The effect of tranexamic acid on synovium of patients undergoing arthroplasty and anterior cruciate ligament reconstruction surgery.

Preoperative hemorrhage can be reduced using anti-fibrinolytic medicine tranexamic acid (TXA). During surgical procedures, local administration is being used more and more frequently, either as an intra-articular infusion or as a perioperative rinse. Serious harm to adult soft tissues can be detrimental to the individual since they possess a weak ability for regeneration. Synovial tissues and primary fibroblast-like synoviocytes (FLS) isolated from patients were examined using TXA treatment in this investigation. FLS is obtained from rheumatoid arthritis (RA), osteoarthritis (OA), and anterior cruciate ligament (ACL)-ruptured patients. The in vitro effect of TXA on primary FLS was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assays for cell death, annexin V/propidium iodide (PI) staining for apoptotic rate, real-time PCR for p65 and MMP-3 expression, and enzyme-linked immunosorbent assay (ELISA) for IL-6 measurement. MTT assays revealed a significant decrease in cell viability in FLS of all groups of patients following treatment with 0.8-60 mg/ml of TXA within 24 h. There was a significant increase in cell apoptosis after 24 h of exposure to TXA (15 mg/ml) in all groups, especially in RA-FLS. TXA increases the expression of MMP-3 and p65 expression. There was no significant change in IL-6 production after TXA treatment. An increase in receptor activator of nuclear factor kappa-Β ligand (RANK-L) production was seen only in RA-FLS. This study demonstrates that TXA caused significant synovial tissue toxicity via the increase in cell death and elevation of inflammatory and invasive gene expression in FLS cells.

Tranexamic acid may promote melanocores clustering in keratinocytes through upregulation of Rab5b.

Tranexamic acid (TXA) is a promising therapeutic agent in melasma that can act on multiple pathophysiologic mechanisms of melasma. However, it is unclear whether TXA affects melanin in keratinocytes. To explore the effect of TXA on melanocores in keratinocytes. The melanocore-incorporated keratinocytes were constructed by co-incubating normal human epidermal keratinocytes (NHEK) with melanocores. After being treated with TXA, autophagy- and melanin-related protein expressions were detected. Then, transcriptome sequencing was used to compare the genetic changes in melanocore-incorporated keratinocytes before and after TXA treatment and further verified the differentially expressed genes. At the same time, the distribution of melanocores in human keratinocytes was observed by transmission electron microscopy. We found that TXA does not promote melanin degradation in primary keratinocytes by inducing autophagy. Protein transport and intracellular protein transport-related genes were enriched after TXA treatment, and Rab5b was significantly upregulated. Transmission electron microscopy showed that the percentage of melanocores distributed in clusters increased after treatment with TXA, which was reduced after Rab5b silencing. In addition, results suggested that melanocores could colocalize with Rab5b and lysosome-associated membrane protein1 (LAMP1). Our study found that Rab5b may be involved in the melanocore distribution in keratinocytes. TXA may promote the clustering distribution of endocytic melanocores through upregulation of Rab5b, representing a potential mechanism of TXA treatment against melasma.

Multiplexed Organelles Portrait Barcodes for Subcellular MicroRNA Array Detection in Living Cells.

Multiplexed profiling of microRNAs' subcellular expression and distribution is essential to understand their spatiotemporal function information, but it remains a crucial challenge. Herein, we report an encoding approach that leverages combinational fluorescent dye barcodes, organelle targeting elements, and an independent quantification signal, termed Multiplexed Organelles Portrait Barcodes (MOPB), for high-throughput profiling of miRNAs from organelles. The MOPB barcodes consist of heterochromatic fluorescent dye-loaded shell-core mesoporous silica nanoparticles modified with organelle targeting peptides and molecular beacon detection probes. Using mitochondria and endoplasmic reticulum as models, we encoded four Cy3/AMCA ER-MOPB and four Cy5/AMCA Mito-MOPB by varying the Cy3 and Cy5 intensity for distinguishing eight organelles' miRNAs. Significantly, the MOPB strategy successfully and accurately profiled eight subcellular organelle miRNAs' alterations in the drug-induced Ca2+ homeostasis breakdown. The approach should allow more widespread application of subcellular miRNAs and multiplexed subcellular protein biomarkers' monitoring for drug discovery, cellular metabolism, signaling transduction, and gene expression regulation readout.

Tranexamic acid integrated into platelet-rich fibrin produces a robust and resilient antihemorrhagic biological agent: a human cohort study.

