Regulation of p53 Activity by (+)-Epiloliolide Isolated from Ulva lactuca.

Ulva lactuca (U. lactuca) is a green alga distributed worldwide and used as a food and cosmetic material. In our previous study, we determined the effects of U. lactuca methanol extracts on the UVB-induced DNA repair. In the present study, we fractionated U. lactuca methanol extracts to identify the effective compound for the DNA repair. MTT assay demonstrated that (+)-epiloliolide showed no cytotoxicity up to 100 µM in BJ-5ta human dermal fibroblast. Upon no treatment, exposure to UVB 400 J/m2 decreased cell viability by 45%, whereas (+)-epiloliolide treatment for 24 h after UVB exposure significantly increased the cell viability. In GO and GESA analysis, a number of differentially expressed genes were uniquely expressed in (+)-epiloliolide treated cells, which were enriched in the p53 signaling pathway and excision repair. Immunofluorescence demonstrated that (+)-epiloliolide increased the nuclear localization of p53. Comet assay demonstrated that (+)-epiloliolide decreased tail moment increased by UVB. Western blot analysis demonstrated that (+)-epiloliolide decreased the levels of p-p53, p21, Bax, and Bim, but increased that of Bcl-2. Reverse transcription PCR (RT-PCR) demonstrated that (+)-epiloliolide decreased the levels of MMP 1, 9, and 13, but increased that of COL1A1. These results suggest that (+)-epiloliolide regulates p53 activity and has protective effects against UVB.

Alpha-neoendorphin can reduce UVB-induced skin photoaging by activating cellular autophagy.

Skin aging is influenced by several genetic, physiological, and environmental factors. In particular, ultraviolet (UV) exposure is an important factor involved in inducing skin photoaging. Autophagy controlling homeostatic balance between the synthesis, degradation, and recycling of cellular organelles and proteins plays important regulatory roles in several biological processes, including aging. The opioid neuropeptide α-neoendorphin (named NEP) is an endogenous decapeptide (N-YGGFLRKYPK-C) that activates the kappa opioid receptor and exhibits certain anti-aging and anti-wrinkling effects on skin cells; however, its action mechanism has not yet been elucidated. Therefore, the aim of this study was to determine the effects of NEP on anti-skin aging and autophagy activation in human dermal fibroblast cells. Western blot results showed that NEP down-regulates the production of phospho-mammalian target of rapamycin (p-mTOR), whereas increases the expression of key autophagy-related molecules such as Beclin-1, Atg5-Atg12, and LC3-II. The immunocytochemical analysis performed with anti-LC3-II antibody also showed that the autophagic indicators, autophagosomes are formed by NEP. These results suggest that NEP can activate cellular autophagy through mTOR-Beclin-1-mediated signaling pathway. It was also revealed by CM-H2DCF-DA assay and Western blottings that NEP can reduce the production of ultraviolet B (UVB)-induced reactive oxygen species (ROS) like with N-acetylcysteine (NAC), resulting in decreasing the expression levels of skin aging-related proteins, such as phospho-ERK (p-ERK), phospho-p38 (p-p38), and phospho-JNK (p-JNK). Furthermore, NEP could increase the type I procollagen production, while decreasing MMP-1, MMP-2, and MMP-9 activities. Taken together, the results demonstrate that NEP can reduce UVB-induced photoaging by activating autophagy.

Pleiotropic effects of a vibrio extracellular protease on the activation of contact system.

Many proteases secreted by pathogenic bacteria can affect seriously on hemostatic system. We have reported that an extracellular zinc metalloprotease (named vEP-45) from Vibrio vulnificus ATCC29307 activates prothrombin to active thrombin, leading the formation of fibrin clot. In this study, the effects of vEP-45 on the intrinsic pathway of coagulation and the kallikrein/kinin system were examined. The protease could activate proteolytically clotting factor zymogens, including FXII, FXI, FX, and prothrombin, to their functional enzymes in vitro and plasma milieu. In addition, it could cleave plasma prekallikrein (PPK) to form an active kallikrein as well as actively digest high-molecular weight kininogen (HK), probably producing bradykinin. In fact, vEP-45 could induce a vascular permeability in a dose-dependent manner in vivo. Taken together, the results demonstrate that vEP-45 can activate plasma contact system by cleaving key zymogen molecules, participating in the intrinsic pathway of coagulation and the kallikrein/kinin system.

