vaccines are urgently needed to prevent recurrent influenza epidemics and inevitable pandemics. We generated double-layered protein nanoparticles incorporating two conserved influenza antigens-nucleoprotein and neuraminidase-through a two-step desolvation-crosslinking method. These protein nanoparticles displayed immunostimulatory properties to antigen-presenting cells by promoting inflammatory cytokine (IL-6 and TNF-α) secretion from JAWS II dendric cells. The nanoparticle immunization induced significant antigen-specific humoral and cellular responses, including antigen-binding and neutralizing antibodies, antibody- and cytokine (IFN-γ and IL-4)-secreting cells, and NP 147 - 155 tetramer-specific cytotoxic T lymphocyte (CTL) responses. Co-administration of monophosphoryl lipid A (MPLA, a toll-like receptor 4 agonist) with the protein nanoparticles further improved immune responses and conferred heterologous and heterosubtypic influenza protection. The MPLA-adjuvanted nanoparticles reduced lung inflammation post-infection. The results demonstrated that the combination of MPLA and conserved protein nanoparticles could be developed into an improved universal influenza vaccine strategy., Competing Interests: Conflict of interest All authors have declared no financial or other potential conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Cysteine (Cys), one of the biological thiols, which plays critical roles in biological system regulating the balance of redox homeostasis. In order to monitor the level of Cys in the living cells and organisms, a chromogenic fluorescence probe Rhocl-Cys based on Rhodamine chloride exhibiting the preferable performance of fluorescence turn-on response reacting with Cys was presented. Rhocl-Cys responded rapidly to Cys within 20 min, and had stable fluorescence intensity within pH 6.0-10.0, high selectivity towards Cys and the anti-inference capability with a low detection limit of 0.80 μM. In particular, Rhocl-Cys could qualitatively and quantitatively monitor the level of endogenous and exogenous Cys in living cells and successfully apply to zebrafish detecting Cys. Therefore, these results might further provide the basis exploring the role of Cys in biological system and facilitate as clinical diagnostic molecular tools., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Sulfite (SO 3 2- ) is considered as a monitor of a wide range of physiological processes. However, cells and tissues are adversely affected when the body ingests high level of sulfite. Here, we designed and synthesized a "turn on" fluorescent probe ImiSft-1 with 2-cyano-N-methylacetamide as the specific recognition site of SO 3 2- . This probe predominantly achieved high response intensity to SO 3 2- and desirable properties such as large Stokes shift (∼180 nm), fast response time (within 15 s), and high sensitivity (LOD = 0.12 μM). Importantly, the probe was highly selective for sulfite from other bio-species including biological thiols. Other functional properties included broad pH adaptability (5.0-10.0) and low cytotoxicity. Given these advantages and the fluorescence imaging in living MCF-7 cells, it was demonstrated that probe ImiSft-1 could monitor the changes of sulfite concentration in living cells., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The fluorescent probe, GXY-ADP-2, with xanthene structure as the fluorescent core was designed and prepared for the selective detection of peroxynitrite (ONOO - ). ONOO - can be produced endogenously and exogenously and is a strong oxidant with a short half-life. Oxidative modifications of biomolecules, that can be attributed to the formation of ONOO - , occur in the reactions of biomolecules with secondary ONOO - -derived radical oxidants. Therefore, it is very important to develop a specific fluorescent probe for detecting ONOO - to monitor oxidative stress state. The excitation wavelength and emission wavelength of the probe are 689 nm and 739 nm respectively. In the process of co-incubation with ONOO - , generate a new substance with two internal conjugated structures through a special reaction mechanism, one giving the fluorescence with the excitation wavelength of 347 nm and the emission wavelength of 484 nm with the detection limit of 0.12 μM, and the other that with the excitation wavelength of 433 nm and the emission wavelength of 583 nm with the detection limit of 0.077 μM. The linear dynamic range of the probe is 0-5 μM. Its response is not affected by the other reactive oxygen species, thus can sensitively detect ONOO - . In bioimaging experiments with HepG2 cells, the green and blue cell fluorescence signals (583 nm and 433 nm, respectively) were increased, while the red one (739 nm) was significantly reduced, under lipopolysaccharide (LPS) induced oxidative stress, proving that the probe could sensitively detect ONOO - in living cells. This work provides a new tool for the dynamic changes of ONOO - and oxidative stress processes in biological systems., (Copyright © 2022. Published by Elsevier B.V.)

