Information transfer from the optical phase to a plasmonic digital encoder composed of a gold nanorod pair.

Small all-optical devices are central to the optical computing. Plasmonic digital encoders (PDEs) with a featured dimension of ∼1µm hold the key for transferring information from far field to photonic processing systems. Here we propose a PDE design composed of two gold nanorods (AuNRs), whose pattern represents 2-bit digital information. We implanted information into the spectral phase of a femtosecond pulse by pulse shaping and controlled the two-photon photoluminescence pattern of an AuNR pair. The high contrast ratios were achieved with 13.01 and 6.02 dB for binary codes "1-0" and "0-1", respectively.

RNA sequencing analysis reveals quiescent microglia isolation methods from postnatal mouse brains and limitations of BV2 cells.

BACKGROUND: Microglia play key roles in neuron-glia interaction, neuroinflammation, neural repair, and neurotoxicity. Currently, various microglial in vitro models including primary microglia derived from distinct isolation methods and immortalized microglial cell lines are extensively used. However, the diversity of these existing models raises difficulty in parallel comparison across studies since microglia are sensitive to environmental changes, and thus, different models are likely to show widely varied responses to the same stimuli. To better understand the involvement of microglia in pathophysiological situations, it is critical to establish a reliable microglial model system. METHODS: With postnatal mouse brains, we isolated microglia using three general methods including shaking, mild trypsinization, and CD11b magnetic-associated cell sorting (MACS) and applied RNA sequencing to compare transcriptomes of the isolated cells. Additionally, we generated a genome-wide dataset by RNA sequencing of immortalized BV2 microglial cell line to compare with primary microglia. Furthermore, based on the outcomes of transcriptional analysis, we compared cellular functions between primary microglia and BV2 cells including immune responses to LPS by quantitative RT-PCR and Luminex Multiplex Assay, TGFß signaling probed by Western blot, and direct migration by chemotaxis assay. RESULTS: We found that although the yield and purity of microglia were comparable among the three isolation methods, mild trypsinization drove microglia in a relatively active state, evidenced by high amount of amoeboid microglia, enhanced expression of microglial activation genes, and suppression of microglial quiescent genes. In contrast, CD11b MACS was the most reliable and consistent method, and microglia isolated by this method maintained a relatively resting state. Transcriptional and functional analyses revealed that as compared to primary microglia, BV2 cells remain most of the immune functions such as responses to LPS but showed limited TGFß signaling and chemotaxis upon chemoattractant C5a. CONCLUSIONS: Collectively, we determined the optimal isolation methods for quiescent microglia and characterized the limitations of BV2 cells as an alternative of primary microglia. Considering transcriptional and functional differences, caution should be taken when extrapolating data from various microglial models. In addition, our RNA sequencing database serves as a valuable resource to provide novel insights for appropriate application of microglia as in vitro models.

DNA Origami Directed Assembly of Gold Bowtie Nanoantennas for Single-Molecule Surface-Enhanced Raman Scattering.

Metallic bowtie nanoarchitectures can produce dramatic electric field enhancement, which is advantageous in single-molecule analysis and optical information processing. Plasmonic bowtie nanostructures were successfully constructed using a DNA origami-based bottom-up assembly strategy, which enables precise control over the geometrical configuration of the bowtie with an approximate 5 nm gap. A single Raman probe was accurately positioned at the gap of the bowtie. Single-molecule surface-enhanced Raman scattering (SM-SERS) of individual nanostructures, including ones containing an alkyne group, was observed. The design achieved repeatable local field enhancement of several orders of magnitude. This method opens the door on a novel strategy for the fabrication of metal bowtie structures and SM-SERS, which can be utilized in the design of highly-sensitive photonic devices.

The role of microglial P2X7: modulation of cell death and cytokine release.