Alzheimer's disease risk associated with changes in Epstein-Barr virus nuclear antigen 1-specific epitope targeting antibody levels.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder influenced by age, sex, genetic factors, immune alterations, and infections. Multiple lines of evidence suggest that changes in antibody response are linked to AD pathology. METHODS: To elucidate the mechanisms underlying AD development, we investigated antibodies that target autoimmune epitopes using high-resolution epitope microarrays. Our study compared two groups: individuals with AD (n = 19) and non-demented (ND) controls (n = 19). To validate the results, we measured antibody levels in plasma samples from AD patients (n = 96), mild cognitive impairment (MCI; n = 91), and ND controls (n = 97). To further explore the invlovement of EBV, we performed epitope masking immunofluorescence microscopy analysis and tests to induce lytic replication using the B95-8 cell line. RESULTS: In this study, we analyzed high-resolution epitope-specific serum antibody levels in AD, revealing significant disparities in antibodies targeting multiple epitopes between the AD and control groups. Particularly noteworthy was the significant down-regulation of antibody (anti-DG#29) targeting an epitope of Epstein-Barr virus nuclear antigen 1 (EBNA1). This down-regulation increased AD risk in female patients (odds ratio up to 6.6), but not in male patients. Our investigation further revealed that the down-regulation of the antibody (anti-DG#29) is associated with EBV reactivation in AD, as indicated by the analysis of EBV VCA IgG or IgM levels. Additionally, our data demonstrated that the epitope region on EBNA1 for the antibody is hidden during the EBV lytic reactivation of B95-8 cells. CONCLUSION: Our findings suggest a potential relationship of EBV in the development of AD in female. Moreover, we propose that antibodies targeting the epitope (DG#29) of EBNA1 could serve as valuable indicators of AD risk in female.

Solution structure and dynamics of C-terminal regulatory domain of Vibrio vulnificus extracellular metalloprotease.

An extracellular metalloprotease (vEP) secreted by Vibrio vulnificus ATCC29307 is a 45-kDa proteolytic enzyme that has prothrombin activation and fibrinolytic activities during bacterial infection. The action of vEP could result in clotting that could serve to protect the bacteria from the host defense machinery. Very recently, we showed that the C-terminal propeptide (C-ter100), which is unique to vEP, is involved in regulation of vEP activity. To understand the structural basis of this function of vEP C-ter100, we have determined the solution structure and backbone dynamics using multidimensional nuclear magnetic resonance spectroscopy. The solution structure shows that vEP C-ter100 is composed of eight anti-parallel ß-strands with a unique fold that has a compact ß-barrel formation which stabilized by hydrophobic and hydrogen bonding networks. Protein dynamics shows that the overall structure, including loops, is very rigid and stabilized. By structural database analysis, we found that vEP C-ter100 shares its topology with that of the collagen-binding domain of collagenase, despite low sequence homology between the two domains. Fluorescence assay reveals that vEP C-ter100 interacts strongly with iron (Fe(3+)). These findings suggest that vEP protease might recruit substrate molecules, such as collagen, by binding at C-ter100 and that vEP participates in iron uptake from iron-withholding proteins of the host cell during infection.

Bcl-2 expression suppresses mismatch repair activity through inhibition of E2F transcriptional activity.

Bcl-2 stimulates mutagenesis after the exposure of cells to DNA-damaging agents. However, the biological mechanisms of Bcl-2-mediated mutagenesis have remained largely obscure. Here we demonstrate that the Bcl-2-mediated suppression of hMSH2 expression results in a reduced cellular capacity to repair mismatches. The pathway linking Bcl-2 expression to the suppression of mismatch repair (MMR) activity involves the hypophosphorylation of pRb, and then the enhancement of the E2F-pRb complex. This is followed by a decrease in hMSH2 expression. MMR has a key role in protection against deleterious mutation accumulation and in maintaining genomic stability. Therefore, the decreased MMR activity by Bcl-2 may be an underlying mechanism for Bcl-2-promoted oncogenesis.