Influenza viral infection causes acute upper respiratory diseases in humans, posing severe risks to global public health. However, current vaccines provide limited protection against mismatched circulating influenza A viruses. Here, the immune responses induced in mice by novel double-layered protein nanoparticles were investigated. The nanoparticles were composed of influenza nucleoprotein (NP) cores and hemagglutinin (HA) or matrix 2 protein ectodomain (M2e) shells. Vaccination with the nanoparticles significantly enhanced M2e-specific serum antibody titers and concomitant ADCC responses. Robust NP-specific T cell responses and robust HA neutralization were also detected. Moreover, vaccination with a trivalent nanoparticle combination containing two routinely circulated HA, conserved M2e, and NP reduced lung virus titers, pulmonary pathologies, and weight loss after homologous virus challenge. This combination also improved survival rates against heterologous and heterosubtypic influenza virus challenges. Our results demonstrate that the trivalent combination elicited potent and long-lasting immune responses conferring influenza viral cross-protection., (Copyright © 2021 Elsevier Inc. All rights reserved.)

H 2 S has been reported to play essential roles in a variety of physiological and pathological procedures. In this work, a novel fluorescent probe, Rho-HS, for detecting H 2 S was developed by introducing the ortho-halogen to activate the least reactive recognition group 2,4-dinitrophenyl moiety. In combination of the structures from both Rhodamine B and fluorescein, Rho-HS could generate both the colorimetric and fluorescent responses. This feature was not frequently achieved and could lead to the quantitative and convenient for the end-user. In comparison with recent probes for H 2 S, the major advantages of Rho-HS included suiting wide pH range (6.0-10.0), relatively rapid response (within 15 min) and the high selectivity among the competing species including the biothiols. With low cytoxicity, Rho-HS was further applied in the biological imaging in living MCF-7 cells and Caenorhabditis elegans. We hope that the designing strategy in this work might provide useful information for more preferable implements in this field., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Bone metastasis of malignant solid tumors has become one of the most serious complications, especially in breast cancer, which was particularly challenging for early detection and treatment in clinical practice. In this work, we reported a new fluorescently labeled bisphosphonate for bone metastasis detection of breast cancer. The designed probes were based on Rhodamine B and bisphosphonate as recognition group, which can specifically target hydroxyapatite (HA) existed in bone tissue. After the osteoclasts were adsorbed on the bone surface, the surrounding microenvironment was acidified, causing the HA to locally dissolve. The probe bound to the HA was then released, and realized the fluorescence turn on under acidic conditions. In vitro experiments showed that G0 was more excellent than G2 owing to shorter connecting arm. Subsequently, we proved that G0 could combine with HA rapidly and exhibit excellent response in solid state. More importantly, we established a model of bone metastasis with MDA-MB-231 cells which was similar to the clinical cases and evaluated the theranostics value of G0 prospectively, which provide the potential application prospect in clinical., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cysteine (Cys) is an indispensable small organic molecule containing sulfhydryl groups, which has essential regulatory effects on the physiological process of human body. In this work, a red emission fluorescent probe TCFQ-Cys was designed and exploited based on 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-ylidene) malononitrile-derivatives. The probe could effectively monitor Cys through the typical acrylate cleavage. The detecting system showed a red emission at 633 nm and the fluorescence was stable within the pH range of 6-9. The detection could be completed in 30 min. TCFQ-Cys presented high sensitivity with a detection limit of 0.133 μM and high selectivity towards Cys from other biological mercaptans. The most important feature was that the system had a wide linear range of 0-300 μM, which covered the physiological requirements of Cys detection. Subsequently, we conducted the biological imaging of Cys in MCF-7 cells and Caenorhabditis elegans (C. elegans). Therefore, TCFQ-Cys had a practical application prospect for further investigating the physiological function of Cys., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Hypochlorite, as one of reactive oxygen species, has drawn much attention due to its essential roles in special biological events and disorders. The exogenous hypochlorite remains a risk for human, animals and plants. In this work, a novel water soluble quinolin-containing nitrone derivative T has been developed for fluorometric sensing hypochlorite. The response mechanism of T towards ClO - was reported for the first time. In comparison with the reported sensors for ClO - , the sensor T in this work exhibited advantages including high selectivity (80 fold over other analytes), rapid response (within 5 s) and lipid-water distribution transformation (LogP from 2.979 to 6.131). Further biological applications suggested that T was capable of