Effect of Nd:YAG laser on bone formation in rat tibia defects: three-dimensional micro-computed tomography image analysis.

Nd:YAG laser is in common clinical use for the treatment of tissue incision, transpiration, and haemostasis in soft tissues. However, few studies have reported the effects of low-level laser therapy (LLLT) from Nd:YAG laser on bone healing. The aim of this study was to perform three-dimensional (3D) morphological evaluation of the photobiomodulation of Nd:YAG laser in bone defects in rat tibiae using micro-computed tomography CT (micro-CT) imaging. A bone defect was created in each tibia of 30 rats. The right side was treated with LLLT from Nd:YAG laser (LT group) daily until sacrifice and the left tibiae served as controls (control group). All tibiae underwent micro-CT imaging at 7, 14, and 21 days after the operation. Three-dimensional image analysis of bone volume (BV) and bone surface area (BS) of new bone formation in the defects was performed and histologic analysis was conducted for all tibiae. Tibial BV and BS values were highest in both groups at 7 days after the operation and decreased at 14 days after operation. BV and BS values were both significantly higher in the LT group than in the control group at 7 and 14 days. There was no significant difference between the groups for either metric at 21 days. The present findings demonstrate that Nd:YAG laser simulates bone formation during the early healing period.

Methodological considerations of the acetaminophen Detection Kit®: Involvement of molecular oxygen (O2) in an indophenol reaction.

The Acetaminophen Detection Kit® (Kanto Chemical Company Co. Inc., Tokyo, Japan) is a colorimetric test based on an indophenol reaction. The test involves three reactions: deproteination of the sample, hydrolysis of acetaminophen to yield p-aminophenol, and coupling p-aminophenol with a derivative of phenol in alkali conditions to form a blue-colored indophenol dye. The kit was devised to accomplish these three reactions with only two reagents, allowing the prompt diagnosis of acetaminophen overdose in emergency medicine. In the user instructions included with the kit and in reports introducing the kit, the chemical composition of the two reagents was not disclosed. Details about the composition can be found in the Safety Data Sheet from the manufacturer; however, there is little explanation about the principle (mechanism) of the coupling reaction. This lack of information appears to have hampered the use of this kit in forensic medicine. In this report, we conducted the coupling reaction by successively adding the two reagents to a p-aminophenol (intermediate molecule) solution with the reaction vessel open to the air and under an anaerobic condition. Development of the blue color was inhibited in the absence of air but gradually developed when the reaction vessel was opened to air. Thus, the coupling reaction is an oxidation-reduction (redox) reaction that requires molecular oxygen (O2) dissolved from the air to act as an oxidant. This finding corroborates statements in previous reports and will hopefully facilitate the use of the kit for forensic purposes.

Diversity in Infection Specificity between the Bloom-forming Microalga Heterosigma akashiwo and Its dsDNA Virus, Heterosigma akashiwo Virus.

Heterosigma akashiwo virus (HaV) is a dsDNA virus that infects the bloom-forming raphidoflagellate Heterosigma akashiwo. Both the host and its virus are phenotypically diverse in terms of infection specificity. Their relationships have been examined based on the occurrence or absence of algal lysis following virus inoculation; however, variations in the strain-level host-virus relationship regarding infectivity and lysis rates remain unclear. Therefore, we performed a series of cross-infectivity tests using 60 H. akashiwo and 22 HaV strains isolated from the coastal waters of western Japan. The host strains were divided into 5 different groups and viruses into 4 groups. Using a representative strain from each group, algal lysis was observed in 14 of the (5×4=) 20 host-virus combinations; the concentration of infectious units in each HaV suspension was then assessed using the most probable number (MPN) assay on the five host strains. Virus titers ranged between 1.1×101 and 2.1×107 infectious units mL-1; the titer of each viral lysate was differently estimated using distinct H. akashiwo strains as hosts. These results suggest that (1) a clonal viral lysate comprises virions with different intraspecific infection specificities and/or (2) the efficiency and error rates of each intracellular replication process vary in each host-virus combination.

Neutron crystallography and quantum chemical analysis of bilin reductase PcyA mutants reveal substrate and catalytic residue protonation states.