BACKGROUND: ATP-gated P2X7 is a non-selective cation channel, which participates in a wide range of cellular functions as well as pathophysiological processes including neuropathic pain, immune response, and neuroinflammation. Despite its abundant expression in microglia, the role of P2X7 in neuroinflammation still remains unclear. METHODS: Primary microglia were isolated from cortices of P0-2 C57BL/6 wild-type or P2X7 knockout (P2X7-/-) mouse pups. Lipopolysaccharide, lipopolysaccharide plus IFNγ, or IL4 plus IL13 were used to polarize microglia to pro-inflammatory or anti-inflammatory states. P2rx7 expression level in resting or activated mouse and human microglia was measured by RNA-sequencing and quantitative real-time PCR. Microglial cell death was measured by cell counting kit-8 and immunocytochemistry, and microglial secretion in wild-type or P2X7-/- microglia was examined by Luminex multiplex assay or ELISA using P2X7 agonist BzATP or P2X7 antagonist A-804598. P2X7 signaling was analyzed by Western blot. RESULTS: First, we confirmed that P2rx7 is constitutively expressed in mouse and human primary microglia. Moreover, P2rx7 mRNA level was downregulated in mouse microglia under both pro- and anti-inflammatory conditions. Second, P2X7 agonist BzATP caused cell death of mouse microglia, while this effect was suppressed either by P2X7 knockout or by A-804598 under both basal and pro-inflammatory conditions, which suggests the mediating role of P2X7 in BzATP-induced microglial cell death. Third, BzATP-induced release of IL1 family cytokines including IL1α, IL1ß, and IL18 was blocked in P2X7-/- microglia or by A-804598 in pro-inflammatory microglia, while the release of other cytokines/chemokines was independent of P2X7 activation. These findings support the specific role of P2X7 in IL1 family cytokine release. Finally, P2X7 activation was discovered to be linked to AKT and ERK pathways, which may be the underlying mechanism of P2X7 functions in microglia. CONCLUSIONS: These results reveal that P2X7 mediates BzATP-induced microglial cell death and specific release of IL1 family cytokines, indicating the important role of P2X7 in neuroinflammation and implying the potential of targeting P2X7 for the treatment of neuroinflammatory disorders.

Surface enhanced fluorescence by metallic nano-apertures associated with stair-gratings.

Metallic nano-apertures associated with stair-gratings are proposed for surface enhanced fluorescence with high excitation enhancement and narrow emission beaming effect. Fluorescence correlation spectroscopy method was utilized to analyze the fluorescence trace and fluorescence enhancement, and the angular patterns of fluorescent emission were measured with the back focal plane imaging method. The stair-grating presents a strong optical response which covering well both the excitation and the emission bands of the photoluminescence process. Such high enhancement and narrow directionality by the stair-gratings would enable the detection of single molecules with low numerical aperture objective effectively.

2D Free-Standing GeS1-xSex with Composition-Tunable Bandgap for Tailored Polarimetric Optoelectronics.

Germanium-based monochalcogenides (i.e., GeS and GeSe) with desirable properties are promising candidates for the development of next-generation optoelectronic devices. However, they are still stuck with challenges, such as relatively fixed electronic band structure, unconfigurable optoelectronic characteristics, and difficulty in achieving free-standing growth. Herein, it is demonstrated that two-dimensional (2D) free-standing GeS1-xSex (0 ≤ x ≤ 1) nanoplates can be grown by low-pressure rapid physical vapor deposition (LPRPVD), fulfilling a continuously composition-tunable optical bandgap and electronic band structure. By leveraging the synergistic effect of composition-dependent modulation and free-standing growth, GeS1-xSex-based optoelectronic devices exhibit significantly configurable hole mobility from 6.22 × 10-4 to 1.24 cm2V-1s⁻1 and tunable responsivity from 8.6 to 311 A W-1 (635 nm), as x varies from 0 to 1. Furthermore, the polarimetric sensitivity can be tailored from 4.3 (GeS0.29Se0.71) to 1.8 (GeSe) benefiting from alloy engineering. Finally, the tailored imaging capability is also demonstrated to show the application potential of GeS1-xSex alloy nanoplates. This work broadens the functionality of conventional binary materials and motivates the development of tailored polarimetric optoelectronic devices.

Single and combined associations of blood lead and essential metals with serum lipid profiles in community-dwelling adults.