monitoring both exogenous and endogenous ClO - in living cells. The imaging in Arabidopsis thaliana indicated that the absorption and transmission of ClO - in plant could be monitored by this sensor through the chlorine-related mechanism. This work might raise referable information for further investigations in the physiological and pathological events in both tumor and plants., Competing Interests: Declaration of competing interest All authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Sulfur-containing species are essential in the composition and the metabolism of the organisms, thus developing a full set of implements to cover all of them is still a favorable choice. Herein, we chose imidazo [1,5-α]pyridine moiety as the basic fluorophore for the detection of sulfite, and preliminarily completed the toolset since biothiols (GSH, Cys, Hcy), H 2 S, and PhSH could be detected by sensors based on the same backbone. The designed sensor, IPD-SFT, with structural novelty and large Stokes shift (130 nm), indicated the most attractive advantages of remarkably rapid response period (within 1 min) and high selectivity for sulfite from all the sulfur-containing species. Other practical properties included high sensitivity (LOD = 50 nM) and wide pH adaptability (5.0-11.0). Furthermore, IPD-SFT could monitor both exogenous and endogenous sulfite. It not only raised a potential tool for sulfite detection, but also preliminarily completed the toolset for all the sulfur-containing species. The development of such toolsets might reveal the sulfur-containing metabolism and corresponding physiology and pathological procedures., Competing Interests: Declaration of competing interest All authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In this work, a primary method was constructed for detecting hydrazine in plant, thus accomplished the closed-loop monitoring of hydrazine circulation within manufacture, environment, plants, animals and human. From a series of sensors, QYL-1 was selected to present the hydrazine sensing properties. As a preliminary tool, QYL-1 suggested the ultra-wide linear range (0-20.0 equivalent) and high selectivity, which were extremely essential for linking the monitoring in various scale and field. For the first time, concentration-dependent tracking of hydrazine was successfully performed in Arabidopsis Thaliana root tips. Afterwards applications in water samples and living MCF-7 cells then fulfilled the demonstration of closing the loop by linking both the upstream and downstream nodes. More than raising a practical method, this work offered initial information for the closed-loop monitoring of hydrazine circulation, which might be significant for the ideal systematic managing in future., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Palladium (Pd) has drawn worldwide attentions because its connections to industry, chemistry, biological material and public health. Quantitative and selective detection tools for Pd and its ion forms are in urgent necessity. Here an umbelliferone derivative Umb-Pd2 was provided as a small, steady, safe and selective sensor for detecting Pd(II). It indicated advantages including sensitive (LOD 1.1 nM), wide pH tolerance (5.0-10.0), applicable linear range (0-1.8 equivalent) and low toxicity. The most attractive point was its explicit selectivity towards Pd(II) from Pd(0) in both independent and coexistence systems. This distinguishing ability was further utilized in imaging in living cells, raising this work as a rare and important example among all the published papers on palladium sensing. Thus, Umb-Pd2 supplied a potential approach for further improvement and applications in both daily chemistry and public health., (Copyright © 2019. Published by Elsevier B.V.)

Background: Central hepatectomy (CH) is more difficult than extended hepatectomy (EH) and is associated with greater morbidity. In this modern era of liver management with aims to prevent post-hepatectomy liver failure (PHLF), there is a need to assess outcomes of CH as a parenchyma-sparing procedure for centrally located liver tumors., Methods: A total of 178 major liver resections performed by specialist surgeons from two Australian tertiary institutions between June 2009 and March 2017 were reviewed. Eleven patients had CH and 24 had EH over this study period. Indications and perioperative outcomes were compared between the groups., Results: The main indication for performing CH was colorectal liver metastases. There was no perioperative mortality in the CH group and four (16.7%) in the EH group (P = 0.285). No group differences were found in median operative time [CH vs. EH: 450 min (290-840) vs. 523 min (310-860), P = 0.328], intraoperative blood loss [850 mL (400-1500) vs. 650 mL (100-2000), P = 0.746] or patients requiring intraoperative blood transfusion [1 (9.1%) vs. 7 (30.4%), P = 0.227]. There was a trend towards fewer hepatectomy-specific complications in the CH group [3 (27.3%) vs. 13 (54.2%), P = 0.167], including PHLF (CH vs. EH: 0 vs. 29.2%, P = 0.072). Median length of stay was similar between groups [CH vs. EH: 9 days (5-23) vs. 12 days (4-85), P = 0.244]., Conclusions: CH has equivalent postoperative outcomes to EH. There is a trend towards fewer hepatectomy-specific complications, including PHLF. In appropriate patients, CH may be considered as a safe parenchyma-sparing alternative to EH., (Copyright © 2019. Published by Elsevier B.V.)