PcyA, a ferredoxin-dependent bilin pigment reductase, catalyzes the site-specific reduction of the two vinyl groups of biliverdin (BV), producing phycocyanobilin. Previous neutron crystallography detected both the neutral BV and its protonated form (BVH+) in the wildtype (WT) PcyA-BV complex, and a nearby catalytic residue Asp105 was found to have two conformations (protonated and deprotonated). Semiempirical calculations have suggested that the protonation states of BV are reflected in the absorption spectrum of the WT PcyA-BV complex. In the previously determined absorption spectra of the PcyA D105N and I86D mutants, complexed with BV, a peak at 730 nm, observed in the WT, disappeared and increased, respectively. Here, we performed neutron crystallography and quantum chemical analysis of the D105N-BV and I86D-BV complexes to determine the protonation states of BV and the surrounding residues and study the correlation between the absorption spectra and protonation states around BV. Neutron structures elucidated that BV in the D105N mutant is in a neutral state, whereas that in the I86D mutant is dominantly in a protonated state. Glu76 and His88 showed different hydrogen bonding with surrounding residues compared with WT PcyA, further explaining why D105N and I86D have much lower activities for phycocyanobilin synthesis than the WT PcyA. Our quantum mechanics/molecular mechanics calculations of the absorption spectra showed that the spectral change in D105N arises from Glu76 deprotonation, consistent with the neutron structure. Collectively, our findings reveal more mechanistic details of bilin pigment biosynthesis.

Degenerate PCR Targeting the Major Capsid Protein Gene of HcRNAV and Related Viruses.

Heterocapsa circularisquama RNA virus (HcRNAV) is the only dinoflagellate-infecting RNA virus that has been isolated to date. We herein investigated the diversity of the major capsid protein gene of HcRNAV and related viruses using degenerate PCR and in silico ana-lyses. Diverse sequences related to HcRNAV were successfully amplified from marine sediments. Amplicons contained conserved and variable regions; the latter were predicted to be located on the outer surface of the capsid. Our approach provides insights into the diversity of viruses that are difficult to isolate in the environment and will enhance rapidly growing metagenome sequence repositories.

Effect of Low-intensity Pulsed Ultrasound on Healing of Bone Defects in Rat Tibia as Measured by Reconstructed Three-dimensional Analysis of Micro CT Images.

BACKGROUND/AIM: The aim of this study was to evaluate the effect of low-intensity pulsed ultrasound (LIPUS) on bone metabolism during the healing period in rat tibiae bone defects using micro-computed tomography (micro CT) imaging for three-dimensional morphological evaluation. MATERIALS AND METHODS: The right tibia received ultrasound exposure (US group) every day, whereas the opposite side served as a control (Control group). At 1, 2, and 3 weeks after the operation, micro CT was performed, and the volume and surface area of new bone formation in the bone defects was evaluated three-dimensionally. RESULTS: Bone volume (BV) and bone surface (BS) in the tibiae of both the US and Control groups demonstrated the highest values 1 week after the operation, with no significant differences between the groups. At 2 weeks after the operation, the BV and BS values in both groups had decreased, but the decrease was smaller in the US group than the Control group. At 3 weeks after the operation, the BV and BS values in the Control group were significantly lower than those in the US group. CONCLUSION: LIPUS stimulation can prevent bone loss during the healing of bone defects.

Structural basis of the protochromic green/red photocycle of the chromatic acclimation sensor RcaE.

Cyanobacteriochromes (CBCRs) are bilin-binding photosensors of the phytochrome superfamily that show remarkable spectral diversity. The green/red CBCR subfamily is important for regulating chromatic acclimation of photosynthetic antenna in cyanobacteria and is applied for optogenetic control of gene expression in synthetic biology. It is suggested that the absorption change of this subfamily is caused by the bilin C15-Z/C15-E photoisomerization and a subsequent change in the bilin protonation state. However, structural information and direct evidence of the bilin protonation state are lacking. Here, we report a high-resolution (1.63Å) crystal structure of the bilin-binding domain of the chromatic acclimation sensor RcaE in the red-absorbing photoproduct state. The bilin is buried within a "bucket" consisting of hydrophobic residues, in which the bilin configuration/conformation is C5-Z,syn/C10-Z,syn/C15-E,syn with the A- through C-rings coplanar and the D-ring tilted. Three pyrrole nitrogens of the A- through C-rings are covered in the α-face with a hydrophobic lid of Leu249 influencing the bilin pKa, whereas they are directly hydrogen bonded in the ß-face with the carboxyl group of Glu217. Glu217 is further connected to a cluster of waters forming a hole in the bucket, which are in exchange with solvent waters in molecular dynamics simulation. We propose that the "leaky bucket" structure functions as a proton exit/influx pathway upon photoconversion. NMR analysis demonstrated that the four pyrrole nitrogen atoms are indeed fully protonated in the red-absorbing state, but one of them, most likely the B-ring nitrogen, is deprotonated in the green-absorbing state. These findings deepen our understanding of the diverse spectral tuning mechanisms present in CBCRs.

Crystal structures of hydroxymethylbilane synthase complexed with a substrate analog: a single substrate-binding site for four consecutive condensation steps.