Background: Although several studies have examined the relationships between lead (Pb) exposure and serum lipid profiles, the associations of the metal mixture, including lead (Pb) and essential metals with lipid profiles, remain unclear. Objective: To investigate the associations of the metal mixture including Pb and essential metals [magnesium (Mg), manganese (Mn), copper (Cu), iron (Fe), zinc (Zn), and calcium (Ca)] with serum lipid profiles [total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C)], as well as the potential interactions among the metals. Methods: Nine hundred and ninety-eight Chinese community-dwelling adults completed a questionnaire and underwent checkups of anthropometric parameters, serum lipid profile levels (TC, TG, LDL-C, and HDL-C), and blood metal concentrations (Pb, Mg, Mn, Cu, Fe, Zn, and Ca). The multivariable linear regression, weighted quantile sum (WQS) regression, and Bayesian kernel machine regression (BKMR) were applied to evaluate the single and combined associations of blood Pb and essential metals with serum lipid profiles. Results: In the multivariable linear regression model, the blood Pb was positively associated with TC, LDL-C, and HDL-C (p < 0.05, all), and the blood Mg were positively associated with serum TC, LDL-C, and Ln TG (p < 0.05, all). In the WQS regression and BKMR models, the metal mixture of blood Pb and the essential metals was positively associated with all of the serum lipid profiles. In addition, an inverse U-shaped association of Pb with Ln TG and the positive interactive effect between blood Pb and Mg levels on TC and LDL-C were found. Conclusion: The levels of blood Pb, together with the essential metals, especially Mg levels, are suggested to be considered when assessing dyslipidemia risk. However, more evidence is still needed to validate the conclusions.

Complement receptor 2 is expressed in neural progenitor cells and regulates adult hippocampal neurogenesis.

Injury and inflammation are potent regulators of adult neurogenesis. As the complement system forms a key immune pathway that may also exert critical functions in neural development and neurodegeneration, we asked whether complement receptors regulate neurogenesis. We discovered that complement receptor 2 (CR2), classically known as a coreceptor of the B-lymphocyte antigen receptor, is expressed in adult neural progenitor cells (NPCs) of the dentate gyrus. Two of its ligands, C3d and interferon-α (IFN-α), inhibited proliferation of wild-type NPCs but not NPCs derived from mice lacking Cr2 (Cr2(-/-)), indicating functional Cr2 expression. Young and old Cr2(-/-) mice exhibited prominent increases in basal neurogenesis compared with wild-type littermates, whereas intracerebral injection of C3d resulted in fewer proliferating neuroblasts in wild-type than in Cr2(-/-) mice. We conclude that Cr2 regulates hippocampal neurogenesis and propose that increased C3d and IFN-α production associated with brain injury or viral infections may inhibit neurogenesis.

Genetic ablation of apolipoprotein A-IV accelerates Alzheimer's disease pathogenesis in a mouse model.

The link between lipoprotein metabolism and Alzheimer's disease (AD) has been established. Apolipoprotein A-IV (apoA-IV), a component of lipoprotein particles similar to apolipoprotein E, has been suggested to play an important role in brain metabolism. Although there are clinical debates on the function of its polymorphism in AD, the pathologic role of apoA-IV in AD is still unknown. Here, we report that genetic ablation of apoA-IV is able to accelerate AD pathogenesis in mice. In a mouse model that overexpresses human amyloid precursor protein (APP) and presenilin 1, genetic reduction of apoA-IV augments extracellular amyloid-ß peptide (Aß) burden and aggravates neuron loss in the brain. In addition, genetic ablation of apoA-IV also accelerates spatial learning deficits and increases the mortality of mice. We have found that apoA-IV colocalizes within Aß plaques in APP/presenilin 1 transgenic mice and binds to Aß in vitro. Subsequent studies show that apoA-IV in this model facilitates Aß uptake in the Aß clearance pathway mediated by astrocytes rather than the amyloidogenic pathway of APP processing. Taken together, we conclude that apoA-IV deficiency increases Aß deposition and results in cognitive damage in the mouse model. Enhancing levels of apoA-IV may have therapeutic potential in AD treatment.

Enhanced delivery of antibodies across the blood-brain barrier via TEMs with inherent receptor-mediated phagocytosis.

BACKGROUND: The near impermeability of the blood-brain barrier (BBB) and the unique neuroimmune environment of the CNS prevents the effective use of antibodies in neurological diseases. Delivery of biotherapeutics to the brain can be enabled through receptor-mediated transcytosis via proteins such as the transferrin receptor, although limitations such as the ability to use Fc-mediated effector function to clear pathogenic targets can introduce safety liabilities. Hence, novel delivery approaches with alternative clearance mechanisms are warranted. METHODS: Binders that optimized transport across the BBB, known as transcytosis-enabling modules (TEMs), were identified using a combination of antibody discovery techniques and pharmacokinetic analyses. Functional activity of TEMs were subsequently evaluated by imaging for the ability of myeloid cells to phagocytose target proteins and cells. FINDINGS: We demonstrated significantly enhanced brain exposure of therapeutic antibodies using optimal transferrin receptor or CD98 TEMs. We found that these modules also mediated efficient clearance of tau aggregates and HER2+ tumor cells via a non-classical phagocytosis mechanism through direct engagement of myeloid cells. This mode of clearance potentially avoids the known drawbacks of FcγR-mediated antibody mechanisms in the brain such as the neurotoxic release of proinflammatory cytokines and immune cell exhaustion. CONCLUSIONS: Our study reports a new brain delivery platform that harnesses receptor-mediated transcytosis to maximize brain uptake and uses a non-classical phagocytosis mechanism to efficiently clear pathologic proteins and cells. We believe these findings will transform therapeutic approaches to treat CNS diseases. FUNDING: This research was funded by Janssen, Pharmaceutical Companies of Johnson & Johnson.