The immunogenicity of subunit vaccines can be augmented by formulating them into nanoparticles. We conjugated recombinant trimetric influenza A/Aichi/2/68(H3N2) hemagglutinin (HA) onto functionalized gold nanoparticle (AuNP) surfaces in a repetitive, oriented configuration. To further improve the immunogenicity, we generated Toll-like receptor 5 (TLR5) agonist flagellin (FliC)-coupled AuNPs as particulate adjuvants. Intranasal immunizations with an AuNP-HA and AuNP-FliC particle mixture elicited strong mucosal and systemic immune responses that protected hosts against lethal influenza challenges. Compared with the AuNP-HA alone group, the addition of AuNP-FliC improved mucosal B cell responses as characterized by elevated influenza specific IgA and IgG levels in nasal, tracheal, and lung washes. AuNP-HA/AuNP-FliC also stimulated antigen-specific interferon-γ (IFN-γ)-secreting CD4 + cell proliferation and induced strong effector CD8 + T cell activation. Our results indicate that intranasal co-delivery of antigen and adjuvant-displaying AuNPs enhanced vaccine efficacy by inducing potent cellular immune responses., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Vaccinations are expected to aid in building immunity against pathogens. This objective often requires the addition of an adjuvant with certain vaccine formulations containing weakly immunogenic antigens. Adjuvants can improve antigen processing, presentation, and recognition, thereby improving the immunogenicity of a vaccine by simulating and eliciting an immune response. Chemokines are a group of small chemoattractant proteins that are essential regulators of the immune system. They are involved in almost every aspect of tumorigenesis, antitumor immunity, and antimicrobial activity and also play a critical role in regulating innate and adaptive immune responses. More recently, chemokines have been used as vaccine adjuvants due to their ability to modulate lymphocyte development, priming and effector functions, and enhance protective immunity. Chemokines that are produced naturally by the body's own immune system could serve as potentially safer and more reliable adjuvant options versus synthetic adjuvants. This review will primarily focus on chemokines and their immunomodulatory activities against various infectious diseases and cancers., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Recurring influenza viruses pose an annual threat to public health. A time-saving, cost-effective and egg-independent influenza vaccine approach is important particularly when responding to an emerging pandemic. We fabricated coated, two-layer protein nanoclusters from recombinant trimeric hemagglutinin from an avian-origin H7N9 influenza A virus as an approach for vaccine development in response to an emerging pandemic. Assessment of the virus-specific immune responses and protective efficacy in mice immunized with the nanoclusters demonstrated that the vaccine candidates were highly immunogenic, able to induce protective immunity and long-lasting humoral antibody responses to this virus without the use of adjuvants. Because the advantages of the highly immunogenic coated nanoclusters also include rapid productions in an egg-independent system, this approach has great potential for influenza vaccine production not only in response to an emerging pandemic, but also as a replacement for conventional seasonal influenza vaccines., Competing Interests: There are no known conflicts of interests., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Influenza vaccines with broad cross-protection are urgently needed. The highly conserved ectodomain of the influenza matrix protein 2 (M2e) can be a promising candidate if its low immunogenicity was overcome. In this study, we generated protein nanoclusters self-assembled from conformation-stabilized M2e tetramers (tM2e) to improve its immunogenicity. The resulting nanoclusters showed an average hydrodynamic diameter of 227 nm. Vaccination with the nanoclusters by an intranasal route elicited high levels of serum antigen-specific IgG in mice (approximately 100-fold higher than that obtained with soluble tM2e), as well as antigen-specific T cell and mucosal antibody responses. The immunity conferred complete protection against lethal challenge with homo- as well as heterosubtypic viruses. These results demonstrate that nanoclusters assembled from conformation-stabilized M2e are promising as a potential universal influenza A vaccine. Self-assembly into nanoclusters represents a novel approach for increasing the immunogenicity of vaccine antigens., From the Clinical Editor: In order to develop more effective influenza vaccination, the highly conserved ectodomain of M2e could be a promising candidate. Unfortunately, it is a weak antigen for vaccination purposes. In this study, self-assembled protein nanoclusters of tM2e were generated and tested. The nanoclusters demonstrated superior vaccination properties, with complete protection against lethal challenge in the studied rodent model, raising hope for the introduction of similar vaccines to challenge human influenza outbreaks., (© 2014.)