Diagnostic Blood Biomarkers in Alzheimer's Disease.

Potential biomarkers for Alzheimer's disease (AD) include amyloid ß1-42 (Aß1-42), t-Tau, p-Tau181, neurofilament light chain (NFL), and neuroimaging biomarkers. Their combined use is useful for diagnosing and monitoring the progress of AD. Therefore, further development of a combination of these biomarkers is essential. We investigated whether plasma NFL/Aß1-42 can serve as a plasma-based primary screening biomarker reflecting brain neurodegeneration and amyloid pathology in AD for monitoring disease progression and early diagnosis. We measured the NFL and Aß1-42 concentrations in the CSF and plasma samples and performed correlation analysis to evaluate the utility of these biomarkers in the early diagnosis and monitoring of AD spectrum disease progression. Pearson's correlation analysis was used to analyse the associations between the fluid biomarkers and neuroimaging data. The study included 136 participants, classified into five groups: 28 cognitively normal individuals, 23 patients with preclinical AD, 22 amyloid-negative patients with amnestic mild cognitive impairment, 32 patients with prodromal AD, and 31 patients with AD dementia. With disease progression, the NFL concentrations increased and Aß1-42 concentrations decreased. The plasma and CSF NFL/Aß1-42 were strongly correlated (r = 0.558). Plasma NFL/Aß1-42 was strongly correlated with hippocampal volume/intracranial volume (r = 0.409). In early AD, plasma NFL/Aß1-42 was associated with higher diagnostic accuracy than the individual biomarkers. Moreover, in preclinical AD, plasma NFL/Aß1-42 changed more rapidly than the CSF t-Tau or p-Tau181 concentrations. Our findings highlight the utility of plasma NFL/Aß1-42 as a non-invasive plasma-based biomarker for early diagnosis and monitoring of AD spectrum disease progression.

Cloning and biochemical characterization of Staphylococcus aureus type IA DNA topoisomerase comprised of distinct five domains.

DNA topoisomerases play critical roles in regulating DNA topology and are essential enzymes for cell survival. In this study, a gene encoding type IA DNA topoisomerase was cloned from Staphylococcus aureus (S. aureus) sp. strain C-66, and the biochemical properties of recombinant enzyme was characterized. The nucleotide sequence analysis showed that the cloned gene contained an open reading frame (2070 bp) that could encode a polypeptide of 689 amino acids. The cloned gene actually produced 79.1 kDa functional enzyme (named Sau-TopoI) in Escherichia coli (E. coli). Sau-TopoI enzyme purified from E. coli showed ATP-independent and Mg(2+)-dependent manners for relaxing negatively supercoiled DNA. The relaxation activity of Sau-TopoI was inhibited by camptothecin, but not by nalidixic acid and etoposide. Cleavage site mapping showed that the enzyme could preferentially bind to and cleave the sequence GGNN↓CAT (N and ↓ represent any nucleotide and cleavage site, respectively). All these results suggest that the purified enzyme is type IA DNA topoisomerase. In addition, domain mapping analysis showed that the enzyme was composed of conserved four domains (I through IV), together with a variable C-terminal region containing a unique domain V.

Electron spin multiplicities of transition-metal aromatic radicals and ions: M[C6(CH3)6] and M(+)[(C6(CH3)6] (M = Ti, V, and Co).

Determination of electron spin multiplicities of transition-metal radicals and ions challenges both experimentalists and theoreticians. In this work, we report preferred electron spin states of M[C(6)(CH(3))(6)] and M(+)(C(6)(CH(3))(6)], where M = Ti, V, and Co. The neutral radicals were formed in a supersonic metal cluster beam source, and their masses were measured with time-of-flight mass spectrometry. Precise ionization energies of the radicals and metal-ligand stretching frequencies of the ions were measured by pulsed field ionization zero electron kinetic energy spectroscopy. C-H stretching frequencies of the methyl group in the radicals were obtained by infrared-ultraviolet two-photon ionization. Electron spin multiplicities of the radicals and ions were investigated by combining the spectroscopic measurements, density functional theory, and Franck-Condon factor calculations. The preferred spin states are quintet, sextet, and quartet for the neutral Ti, V, and Co radicals, respectively; for the corresponding singly charged cations, they are quartet, quintet, and triplet. In these high-spin states, the aromatic ring remains nearly planar. This finding contrasts to the previous study of Sc(hmbz), for which low-spin states are favored, and the aromatic ring is severely bent.