Mouse primary microglia respond differently to LPS and poly(I:C) in vitro.

Microglia, CNS resident innate immune cells, respond strongly to activation of TLR3 and TLR4, which recognize viral dsRNA poly(I:C) and bacterial endotoxin LPS, respectively. However, few studies have thoroughly and parallelly compared functional phenotypes and downstream mechanisms between LPS- and poly(I:C)-exposed primary microglia. Here, we investigated the responses of mouse primary microglia upon LPS and poly(I:C) stimulation by detecting various phenotypes ranging from morphology, proliferation, secretion, chemotaxis, to phagocytosis. Furthermore, we explored their sequential gene expression and the downstream signal cascades. Interestingly, we found that the microglial activation pattern induced by LPS was distinguished from that induced by poly(I:C). Regarding microglial morphology, LPS caused an ameboid-like shape while poly(I:C) induced a bushy shape. Microglial proliferation was also facilitated by LPS but not by poly(I:C). In addition, LPS and poly(I:C) modulated microglial chemotaxis and phagocytosis differently. Furthermore, genome-wide analysis provided gene-level support to these functional differences, which may be associated with NF-κb and type I interferon pathways. Last, LPS- and poly(I:C)-activated microglia mediated neurotoxicity in a co-culture system. This study extends our understanding of TLR roles in microglia and provides insights into selecting proper inflammatory microglial models, which may facilitate identification of new targets for therapeutic application.

Fibrinogen has chaperone-like activity.

Partially or completely unfolded polypeptides are highly prone to aggregation due to nonspecific interactions between their exposed hydrophobic surfaces. Extracellular proteins are continuously subjected to stresses conditions, but the existence of extracellular chaperones remains largely unexplored. The results presented here demonstrate that one of the most abundant extracellular proteins, fibrinogen has chaperone-like activity. Fibrinogen can specifically bind to nonnative form of citrate synthase and inhibit its thermal aggregation and inactivation in an ATP-independent manner. Interestingly, fibrinogen maintains thermal-denatured luciferase in a refolding competent state allowing luciferase to be refolded in cooperation with rabbit reticulocyte lysate. Fibrinogen also inhibits fibril formation of yeast prion protein Sup35 (NM). Furthermore, fibrinogen rescues thermal-induced protein aggregation in the plasma of fibrinogen-deficient mice. Our studies demonstrate the chaperone-like activity of fibrinogen, which not only provides new insights into the extracellular chaperone protein system, but also suggests potential diagnostic and therapeutic approaches to fibrinogen-related pathological conditions.

Isolation and Culture of Astrocytes from Postnatal and Adult Mouse Brains.

Astrocytes play fundamental roles in development and plasticity of the central nervous system. Dysfunction of astrocytes contributes to most of neurological diseases ranging from neurodegenerative diseases to psychological disorders. To better understand the involvement of astrocytes in both physiological and pathological conditions, and further elucidate their underlying mechanisms, it is critical to establish a reliable isolation and culture method for the cells. In this chapter, we describe the isolation and culture protocols of astrocytes from postnatal and adult mouse brains.

PET Imaging of the P2X7 Ion Channel with a Novel Tracer [18F]JNJ-64413739 in a Rat Model of Neuroinflammation.

