Synthetic inhibitors of matrix metalloproteinases (MMPs) designed previously as well as tissue inhibitors of metalloproteinases (TIMPs) lack enzyme selectivity, which has been a major obstacle for developing the inhibitors into safe and effective MMP-targeted drugs. Here we designed a fusion protein named APP-IP-TIMP-2, in which the ten amino acid residues sequence of APP-derived MMP-2-selective inhibitory peptide (APP-IP) is added to the N terminus of TIMP-2. The APP-IP and TIMP-2 regions of the fusion protein are designed to interact with the active site and the hemopexin-like domain of MMP-2, respectively. The reactive site of the TIMP-2 region, which has broad specificity against MMPs, is blocked by the APP-IP adduct. The recombinant APP-IP-TIMP-2 showed strong inhibitory activity toward MMP-2 (Ki(app) = 0.68 pM), whereas its inhibitory activity toward MMP-1, MMP-3, MMP-7, MMP-8, MMP-9 or MT1-MMP was six orders of magnitude or more weaker (IC(50) > 1 μM). The fusion protein inhibited the activation of pro-MMP-2 in the concanavalin A-stimulated HT1080 cells, degradation of type IV collagen by the cells, and the migration of the stimulated cells. As compared with the decapeptide APP-IP (t1/2 = 30 min), APP-IP-TIMP-2 (t1/2 > 96 h) showed much longer half-life in cultured tumor cells. Therefore, the fusion protein may be a useful tool to evaluate contributions of proteolytic activity of MMP-2 in various pathophysiological processes. It may also be developed as an effective anti-tumor drug that has restricted side effects.
- Current opinion in clinical nutrition and metabolic care
- Published over 7 years ago
Here, we discuss the recent data on the role of different N-methyl D-aspartate receptor (NMDAR) coagonists, D-serine and glycine, in regulating NMDAR activity and neurotoxicity.
The N-methyl-D-aspartate receptor (NMDAR) is crucial for pain-related behaviors. D-Serine is synthesized from L-serine by serine racemase (SR) and modulates NMDAR functions by acting as an agonist at the glycine-binding site. We analyzed noxious stimulus-induced ultrasonic vocalization and locomotor activity in the open-field test using SR knockout (SR-KO) mice to examine the role of endogenous D-serine in mammalian behaviors. SR-KO mice emitted less ultrasonic vocalization after noxious stimulation (VAS) than wild-type (WT) mice. The locomotor activity of WT mice decreased with repeated daily exposures to the open field, whereas that of SR-KO mice remained unchanged. VAS was significantly enhanced during arthritis in WT mice, whereas it was not enhanced during arthritis in SR-KO mice. These results indicate that mice lacking the ability to produce D-serine endogenously in the brain differ from normal mice with respect to the chronic pain-induced behavioral changes.
AIM: To characterize novel multiple bacteriocins produced by Lactobacillus sakei D98. METHODS AND RESULTS: Lactobacillus sakei D98 isolated from Shubo (rice malt) produced at least three bacteriocins. Using three purification steps, three novel antimicrobial peptides termed sakacin D98a, sakacin D98b, and sakacin D98c were purified from the culture supernatant. Amino acid and DNA sequencing analysis revealed that the sakacins D98a, D98b, and D98c are novel class IIa-like or class IId bacteriocins. In particular, sakacin D98b has a variant pediocin box sequence, YANGVXC (with Ala instead of Gly), and a different location for the disulfide bridge (Cys(11) and Cys(18) ) from that found in other class IIa bacteriocins. CONCLUSIONS: Three novel bacteriocins were identified from Lactobacillus sakei D98. Their antimicrobial spectra and intensities indicate that these sakacins would have different modes of action. In addition, sakacin D98b showed low inhibitory activity against Listeria, probably due to the differences in amino acids and position of the disulfide bridge compared to the other class IIa bacteriocins. SIGNIFICANCE AND IMPACT OF STUDY: Sakacins D98a and D98c are novel bacteriocins belonging to class IId bacteriocins. On the other hand, sakacin D98b, a class IIa-like bacteriocin, has a unique internal structure and activity spectrum. This article is protected by copyright. All rights reserved.