High-resolution electron spectroscopy, preferential metal-binding sites, and thermochemistry of lithium complexes of polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons are model systems for studying the mechanisms of lithium storage in carbonaceous materials. In this work, Li complexes of naphthalene, pyrene, perylene, and coronene were synthesized in a supersonic metal-cluster beam source and studied by zero-electron-kinetic-energy (ZEKE) electron spectroscopy and density functional theory calculations. The adiabatic ionization energies of the neutral complexes and frequencies of up to nine vibrational modes in the singly charged cations were determined from the ZEKE spectra. The metal-ligand bond energies of the neutral complexes were obtained from a thermodynamic cycle. Preferred Li∕Li(+) binding sites with the aromatic molecules were determined by comparing the measured spectra with theoretical calculations. Li and Li(+) prefer the ring-over binding to the benzene ring with a higher π-electron content and aromaticity. Although the ionization energies of the Li complexes show no clear correlation with the size of the aromatic molecules, the metal-ligand bond energies increase with the extension of the π-electron network up to perylene, then decrease from perylene to coronene. The trends in the ionization and metal-ligand bond dissociation energies of the complexes are discussed in terms of the orbital energies, local quadrupole moments, and polarizabilities of the free ligands and the charge transfer between the metal atom and aromatic molecules.

Effect of Vibrio-Derived Extracellular Protease vEP-45 on the Blood Complement System.

Vibrio vulnificus is a pathogenic bacterium that can causes wound infections and fetal septicemia. We have reported that V. vulnificus ATCC29307 produces an extracellular zinc-metalloprotease (named vEP-45). Our previous results showed that vEP-45 can convert prothrombin to active thrombin and also activate the plasma kallikrein/kinin system. In this study, the effect of vEP-45 on the activation of the complement system was examined. We found that vEP-45 could proteolytically convert the key complement precursor molecules, including C3, C4, and C5, to their corresponding active forms (e.g., C3a, C3b, C4a, C4b, and C5a) in vitro cleavage assays. C5b production from C5 cleavage mediated by vEP-45 was not observed, whereas the level of C5a was increased in a dose-dependent manner compared to that of the non-treated control. The cleavage of the complement proteins in human plasma by vEP-45 was also confirmed via Western blotting. Furthermore, vEP-45 could convert C3 and C5 to active C3a and C5a as a proinflammatory mediator, while no cleavage of C4 was observed. These results suggest that vEP-45 can activate the complement system involved in innate immunity through an alternative pathway.

Effect of 7-Methylsulfinylheptyl Isothiocyanate on the Inhibition of Melanogenesis in B16-F1 Cells.

Skin aging, characterized by hyperpigmentation, inflammation, wrinkles, and skin cancer, is influenced by intrinsic and extrinsic factors with synergistic effects. Autophagy maintains the homeostatic balance between the degradation, synthesis, and recycling of cellular proteins and organelles, and plays important roles in several cellular and biological processes, including aging. The compound 7-methylsulfinylheptyl isothiocyanate (7-MSI) is a sulfur-containing phytochemical produced by various plants, particularly cruciferous vegetables, with reported anti-inflammatory properties and a role in pathogen defense; however, its effects on skin whitening have not been studied in detail. The purpose of this study was to observe the effects of 7-MSI on skin whitening and autophagy in cultured murine melanoma (B16-F1) cells. Western blotting was used to evaluate the impact of 7-MSI on melanogenesis-, tyrosinase-, and autophagy-associated proteins. The levels of the melanogenesis-associated protein's microphthalmia-associated transcription factor (MITF) and tyrosinase and tyrosinase-related protein-1 were decreased by treatment with 7-MSI under melanogenesis induction. Melanin synthesis also decreased by approximately 63% after treatment with 7-MSI for 73 h, compared with that non-treated controls. In addition, autophagosome formation and the expression levels of the autophagy-related proteins mTOR, p-mTOR, Beclin-1, Atg12, and LC3 were higher in 7-MSI-treated B16-F1 cells than in non-treated cells. These results indicate that 7-MSI can inhibit melanin synthesis in B16-F1 cells by suppressing melanogenesis and autophagy activation and thus can potentially be used as a novel multifunctional cosmetic agent.