The influenza M2 ectodomain (M2e) is poorly immunogenic and has some amino acid changes among isolates from different host species. We expressed a tandem repeat construct of heterologous M2e sequences (M2e5x) derived from human, swine, and avian origin influenza A viruses on virus-like particles (M2e5x VLPs) in a membrane-anchored form. Immunization of mice with M2e5x VLPs induced protective antibodies cross-reactive to antigenically different influenza A viruses and conferred cross protection. Anti-M2e antibodies induced by heterologous M2e5x VLPs showed a wider range of cross reactivity to influenza A viruses at higher levels than those by live virus infection, homologous M2e VLPs, or M2e monoclonal antibody 14C2. Fc receptors were found to be important for mediating protection by immune sera from M2e5x VLP vaccination. The present study provides evidence that heterologous recombinant M2e5x VLPs can be more effective in inducing protective M2e immunity than natural virus infection and further supports an approach for developing an effective universal influenza vaccine., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The cytotoxicity of intact cinnamomin (a type II ribosome-inactivating protein, RIP) and the RNA N-glycosidase activity of cinnamomin A-chain have been studied and compared with those of ricin. Cinnamomin A-chain exhibits a similar RNA N-glycosidase activity in inhibiting in vitro protein synthesis compared with that of ricin, whereas the cytotoxicity to BA/F3beta cells of intact cinnamomin is markedly lower than intact ricin. In order to demonstrate that it is the B-chains of the two RIPs that bear the difference in cytotoxicity, two hybrid RIPs are prepared from the purified A-/B-chains of cinnamomin and ricin by the disulfide exchange reaction. It has been found that hybrid RIP constructed from cinnamomin A-chain and ricin B-chain is more toxic to BA/F3beta cells than the native cinnamomin, and equivalent to the native ricin. However, the cytotoxicity to BA/F3beta cells of the hybrid RIP constructed from the ricin A-chain and cinnamomin B-chain is lower than ricin, equivalent to the native cinnamomin. Furthermore, the bound amounts of two B-chains on the cell surface are determined by the method of direct cellular ELISA and Scatchard analysis of the binding of the two B-chains indicates that cinnamomin and ricin share similar binding sites with different affinity.

The pure cinnamomin A-chain is unstable compared to that in the mixture of A- and B-chain or in intact cinnamomin molecule either being stored at 4 degrees C or being heated. When being heated at 45 degrees C for 20min, the A-chain generates partially unfolded intermediate and loses its tertiary structure as monitored by circular dichroism (CD) and tryptophan fluorescence, thus resulting in the inactivity of its RNA N-glycosidase albeit it retains most of its secondary structures. This partially unfolded intermediate is sensitive to protease, exhibiting property of a molten globule. The changes in conformation and activity are irreversible upon cooling. The partially unfolded intermediate can fully restore its RNA N-glycosidase activity in the presence of cinnamomin B-chain. The phenomenon, that the cinnamomin B-chain mediates the refolding of partially unfolded A-chain, probably plays an important role in the intracellular transport of the cytotoxic protein, i.e., keeping the structural stability of A-chain and refolding partially unfolded A-chain that occasionally appeared in the process of intracellular transport, to avoid the destiny of proteolysis that occurs in most denatured proteins in cell.

An iron-superoxide dismutase (SOD) was purified and characterized from the mature seeds of camphor tree (Cinnamomum camphora). The ultraviolet and visible absorption spectra of camphor Fe-SOD showed patterns typical of cambialistic Fe-SODs. The inductively coupled plasma assay indicated that there was 0.5-1 atom of Fe(2+) per camphor Fe-SOD subunit. The cDNA of camphor Fe-SOD, including the coding region and the 3' noncoding region, was obtained by reverse transcription polymerase chain reaction using the total RNA from immature seeds of C. camphora as template and then sequenced. The complete amino acid sequence of camphor Fe-SOD was deduced from the cDNA sequence. The correctness of the amino acid sequence was confirmed by directly sequencing five peptide fragments of the enzyme. The molecular mass calculated for the camphor Fe-SOD subunit from its 204 amino acid residues was 22,930.6 Da, The cDNA of camphor Fe-SOD was cloned into the expression vector PMFT7-5 and then expressed in Escherichia coli strain BL21. The reconstructed Fe- or Mn-SOD was purified to homogeneity through column chromatography. Activity of the Fe- or Mn-SOD was found to be almost equal to that of natural camphor Fe-SOD, which is the first cambialistic SOD isolated from eukaryotic cells., (Copyright 2002 Elsevier Science (USA).)