OBJECTIVE: To investigate the incorporation of the antifibrinolytic agent tranexamic acid (TA) during platelet-rich fibrin (PRF) formation to produce a robust fibrin agent with procoagulation properties. STUDY DESIGN: Blood from healthy volunteers was collected. Into 3 tubes, TA was immediately added in 1-mL, 0.4-mL, and 0.2-mL volumes, and the fourth tube was without additions. After PRF preparation, the clots were weighed in their raw (clot) and membrane forms. PRF physical properties were analyzed using a universal testing system (Instron). Protein and TA levels in the PRF were analyzed using a bicinchoninic acid assay and a ferric chloride assay, respectively. RESULTS: The addition of TA to PRF led to a robust weight compared with sham control. PRF weight was greater in females in all tested groups. The addition of TA also led to greater resilience to tears, especially at 1-mL TA addition to the blood. Furthermore, TA addition led to a greater value of total protein within the PRF and entrapment of TA in the PRF. CONCLUSIONS: Addition of TA to a PRF preparation leads to robust PRF with greater protein levels and the amalgamation of TA into the PRF. Such an agent may enhance the beneficial properties of PRF and attribute procoagulation properties to it.

Tranexamic acid inhibits melanogenesis partially via stimulation of TGF-ß1 expression in human epidermal keratinocytes.

Oral tranexamic acid (TA) has been an effective treatment for melasma with unclear mechanism. The present study aimed to demonstrate the effect of TA on melanogenesis via regulation of TGF-ß1 expression in keratinocytes. We firstly determined the expression level of TGF-ß1 in TA-treated keratinocyte-conditioned medium (KCM). Then, the mRNA and protein levels of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR) and tyrosinase-related protein 1 (TRP-1) of human epidermal melanocytes (NHEMs) in the presence of TA-treated KCM were evaluated via RT-PCR and western blot analysis. Moreover, melanin content and tyrosinase activity were quantified. TGF-ß1 gene was knocked down by small interfering RNA (siRNA) in keratinocytes. The mRNA and protein levels of TGF-ß1 in keratinocytes were significantly increased after TA treatment. Melanin contents, tyrosinase activity, protein and mRNA levels of TYR, MITF and TRP-1 were downregulated in NHEMs in the presence of TA-treated KCM. Knockdown of TGF-ß1 in keratinocytes could attenuate the inhibitory effect of TA-treated KCM on melanogenesis. TA could stimulate TGF-ß1 expression in keratinocytes, which further inhibits melanogenesis through the paracrine signalling.

Application of a Thermosensitive In Situ Gel of Chitosan-Based Nasal Spray Loaded with Tranexamic Acid for Localised Treatment of Nasal Wounds.

The integrity of the nasal epithelium plays a crucial role in the airway defence mechanism. The nasal epithelium may be injured as a result of a large number of factors leading to nose bleeds, also known as epistaxis. However, local measures commonly used to treat epistaxis and improve wound healing present several side effects and patient discomfort. Hence, this study aims to address some of these drawbacks by developing a new formulation for nasal epithelial wound healing. Chitosan, a biodegradable and biocompatible polymer, was used to develop a thermosensitive nasal formulation for the delivery of tranexamic acid (TXA), one of the most effective pharmacological options to control bleeding with cost and tolerability advantages. The in situ gelation properties of the formulation upon administration in the nasal cavity were investigated in terms of gelation time and temperature. It was found that the developed formulation can undergo rapid liquid-to-gel phase change within approximately 5 min at 32°C, which is well within the human nasal cavity temperature range. The spray pattern, deposition and droplet size generated by the nasal spray was also characterised and were found to be suitable for nasal drug delivery. It was also observed that the in situ gelation of the formulation prevent nasal runoff, while the majority of drug deposited mainly in the anterior part of the nose with no lung deposition. The developed formulation was shown to be safe on human nasal epithelium and demonstrated six times faster wound closure compared to the control TXA solution.

Comparison of Cytotoxic Effects of Intra-Articular Use of Tranexamic Acid versus Epinephrine on Rat Cartilage.