Plasma contact factors as novel biomarkers for diagnosing Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the most common cause of dementia and most of AD patients suffer from vascular abnormalities and neuroinflammation. There is an urgent need to develop novel blood biomarkers capable of diagnosing Alzheimer's disease (AD) at very early stage. This study was performed to find out new accurate plasma diagnostic biomarkers for AD by investigating a direct relationship between plasma contact system and AD. METHODS: A total 101 of human CSF and plasma samples from normal and AD patients were analyzed. The contact factor activities in plasma were measured with the corresponding specific peptide substrates. RESULTS: The activities of contact factors (FXIIa, FXIa, plasma kallikrein) and FXa clearly increased and statistically correlated as AD progresses. We present here, for the first time, the FXIIa cut-off scores to as: > 26.3 U/ml for prodromal AD [area under the curve (AUC) = 0.783, p < 0.001] and > 27.2 U/ml for AD dementia (AUC = 0.906, p < 0.001). We also describe the cut-off scores from the ratios of CSF Aß1-42 versus the contact factors. Of these, the representative ratio cut-off scores of Aß1-42/FXIIa were to be: < 33.8 for prodromal AD (AUC = 0.965, p < 0.001) and < 27.44 for AD dementia (AUC = 1.0, p < 0.001). CONCLUSION: The activation of plasma contact system is closely associated with clinical stage of AD, and FXIIa activity as well as the cut-off scores of CSF Aß1-42/FXIIa can be used as novel accurate diagnostic AD biomarkers.

Two Opposing Roles of SARS-CoV-2 RBD-Reactive Antibodies in Pre-Pandemic Plasma Samples From Elderly People in ACE2-Mediated Pseudovirus Infection.

A novel coronavirus designated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged and caused an outbreak of unusual viral pneumonia. Several reports have shown that cross-reactive antibodies against SARS-CoV-2 also exist in people unexposed to this virus. However, the neutralizing activity of cross-reactive antibodies is controversial. Here, we subjected plasma samples from SARS-CoV-2-unexposed elderly Korean people (n = 119) to bead-based IgG antibody analysis. SARS-CoV-2 S1 subunit-reactive IgG antibody analysis detected positive signals in some samples (59 of 119, 49.6%). SARS-CoV-2 receptor-binding domain (RBD)-reactive antibody levels were most significantly correlated with human coronavirus-HKU1 S1 subunit-reactive antibody levels. To check the neutralizing activity of plasma samples, the SARS-CoV-2 spike pseudotype neutralizing assay was used. However, the levels of cross-reactive antibodies did not correlate with neutralizing activity. Instead, SARS-CoV-2 pseudovirus infection was neutralized by some RBD-reactive plasma samples (n = 9, neutralization ≥ 25%, P ≤ 0.05), but enhanced by other RBD-reactive plasma samples (n = 4, neutralization ≤ -25%, P ≤ 0.05). Interestingly, the blood plasma groups with enhancing and neutralizing effects had high levels of SARS-CoV-2 RBD-reactive antibodies than the plasma group that had no effect. These results suggest that some SARS-CoV-2 RBD-reactive antibodies from pre-pandemic elderly people exert two opposing functions during SARS-CoV-2 pseudovirus infection. In conclusion, preformed RBD-reactive antibodies may have two opposing functions, namely, protecting against and enhancing viral infection. Analysis of the epitopes of preformed antibodies will be useful to elucidate the underlying mechanism.

Purification of catalytically active caspase-12 and its biochemical characterization.