PURPOSE: The P2X7 receptor, an adenosine triphosphate (ATP)-gated purinoreceptor, has emerged as one of the key players in neuroinflammatory processes. Therefore, developing a positron emission tomography (PET) tracer for imaging of P2X7 receptors in vivo presents a promising approach to diagnose, monitor, and study neuroinflammation in a variety of brain disorders. To fulfill the goal of developing a P2X7 PET ligand as a biomarker of neuroinflammation, [18F]JNJ-64413739 has been recently disclosed. PROCEDURES: We evaluated [18F]JNJ-64413739 in a rat model of neuroinflammation induced by an intracerebral injection of lipopolysaccharide (LPS). In vivo brain uptake was determined by PET imaging. Upregulation of neuroinflammatory biomarkers was determined by quantitative polymerase chain reaction (qPCR). Distribution of the tracer in the brain was determined by ex vivo autoradiography (ARG). The specificity of [18F]JNJ-64413739 was confirmed by performing blocking experiments with the P2X7 antagonist JNJ-54175446. RESULTS: Brain regions of rats injected with LPS had a significantly increased uptake (34 % ± 3 % s.e.m., p = 0.036, t test, standardized uptake value measured over the entire scanning period) of [18F]JNJ-64413739 relative to the corresponding brain regions of control animals injected with phosphate-buffered saline (PBS). The uptake in the contralateral regions and cerebellum was not significantly different between the groups of animals. The increase in uptake of [18F]JNJ-64413739 at the LPS-injected site observed by PET imaging was concordant with ex vivo ARG, upregulation of neuroinflammatory biomarkers, and elevated P2X7 expression levels. CONCLUSIONS: While further work is needed to study [18F]JNJ-64413739 in other types of neuroinflammation, the current results favorably characterize [18F]JNJ-64413739 as a potential PET tracer of central neuroinflammation.

Pharmacology of JNJ-28583113: A novel TRPM2 antagonist.

Transient receptor potential melastatin type 2 (TRPM2) is a cation channel activated by free intracellular ADP-ribose and reactive oxygen species. TRPM2 signaling has been linked to the pathophysiology of CNS disorders such as neuropathic pain, bipolar disorder and Alzheimer's disease. In this manuscript, we describe the discovery of JNJ-28583113, a potent brain penetrant TRPM2 antagonist. Ca2+ flux assays in cells overexpressing TRPM2 and electrophysiological recordings were used to test the pharmacology of JNJ-28583113. JNJ-28583113 was assayed in vitro on GSK-3 phosphorylation levels, cell death, cytokine release in microglia and unbiased morphological phenotypic analysis. Finally, we dosed animals to evaluate its pharmacokinetic properties. Our results showed that JNJ-28583113 is a potent (126 ±â€¯0.5 nM) TRPM2 antagonist. Blocking TRPM2 caused phosphorylation of GSK3α and ß subunits. JNJ-28583113 also protected cells from oxidative stress induced cell death as well as morphological changes induced by non-cytotoxic concentrations of H2O2. In addition, inhibiting TRPM2 blunted cytokine release in response to pro-inflammatory stimuli in microglia. Lastly, we showed that JNJ-28583113 was brain penetrant but not suitable for systemic dosing as it was rapidly metabolized in vivo. While the in-vitro pharmacology of JNJ-28583113 is the best in class, its in-vivo properties would need optimization to assist in further probing key roles of TRPM2 in CNS pathophysiology.

Soluble tissue factor has unique angiogenic activities that selectively promote migration and differentiation but not proliferation of endothelial cells.

The level of circulating tissue factor (TF) is up-regulated in human angiogenesis-related malignancies. However, whether circulating TF has angiogenic activities has not been determined. Soluble TF (sTF) is the main domain of circulating TF. Here, using cell migration, wound healing, and tubule formation assays, human recombinant sTF was found to significantly promote the migration and differentiation of endothelial cells. The stress fiber formation and rearrangement induced by sTF observed through immunofluorescence microscope may be responsible for the stimulatory migration effect of sTF. Nevertheless, sTF had no effects on endothelial cell proliferation. Interestingly, sTF can be internalized by endothelial cells, which implies a novel mechanism for sTF in angiogenesis. These results suggest that sTF has unique angiogenic activities and may serve as a potential therapeutic target to treat diseases associated with angiogenesis such as cancer and rheumatoid arthritis.

Sulfate stabilizes the folding intermediate more than the native structure of endostatin.

Endostatin, a potent angiogenesis inhibitor, is an acid resistant protein with compact tertiary structure. Nuclear magnetic resonance, circular dichroism, and tryptophan emission fluorescence were used to monitor the structural changes of endostatin during acid-, heat-, and urea-induced unfolding processes. Results show that sulfate anions sensitize endostatin to acid, but specifically stabilize it against heat or urea. Moreover, the disappearance of the tertiary structure and the formation of the folding intermediate of endostatin at pH 3.0 are sulfate concentration dependent. These phenomena indicate that sulfate anions stabilize the folding intermediate more than the native structure of endostatin. In addition, heparin shows stronger effect than sodium sulfate on sensitizing endostatin against acid, and very limited stabilizing effect against urea. The loose structure of endostatin upon heparin binding may imply that the physiologically favorable structure for endostatin exerting its biological functions is not as compact as what was reported.