BACKGROUND Adequate visualization is known to be essential to perform arthroscopic procedures effectively and efficiently. We hypothesized that tranexamic acid may be considered as an alternative agent to reduce intra-articular bleeding during arthroscopic procedures, after comparing its potential chondrotoxicity with that of epinephrine. MATERIAL AND METHODS Seventy-two rats were randomized into 3 groups with 24 rats each. The injections were performed in the right knees, as follows: Group 1: 0.25 mL of tranexamic acid solution, Group 2: 0.25 mL of epinephrine solution, and Group 3: 0.25 mL of 0.9% saline, serving as control. One week after the injections, the animals were euthanized. Samples were evaluated histologically based on the Osteoarthritis Research Society International (OARSI) Histopathology Grading and Staging System and the "live/dead" staining technique to determine chondrocyte viability. RESULTS Comparison of epinephrine and tranexamic acid revealed significantly higher OARSI scores in the epinephrine group (epinephrine: 3.42±1.31, TA: 0.92±0.90; P<0.001). The most significant difference between the 2 groups was in the number of joints diagnosed with OARSI grade III. The percentage of viability was significantly higher in the tranexamic acid group when compared with the epinephrine group (tranexamic acid: 79.74±3.343; epinephrine: 63.81±1.914; P<0.05). CONCLUSIONS Based on the histologic parameters and chondrocyte viability, tranexamic acid is less cytotoxic than epinephrine in rat chondrocytes at the doses typically used in irrigation fluid, and may be a good alternative to epinephrine in arthroscopic surgery.

Volatile Decay Products in Breath During Peritonitis Shock are Attenuated by Enteral Blockade of Pancreatic Digestive Proteases.

There is a need to develop markers for early detection of organ failure in shock that can be noninvasively measured at point of care. We explore here the use of volatile organic compounds (VOCs) in expired air in a rat peritonitis shock model. Expired breath samples were collected into Tedlar gas bags and analyzed by standardized gas chromatography. The gas chromatograms were digitally analyzed for presence of peak amounts over a range of Kovach indices. Following the induction of peritonitis, selected volatile compounds were detected within about 1 h, which remained at elevated amounts over a 6 h observation period. These VOCs were not present in control animals without peritonitis. Comparisons with know VOCs indicate that they include 1,4-diaminobutane and trimethylamine N-oxide. When pancreatic digestive proteases were blocked with tranexamic acid in the intestine and peritoneum, a procedure that serves to reduce organ failure in shock, the amounts of VOCs in the breath decreased spontaneously to control values without peritonitis. These results indicate that peritonitis shock is accompanied by development of volatile organic compounds that may be generated by digestive enzymes in the small intestine. VOCs may serve as indicators for detection of early forms of autodigestion by digestive proteases.

A Novel Chemical Enhancer Approach for Transdermal Drug Delivery with C17-Monoglycerol Ester Liquid Crystal-forming Lipid.

Transdermal administration of drugs represents an excellent alternative to conventional pharmaceutical dosage forms. However, insufficient penetration of the active pharmaceutical substance through the skin is a common problem. Thus, in the present study we evaluated the skin permeation enhancing ability of liquid crystal (LC) topical formulations. A recently developed LC-forming lipid, C17- monoglycerol ester (MGE), was evaluated and compared with glycerol monoolate (GMO), which is considered as the gold standard for LC formulations. We initially prepared LC formulations containing drugs with different physiochemical properties (tranexamic acid [TXA], 4-methoxy-salicylic acid [4-MS], catechin [CC], and calcein [Cal]), and confirmed the LC phase structures in the prepared formulations using a polarizing light microscope and a small-angle X-ray scattering (SAXS). The physicochemical properties of these formulations were also assessed using a viscometer and a zetasizer. The release rate of the drugs from the LC formulations was determined using a dialysis release method. The skin penetration-enhancing ability of LC formulations was also investigated in an in vitro skin permeation study. The results showed that both MGE- and GMO-LC-forming lipids shared the same behavior in terms of their birefringence indexes, LC phase structures, particle sizes, and zeta potentials. Both the MGE- and GMO-LC formulations managed to improve the skin permeation for various drugs with a range of physiochemical properties. However, MGE formulations showed lower viscosity, faster drug release rate, and better skin penetration-enhancing ability than GMO formulations, strongly suggesting that the low viscosity of MGELC-forming lipids might influence drug diffusivity and permeability through the skin. The present MGELC formulation might be utilized as a promising new topical formulation for therapeutic drugs and cosmetic ingredients.

α-Enolase Causes Proinflammatory Activation of Pulmonary Microvascular Endothelial Cells and Primes Neutrophils Through Plasmin Activation of Protease-Activated Receptor 2.