Caspase-12, mainly detected in endoplasmic reticulum (ER), has been suggested to play a role in ER-mediated apoptosis and inflammatory caspase activation pathway. Cleavage of the prodomain by caspase-3/-7 at the carboxyl terminus of Asp94 or m-calpain at the carboxyl terminus of Lys158 was reported to be a part of caspase-12-involved apoptosis. We biochemically characterized the prodomain-free forms of caspase-12 and the equivalent enzymes; Deltapro1(G95-D419), rev-Deltapro1[(T319-N419)-(G95-D318), a reverse form of Deltapro1] and rev-Deltapro2[(T319-N419)-(T159-D318)]. The three variants showed comparable activities which were dependent on salt concentration and pH. Auto-proteolytic cleavage was observed at two sites (carboxyl termini of Asp318 and Asp320) in Deltapro1. Constitutively active forms of caspase-12 (rev-Deltapro1 and rev-Deltapro2) could induce cell death in cells transfected with the corresponding expression vectors, but no cleavage of caspase-3, DFF45 or Bid was observed, indicating caspase-12 may mediate a distinct apoptotic pathway rather than caspase-8 or -9-mediated cell death.

Conformational isomers and isomerization of group 6 (Cr, Mo, and W) metal-bis(toluene) sandwich complexes probed by variable-temperature electron spectroscopy.

Group 6 metal (Cr, Mo, and W)-bis(toluene) sandwich complexes are synthesized in a laser-vaporization molecular beam source. Conformational isomers and isomerization of these complexes are studied by variable-temperature pulsed-field-ionization zero-electron-kinetic-energy spectroscopy and density functional theory. For Cr-bis(toluene), four rotational conformers are identified with methyl-group dihedral angles of 0, 60, 120, and 180°. The ground electronic states of these conformers are (1)A(1) (C(2v), 0°), (1)A (C(2), 60 and 120°), and (1)A(g) (C(2h), 180°) in the neutral form and (2)A(1) (C(2v), 0°), (2)A (C(2), 60 and 120°), and (2)A(g) (C(2h), 180°) in the singly charged cationic form. For Mo- and W-bis(toluene), the four rotamers are resolved into three (0, 60/120, and 180°) and two (0 and 60/120/180°) groups, respectively. For all three metal sandwiches, the most stable conformer is in the complete eclipsed configuration (0°) and has the highest ionization energy. The conversion from 60/120/180° to 0° rotamer is observed from helium to argon supersonic expansions and is more efficient for the heavier Mo and W species.

Ring deformation and pi-electron redistribution of methylbenzenes induced by metal coordination.

Scandium (Sc) complexes of p-xylene, mesitylene, and hexamethylbenzene were produced in a laser-vaporization molecular beam source and studied with pulsed-field-ionization zero-electron-kinetic-energy spectroscopy, and density functional theory. In addition, infrared-ultraviolet resonant two-photon ionization spectra were recorded for Sc(hexamethylbenzene) in the C-H stretching region. Adiabatic ionization energies and several vibrational frequencies of these complexes were obtained from the spectroscopic measurements, and electronic transitions were determined by combining the spectra with the theoretical data. The ionization energies of the three complexes decrease with increasing number of the methyl groups, whereas the metal-ligand stretching frequencies of the p-xylene and mesitylene complexes are essentially the same and slightly smaller than that of the hexamethylbenzene species. Unlike benzene, the arene ring of the methylbenzene molecules is bent and the pi-electrons are localized in a 1,4-diene fashion upon Sc coordination. The distortion of the aromatic ring is due to differential metal binding with the ring carbon atoms in the low-spin ground electronic state.

High-resolution electron spectroscopy and structures of lithium-nucleobase (adenine, uracil, and thymine) complexes.