Neuropsychopharmacology of JNJ-55308942: evaluation of a clinical candidate targeting P2X7 ion channels in animal models of neuroinflammation and anhedonia.

Emerging data continues to point towards a relationship between neuroinflammation and neuropsychiatric disorders. ATP-induced activation of P2X7 results in IL-1ß release causing neuroinflammation and microglial activation. This study describes the in-vitro and in-vivo neuropharmacology of a novel brain-penetrant P2X7 antagonist, JNJ-55308942, currently in clinical development. JNJ-55308942 is a high-affinity, selective, brain-penetrant (brain/plasma of 1) P2X7 functional antagonist. In human blood and in mouse blood and microglia, JNJ-55308942 attenuated IL-1ß release in a potent and concentration-dependent manner. After oral dosing, the compound exhibited both dose and concentration-dependent occupancy of rat brain P2X7 with an ED50 of 0.07 mg/kg. The P2X7 antagonist (3 mg/kg, oral) blocked Bz-ATP-induced brain IL-1ß release in conscious rats, demonstrating functional effects of target engagement in the brain. JNJ-55308942 (30 mg/kg, oral) attenuated LPS-induced microglial activation in mice, assessed at day 2 after a single systemic LPS injection (0.8 mg/kg, i.p.), suggesting a role for P2X7 in microglial activation. In a model of BCG-induced depression, JNJ-55308942 dosed orally (30 mg/kg), reversed the BCG-induced deficits of sucrose preference and social interaction, indicating for the first time a role of P2X7 in the BCG model of depression, probably due to the neuroinflammatory component induced by BCG inoculation. Finally, in a rat model of chronic stress induced sucrose intake deficit, JNJ-55308942 reversed the deficit with concurrent high P2X7 brain occupancy as measured by autoradiography. This body of data demonstrates that JNJ-55308942 is a potent P2X7 antagonist, engages the target in brain, modulates IL-1ß release and microglial activation leading to efficacy in two models of anhedonia in rodents.

Insight into the catalytic mechanism of arginine deiminase: functional studies on the crucial sites.

Arginine deiminase (ADI) catalyzes the irreversible hydrolysis of arginine to citrulline and ammonia. It belongs to a newly classified superfamily of guanidino-group-modifying enzymes. Located in the catalytic center of Mycoplasma hominis ADI, some crucial sites (Asp160, Glu212, His268, and Asp270) are highly conserved among these enzymes. Here, we constructed five ADI single mutants D160E, E212D, H268F, H268Y, and D270E, and three double mutants D160E/D270E, D160E/E212D, and E212D/D270E, aiming to evaluate the contributions of these crucial residues to the structure, stability, and enzymatic activity of ADI, and to elucidate their roles in the catalytic process of this family of enzymes. Tryptophan emission fluorescence and circular dichroism were used to analyze the different effects of mutagenesis on these conserved residues on the secondary and tertiary structures of ADI. Urea-induced unfolding and trypsin digestion were applied to measure their stabilities against denaturants and proteases, respectively. Additionally, the enzymatic activities of ADI and its mutants were measured. Here, we report that all the mutations have little effect on the native structure of ADI. However, the substitutions on these crucial sites still interfere with the stability of ADI to different degrees. As these mutations impair both the substrate binding and the substrate induced conformational changes of ADI to different extents, most of the mutants except D160E (preserves about 30% of the enzymatic activity of wild type) have totally lost the enzymatic activity in the hydrolysis of arginine and the inhibitory ability on the proliferation of mouse melanoma cells.

Contributions of Zn(II)-binding to the structural stability of endostatin.

Endostatin has a compact structure with a Zn(II)-binding site (His1, His3, His11, and Asp76) at the N-terminus. In this study, the effects of Zn(II)-binding on the folding and stability of recombinant human endostatin were studied. The results show that Zn(II)-binding largely stabilizes the structure of endostatin at physiological pH. Under some proteolytic conditions, Zn(II)-binding also contributes to the integrity of the N-terminus of endostatin, which is critical for endostatin to maintain a stable structure. Moreover, engineering an extra Zn(II)-binding peptide to the N-terminus of human endostatin makes this molecule more stable and cooperative in the presence of Zn(II).