UNLABELLED: Proinflammatory activation of vascular endothelium leading to increased surface expression of adhesion molecules and neutrophil (PMN) sequestration and subsequent activation is paramount in the development of acute lung injury and organ injury in injured patients. We hypothesize that α-enolase, which accumulates in injured patients, primes PMNs and causes proinflammatory activation of endothelial cells leading to PMN-mediated cytotoxicity. METHODS: Proteomic analyses of field plasma samples from injured versus healthy patients were used for protein identification. Human pulmonary microvascular endothelial cells (HMVECs) were incubated with α-enolase or thrombin, and intercellular adhesion molecule-1 surface expression was measured by flow cytometry. A two-event in vitro model of PMN cytotoxicity HMVECs activated with α-enolase, thrombin, or buffer was used as targets for lysophosphatidylcholine-primed or buffer-treated PMNs. The PMN priming activity of α-enolase was completed, and lysates from both PMNs and HMVECs were immunoblotted for protease-activated receptor 1 (PAR-1) and PAR-2 and coprecipitation of α-enolase with PAR-2 and plasminogen/plasmin. RESULTS: α-Enolase increased 10.8-fold in injured patients (P < 0.05). Thrombin and α-enolase significantly increased intercellular adhesion molecule-1 surface expression on HMVECs, which was inhibited by antiproteases, induced PMN adherence, and served as the first event in the two-event model of PMN cytotoxicity. α-Enolase coprecipitated with PAR-2 and plasminogen/plasmin on HMVECs and PMNs and induced PMN priming, which was inhibited by tranexamic acid, and enzymatic activity was not required. CONCLUSIONS: α-Enolase increases after injury and may activate pulmonary endothelial cells and prime PMNs through plasmin activity and PAR-2 activation. Such proinflammatory endothelial activation may predispose to PMN-mediated organ injury.

Tranexamic acid overdosage-induced generalized seizure in renal failure.

We report a 45-year-old lady with chronic kidney disease stage 4 due to chronic tubulointerstial disease. She was admitted to our center for severe anemia due to menorrhagia and deterioration of renal function. She was infused three units of packed cells during a session of hemodialysis. Tranexamic acid (TNA) 1 g 8-hourly was administered to her to control bleeding per vaginum. Two hours after the sixth dose of TNA, she had an episode of generalized tonic clonic convulsions. TNA was discontinued. Investigations of the patient revealed no biochemical or structural central nervous system abnormalities that could have provoked the convulsions. She did not require any further dialytic support. She had no further episodes of convulsion till dis-charge and during the two months of follow-up. Thus, the precipitating cause of convulsions was believed to be an overdose of TNA.

Protein hydrolysate-induced cholecystokinin secretion from enteroendocrine cells is indirectly mediated by the intestinal oligopeptide transporter PepT1.

Dietary protein is a major stimulant for cholecystokinin (CCK) secretion by the intestinal I cell, however, the mechanism by which protein is detected is unknown. Indirect functional evidence suggests that PepT1 may play a role in CCK-mediated changes in gastric motor function. However, it is unclear whether this oligopeptide transporter directly or indirectly activates the I cell. Using both the CCK-expressing enteroendocrine STC-1 cell and acutely isolated native I cells from CCK-enhanced green fluorescent protein (eGFP) mice, we aimed to determine whether PepT1 directly activates the enteroendocrine cell to elicit CCK secretion in response to oligopeptides. Both STC-1 cells and isolated CCK-eGFP cells expressed PepT1 transcripts. STC-1 cells were activated, as measured by ERK(1/2) phosphorylation, by both peptone and the PepT1 substrate Cefaclor; however, the PepT1 inhibitor 4-aminomethyl benzoic acid (AMBA) had no effect on STC-1 cell activity. The PepT1-transportable substrate glycyl-sarcosine dose-dependently decreased gastric motility in anesthetized rats but had no affect on activation of STC-1 cells or on CCK secretion by CCK-eGFP cells. CCK secretion was significantly increased in response to peptone but not to Cefaclor, cephalexin, or Phe-Ala in CCK-eGFP cells. Taken together, the data suggest that PepT1 does not directly mediate CCK secretion in response to PepT1 specific substrates. PepT1, instead, may have an indirect role in protein sensing in the intestine.

Solution structure and functional characterization of human plasminogen kringle 5.