Li complexes of adenine, uracil, and thymine were produced by laser vaporization of rods made of Li and nucleobase powders in a metal-cluster beam source and studied by pulsed-field-ionization zero-electron-kinetic-energy (ZEKE) spectroscopy and density functional theory calculations. The ZEKE measurements determined the adiabatic ionization energies of the three neutral complexes and frequencies of several vibrational modes for the metal-adenine and -uracil ions. The measured spectra were compared with spectral simulations to determine the preferred metal binding sites. For adenine, the most stable structure is formed by Li/Li(+) bidentately binding to both the N7 atom of the imidazole ring and the NH(2) group of the pyrimidine ring. For uracil and thymine, the ideal site for Li/Li(+) coordination is the O4 atom. Although it has only a small effect on the geometries of uracil and thymine, lithium coordination forces the rotation of the NH(2) group out of the adenine plane. The adiabatic ionization energies of the three complexes follow the trend of uracil (33910+/-5 cm(-1))>thymine (33386+/-5 cm(-1))>adenine (32240+/-5 cm(-1)), whereas their metal-ligand bond dissociation energies are about the same, (92-97) +/-6 kJ mol(-1). For all three complexes, the neutral bond energies are smaller than those of the corresponding ions due to a weaker electrostatic interaction and stronger electron repulsion.

Cloning, purification and characterization of a recombinant protease with novel thrombolytic activity in human plasma and rat thrombosis models.

BACKGROUND: There is a need to identify and develop novel thrombolytic agents that can directly digest fibrin clots from various biological resources. OBJECTIVE: To clone, express, purify, and characterize a recombinant protease named rvFMP capable of cleaving fibrinogen, fibrin polymer, and cross-linked fibrin in human plasma milieu and rat thrombosis model systems. RESULTS: We cloned a vFMP-encoding gene from the genomic DNA of V. furnissii KCCM41679 using polymerase chain reaction (PCR), expressed in Escherichia coli, and purified rvFMP (stands for recombinant vibrio furnissii metalloprotease). The proteolytic activity of purified rvFMP enzyme could be clearly inhibited by 1,10-phenanthroline and ethylene glycol tetraacetic acid, but not by diisopropyl fluorophosphate, suggesting that it can be a typical metalloprotease. rvFMP showed an effective proteolytic activity in cleaving cross-linked fibrins in human plasma milieu. Remarkably, rvFMP exhibited a clear thrombolytic activity in rat thrombosis models such as ferric chloride-exposed rat carotid artery and carrageenan-treated rat tail. However, rvFMP (1.5 mg/kg) evoked no internal bleeding and also showed no lethal effect in mice. The recombinant enzyme also showed no cytotoxicity and had an inability to induce tumour necrosis factor-α (TNF-α) in Raw264.7 cells. CONCLUSION: rvFMP can be a candidate enzyme capable of being developed as a novel direct-acting thrombolytic agent.

Comparison of Two Analytical Platforms in Cerebrospinal Fluid Biomarkers for the Classification of Alzheimer's Disease Spectrum with Amyloid PET Imaging.

BACKGROUND: Cerebrospinal fluid (CSF) amyloid-ß1-42 (Aß1-42), total tau protein (t-Tau), and phosphorylated Tau (p-Tau) are ATN biomarkers for Alzheimer's disease (AD) and reflect pathogenic changes in the brain. CSF biomarkers of AD are considered for inclusion in the diagnostic criteria for research and clinical purposes to reduce the uncertainty of clinical diagnosis and to distinguish among AD stages. OBJECTIVE: This study aims to compare two commercially available analytical platforms with respect to accuracy and the potential for early diagnosis of AD. METHODS: A total of 211 CSF samples from healthy control (HC) and AD subjects were analyzed using two analytical platforms, INNOTEST ELISA and INNOBIA AlzBio3 xMAP kits. The accuracy of diagnosis and AUC values distinguishing study groups were compared between the two analytical platforms. RESULTS: The absolute values for Aß1-42, t-Tau, and p-Tau181 levels differed between the two platforms. The Aß1-42 levels decreased, while t-Tau and p-Tau levels increased according to the AD stages. The AUC of Aß1-42 and t-Tau, which distinguish the early stages of AD (preclinical and prodromal AD), were similar between the two platforms, whereas there were significant differences in p-Tau AUC values. CSF p-Tau using the INNOBIA was highly accurate for distinguishing both preclinical AD (AUC = 0.826, cut-off score≥38.89) and prodromal AD (AUC = 0.862, cut-off score≥41.88) from HC. CONCLUSION: The accuracy of CSF p-Tau levels in the preclinical and prodromal AD is higher for the INNOBIA than the INNOTEST, and the early stage AD can be accurately distinguished from HC.