The N-methyl-d-aspartate receptor (NMDAR) coagonists glycine, d-serine and l-proline play crucial roles in NMDAR-dependent neurotransmission and are associated with a range of neuropsychiatric disorders. We conducted the first genome-wide association study of concentrations of these coagonists and their enantiomers in plasma and cerebrospinal fluid (CSF) of human subjects from the general population (N=414). Genetic variants at chromosome 22q11.2, located in and near PRODH (proline dehydrogenase), were associated with l-proline in plasma (β=0.29; P=6.38 × 10(-10)). The missense variant rs17279437 in the proline transporter SLC6A20 was associated with l-proline in CSF (β=0.28; P=9.68 × 10(-9)). Suggestive evidence of association was found for the d-serine plasma-CSF ratio at the d-amino-acid oxidase (DAO) gene (β=-0.28; P=9.08 × 10(-8)), whereas a variant in SRR (that encodes serine racemase and is associated with schizophrenia) constituted the most strongly associated locus for the l-serine to d-serine ratio in CSF. All these genes are highly expressed in rodent meninges and choroid plexus, anatomical regions relevant to CSF physiology. The enzymes and transporters they encode may be targeted to further construe the nature of NMDAR coagonist involvement in NMDAR gating. Furthermore, the highlighted genetic variants may be followed up in clinical populations, for example, schizophrenia and 22q11 deletion syndrome. Overall, this targeted metabolomics approach furthers the understanding of NMDAR coagonist concentration variability and sets the stage for non-targeted CSF metabolomics projects.Molecular Psychiatry advance online publication, 10 February 2015; doi:10.1038/mp.2014.190.
Energy minima of the 20 natural amino acids (capped by a peptide bond at both the N- and C -termini, CH3-C(=O)-N(H)-(H)Cα®-C(=O)-N(H)-CH3) were obtained by ab initio geometry optimisation. Starting with a large number of minima, quickly generated by MarvinView, geometry optimisation at the HF/6-31G(d,p) level of theory reduced the number of minima, followed by further optimisation at the B3LYP/apc-1 and MP2/cc-pVDZ levels, which caused some minima to disappear and some stable minima to migrate on the Ramachandran map. There is a relation between the number of minima and the size and the flexibility of the side-chain. The energy minima of the 20 amino acids are mainly located in the regions of βL, γL, δL, and αL of the Ramachandran map. Multipole moments of atoms occurring in the fragment [-NH-Cα-C(=O)-] common to all 20 amino acids were calculated at the three levels of theory mentioned above. The near parallelism in behaviour of these moments between levels of theory is beneficial towards estimating moments with the more expensive B3LYP and MP2 methods, from data calculated with the cheaper HF method. Finally, we explored the transferability of properties between different amino acids: the bond length and angles of the common fragment [-NH-Cα(HαCβ)-C'(=O)-] in all amino acids except Gly and Pro. All bond lengths are highly transferable between different amino acids and the standard deviations are small.
The objectives of this study are to systematically assess the bioactive substances and overall antioxidant capacities of commercially fermented soy products and to find the relationships between the presence of beneficial components in different types of soybean fermented products. The results show that phenolic profiles increased significantly after fermentation as compared with raw yellow soybeans. Among all the samples, the douchi and fermented black bean sauce had the highest detected antioxidant profiles. Even though the total isoflavone content was reduced in fermented soybean products (794.84 μg/g on average) as compared with raw yellow soybeans (3477.6 μg/g), there was an obvious trend of conversion of the glucoside form in raw soybeans into the aglycone-form isoflavones in the fermented soybean products. The highest daidzein and genistein values were found in the “Yangfan” black bean douchi, i.e. 860.3 μg/g and 1025.9 μg/g, respectively. The amounts of essential amino acids also were improved in most fermented soybean products. The douchi and black bean fermented products are recommended for consumption due to their abundant bioactive substances.