The ligand binding properties of the kringle 5 (K5) domain of human plasminogen have been investigated via intrinsic tryptophan fluorescence. The oleic acid (OA) affinity for K5 was quantified, yielding an association constant K(a) approximately 2.08 x 10(4) mM(-1). Simultaneously, it was determined that OA and trans-4-(aminomethyl)cyclohexanecarboxylic acid (AMCHA) (K(a) approximately 50 mM(-1)) compete for binding to K5. The solution structure of K5 in the presence of 11 mM AMCHA was solved via NMR spectroscopy (protein heavy atom RMSD approximately 0.93 +/- 0.12 A). The AMCHA binding site was localized via (1)H/(15)N chemical shift perturbation mapping assisted by in silico docking. We have found that AMCHA binds at the canonical kringle lysine binding site (LBS), structured by the Pro54-Gly60 segment plus the neighboring Phe36, Thr37, Trp62, Leu71, and Tyr72 residues. The segment 30-42, encompassing LBS residues, appears to be endowed with a higher degree of structural flexibility as suggested by the relatively lower value of S(2), the generalized order parameter, consistent with a higher backbone heavy atom RMSD of approximately 1.22 A (vs 0.84 A overall) between the two monomeric units in the crystal unit cell, of potential significance for ligand binding. OA was found to perturb the same area of the protein, namely, the LBS, as well as Tyr74. Combined with previous studies, the observation of OA binding expands the range of ligands that interact with kringle 5 while it widens the scope of potential biological functions for kringle domains.

Recombinant activated factor VII and tranexamic acid are haemostatically effective during major surgery in factor XI-deficient patients with inhibitor antibodies.

One-third of patients with severe factor XI (FXI) deficiency caused by homozygosity for null alleles develop inhibitor antibodies following exposure to plasma. Haemostasis during surgery is achievable in such patients by recombinant activated factor VII (rFVIIa) at doses used in haemophilia A patients with an inhibitor to FVIII. However, thrombosis has occurred in three of 12 such patients. In this study we discerned whether low-dose rFVIIa would secure haemostasis and cause no thrombosis in patients with severe FXI deficiency and an inhibitor during surgery. In vitro, a very low concentration of rFVIIa (0.24 microg/ml) induced thrombin generation in FXI-deficient plasma quite similarly to 1.9 microg/ml (a concentration that is achieved in patients with haemophilia A and inhibitor after infusion of 80 microg/kg). Based on this finding, a protocol was designed for four patients with severe FXI deficiency and an inhibitor or immunoglobulin A deficiency who underwent five major surgical procedures. This included administration of tranexamic acid from two hours before surgery until seven to 14 days after, and single infusion of low-dose rFVIIa. No excessive bleeding or thrombosis were observed. In conclusion, a single low dose of rFVIIa and tranexamic acid secure normal haemostasis in patients with severe FXI deficiency who can not receive blood products.

Two families of synthetic peptides that enhance fibrin turbidity and delay fibrinolysis by different mechanisms.

When fibrin clots are formed in vitro in the presence of certain positively charged peptides, the turbidity is enhanced and fibrinolysis is delayed. Here we show that these two phenomena are not always linked and that different families of peptides bring about the delay of lysis in different ways. In the case of intrinsically adhesive peptides corresponding to certain regions of the fibrinogen gammaC and betaC domains, even though these peptides bind to fibrin(ogen) and enhance turbidity, the delay in lysis is mainly due to direct inhibition of plasminogen activation. In contrast, for certain pentapeptides patterned on fibrin B knobs, the delay in lysis is a consequence of how fibrin units assemble. On their own, these B knob surrogates can induce the gelation of fibrinogen molecules. The likely cause of enhanced clot turbidity and delay in fibrinolysis was revealed by a crystal structure of the D-dimer from human fibrinogen cocrystallized with GHRPYam, the packing of which showed the direct involvement of the ligand tyrosines in antiparallel betaC-betaC interactions.

Immunohistochemical localization of aspartoacylase in the rat central nervous system.

Aspartoacylase (ASPA; EC 3.5.1.15) catalyzes deacetylation of N-acetylaspartate (NAA) to generate free acetate in the central nervous system (CNS). Mutations in the gene coding ASPA cause Canavan disease (CD), an autosomal recessive neurodegenerative disease that results in death before 10 years of age. The pathogenesis of CD remains unclear. Our working hypothesis is that deficiency in the supply of the NAA-derived acetate leads to inadequate lipid/myelin synthesis during development, resulting in CD. To explore the localization of ASPA in the CNS, we used double-label immunohistochemistry for ASPA and several cell-specific markers. A polyclonal antibody was generated in rabbit against mouse recombinant ASPA, which reacted with a single band (approximately 37 kD) on Western blots of rat brain homogenate. ASPA colocalized throughout the brain with CC1, a marker for oligodendrocytes, with 92-98% of CC1-positive cells also reactive with the ASPA antibody. Many cells were labeled with ASPA antibodies in white matter, including cells in the corpus callosum and cerebellar white matter. Relatively fewer cells were labeled in gray matter, including cerebral cortex. No astrocytes were labeled for ASPA. Neurons were unstained in the forebrain, although small numbers of large reticular and motor neurons were faintly to moderately stained in the brainstem and spinal cord. Many ascending and descending neuronal fibers were moderately stained for ASPA in the medulla and spinal cord. Microglial-like cells showed faint to moderate staining with the ASPA antibodies throughout the brain by the avidin/biotin-peroxidase detection method, and colocalization studies with labeled lectins confirmed their identity as microglia. The predominant immunoreactivity in oligodendrocytes is consistent with the proposed role of ASPA in myelination, supporting the case for acetate supplementation as an immediate and inexpensive therapy for infants diagnosed with CD.

Enzymatic preparation of D-beta-acetylthioisobutyric acid and cetraxate hydrochloride using a stereo- and/or regioselective hydrolase, 3,4-dihydrocoumarin hydrolase from Acinetobacter calcoaceticus.

3,4-Dihydrocoumarin hydrolase (DCH) from Acinetobacter calcoaceticus F46, which was previously found on screening for aromatic lactone-hydrolyzing enzymes, catalyzes the hydrolysis of several linear esters. The substrate specificity of the enzyme toward linear esters was quite characteristic, i.e., (1) it was specific toward methyl esters, (2) it recognized the configuration at the 2-position, and (3) it hydrolyzed diesters to monoesters. DCH hydrolyzed the methyl esters of beta-acetylthioisobutyrate and cetraxate. The products of these reactions were identified as D-beta-acetylthioisobutyrate and cetraxate, respectively, i.e., the hydrolysis reactions catalyzed by DCH were stereo- and/or regioselective. With recombinant Escherichia coli cells expressing the DCH gene as a catalyst, stereospecific hydrolysis of methyl beta-acetylthioisobutyrate and regioselective hydrolysis of methyl cetraxate proceeded efficiently.

Tranexamic acid, a widely used antifibrinolytic agent, causes convulsions by a gamma-aminobutyric acid(A) receptor antagonistic effect.

Application of 4-(aminomethyl)cyclohexanecarboxylic acid (tranexamic acid; TAMCA) to the central nervous system (CNS) has been shown to result in hyperexcitability and convulsions. However, the mechanisms underlying this action are unknown. In the present study, we demonstrate that TAMCA binds to the gamma-aminobutyric acid (GABA) binding site of GABA(A) receptors in membranes from rat cerebral cortex and does not interfere with N-methyl-D-aspartate receptors. Patch-clamp studies using human embryonic kidney cells transiently transfected with recombinant GABA(A) receptors composed of alpha 1 beta 2 gamma 2 subunits showed that TAMCA did not activate these receptors but dose dependently blocked GABA-induced chloride ion flux with an IC(50) of 7.1 +/- 3.1 mM. Application of TAMCA to the lumbar spinal cord of rats resulted in dose-dependent hyperexcitability, which was completely blocked by coapplication of the GABA(A) receptor agonist muscimol. These results indicate that TAMCA may induce hyperexcitability by blocking GABA-driven inhibition of the CNS.

Binding diversity of a noncovalent-type low-molecular-weight serine protease inhibitor and function of a catalytic water molecule: X-ray crystal structure of PKSI-527--inhibited trypsin.

PKSI-527 is a noncovalent-type low-molecular-weight inhibitor. The X-ray crystal structure of the trypsin-PKSI-527 complex revealed a binding mode (Form II) different from the previously reported one (Form I) [Nakamura, M. et al. (1995) Biochem. Biophys. Res. Commun. 213, 583--587]. In contrast to the previous case, the electron density of the inhibitor revealed the whole structure clearly. Each structural part of the inhibitor in Forms I and II was differently located at the active site, although the modes of binding of the terminal amino group to the Asp189 carboxyl group were similar. This binding diversity, which is a characteristic of the noncovalent-type low-molecular-weight inhibitor, provides a suitable example for estimating the possible mechanism toward the enzymatic inhibition, together with the structural basis necessary for a specific inhibitor. The mode of binding in Form II reflects the inhibitor-specific situation and is in contrast with the substrate-mimetic binding mode for Form I. Based on the generally accepted catalytic mechanism for serine protease, we propose that a water molecule located at the catalytic site plays an important role in blocking the catalytic function of the reactive Ser193 OH group.