Chronic neuropathic pain is a debilitating condition that remains difficult to treat. Diminished synaptic inhibition by GABA and glycine and increased NMDA receptor (NMDAR) activity in the spinal dorsal horn are key mechanisms underlying neuropathic pain. However, the reciprocal relationship between synaptic inhibition and excitation in neuropathic pain is unclear. Here, we show that intrathecal delivery of K(+)-Cl(-) cotransporter-2 (KCC2) using lentiviral vectors produces a complete and long-lasting reversal of pain hypersensitivity induced by nerve injury. KCC2 gene transfer restores Cl(-) homeostasis disrupted by nerve injury in both spinal dorsal horn and primary sensory neurons. Remarkably, restoring Cl(-) homeostasis normalizes both presynaptic and postsynaptic NMDAR activity increased by nerve injury in the spinal dorsal horn. Our findings indicate that nerve injury recruits NMDAR-mediated signaling pathways through the disruption of Cl(-) homeostasis in spinal dorsal horn and primary sensory neurons. Lentiviral vector-mediated KCC2 expression is a promising gene therapy for the treatment of neuropathic pain.
Glyphosate, an herbicidal derivative of the amino acid glycine, was introduced to agriculture in the 1970s. Glyphosate targets and blocks a plant metabolic pathway not found in animals, the shikimate pathway, required for the synthesis of aromatic amino acids in plants. After almost forty years of commercial use, and multiple regulatory approvals including toxicology evaluations, literature reviews, and numerous human health risk assessments, the clear and consistent conclusions are that glyphosate is of low toxicological concern, and no concerns exist with respect to glyphosate use and cancer in humans. This manuscript discusses the basis for these conclusions. Most toxicological studies informing regulatory evaluations are of commercial interest and are proprietary in nature. Given the widespread attention to this molecule, the authors gained access to carcinogenicity data submitted to regulatory agencies and present overviews of each study, followed by a weight of evidence evaluation of tumor incidence data. Fourteen carcinogenicity studies (nine rat and five mouse) are evaluated for their individual reliability, and select neoplasms are identified for further evaluation across the data base. The original tumor incidence data from study reports are presented in the online data supplement. There was no evidence of a carcinogenic effect related to glyphosate treatment. The lack of a plausible mechanism, along with published epidemiology studies, which fail to demonstrate clear, statistically significant, unbiased and non-confounded associations between glyphosate and cancer of any single etiology, and a compelling weight of evidence, support the conclusion that glyphosate does not present concern with respect to carcinogenic potential in humans.
Mutations in the N-methyl-D-aspartate receptor (NMDAR) gene GRIN2A cause epilepsy-aphasia syndrome (EAS), a spectrum of epileptic, cognitive and language disorders. Using bioinformatic and patient data we shortlisted 10 diverse missense mutations for characterisation. We used high-throughput calcium-flux assays and patch clamp recordings of transiently transfected HEK-293 cells for electrophysiological characterization, and Western blotting and confocal imaging to assay expression and surface trafficking. Mutations P79R, C231Y, G483R and M705V caused a significant reduction in glutamate and glycine agonist potency, whilst D731N was non-responsive. These mutants, along with E714K, also showed significantly decreased total protein levels and trafficking to the cell surface, whilst C436R was not trafficked at all. Crucially this reduced surface expression did not cause the reduced agonist response. We were able to rescue the phenotype of P79R, C231Y, G483R and M705V after treatment with a GluN2A-selective positive allosteric modulator. With our methodology we were not able to identify any functional deficits in mutations I814T, D933N and N976S located between the glutamate-binding domain and C-terminus. We show GRIN2A mutations affect the expression and function of the receptor in different ways. Careful molecular profiling of patients will be essential for future effective personalised treatment options.