Hydroxymethylbilane synthase (HMBS), which is involved in the heme biosynthesis pathway, has a dipyrromethane cofactor and combines four porphobilinogen (PBG) molecules to form a linear tetrapyrrole, hydroxymethylbilane. Enzyme kinetic study of human HMBS using a PBG-derivative, 2-iodoporphobilinogen (2-I-PBG), exhibited noncompetitive inhibition with the inhibition constant being 5.4 ± 0.3 µM. To elucidate the reaction mechanism of HMBS in detail, crystal structure analysis of 2-I-PBG-bound holo-HMBS and its reaction intermediate possessing two PBG molecules (ES2), and inhibitor-free ES2 was performed at 2.40, 2.31, and 1.79 Šresolution, respectively. Their overall structures are similar to that of inhibitor-free holo-HMBS, and the differences are limited near the active site. In both 2-I-PBG-bound structures, 2-I-PBG is located near the terminus of the cofactor or the tetrapyrrole chain. The propionate group of 2-I-PBG interacts with the side chain of Arg173, and its acetate group is associated with the side chains of Arg26 and Ser28. Furthermore, the aminomethyl group and pyrrole nitrogen of 2-I-PBG form hydrogen bonds with the side chains of Gln34 and Asp99, respectively. These amino acid residues form a single substrate-binding site, where each of the four PBG molecules covalently binds to the cofactor (or oligopyrrole chain) consecutively, ultimately forming a hexapyrrole chain. Molecular dynamics simulation of the ES2 intermediate suggested that the thermal fluctuation of the lid and cofactor-binding loops causes substrate recruitment and oligopyrrole chain shift needed for consecutive condensation. Finally, the hexapyrrole chain is hydrolyzed self-catalytically to produce hydroxymethylbilane.

Chronological distribution of dinoflagellate-infecting RNA virus in marine sediment core.

A bivalve-killing marine dinoflagellate, Heterocapsa circularisquama, is susceptible to the infectious single-stranded RNA virus, Heterocapsa circularisquama RNA virus (HcRNAV). The ecological relationship between H. circularisquama and HcRNAV was intensively studied from 2001 through 2005; however, only limited data are available for the ecological dynamics of HcRNAV before 2001. In this study, we applied radiometric dating and reverse transcription PCR (RT-PCR) to determine the chronological distribution of HcRNAV in a marine sediment core sampled from the Uranouchi Inlet, Kochi, Japan, where H. circularisquama was first discovered. Our results show that HcRNAV had existed in the inlet long before its first bloom in 1988. Furthermore, five HcRNAV variants, phylogenetically distinguishable based on the nucleotide sequence of the major capsid protein (MCP) gene, were identified. These variants were found to be distributed throughout the core over time, suggesting that the HcRNAV sequences registered in the NCBI database are only a portion of the variants that have emerged in the history of HcRNAV diversification. Herein, we have verified the applicability of the retrospective approach for speculating the distribution of algal RNA viruses over time in aquatic environments.

Pathological Features of Ruptured Coronary Plaque and Thrombus Interfaces: Fibrin and von Willebrand Factor as Platelet Scaffolds on Rupture Sites.

Arterial thrombus formation is thought to be initiated by platelet adhesion to the subendothelial matrix, but ruptured atherosclerotic plaques are characterized by substantial reduction of matrix proteins compared with stable plaques. Intraplaque erythrocytes and/or fibrin have been reported in high-risk coronary plaques. The aims of the current study were to identify factors that provide scaffolds for platelets at the sites of ruptured coronary plaques and investigate depositions of iron and bilirubin as hemoglobin catabolites in the ruptured plaques. Histological characteristics of plaque components and the thrombus interface were examined in 73 acute coronary aspirated thrombi. Necrotic debris (95%), macrophages (95%), and cholesterin clefts (81%) were observed frequently at the ruptured plaque and thrombus interface. A fibrous matrix (47%), calcification (32%), and extracellular deoxyribonucleic acid (15%) were identified as small foci. Tissue factor was localized in the necrotic core and macrophages. Fibrin and von Willebrand factor were consistently deposited within the plaques and beneath platelet aggregations. The citrullinated histone H3-immunopositive area accounted for only 0.5% of the plaque area. Bilirubin and iron depositions were detected in approximately 20% of the plaques in addition to biliverdin reductase and ferritin expression in macrophages. Fibrin and von Willebrand factor rather than matrix proteins and neutrophil extracellular traps may be major adhesive molecules at the sites of ruptured plaques. Iron and bilirubin deposits may be markers for rupture-prone plaques.

Evidence for dynamic in vivo interconversion of the conformational states of IscU during iron-sulfur cluster biosynthesis.

IscU is a central component of the ISC machinery and serves as a scaffold for de novo assembly of Fe-S clusters. The dedicated chaperone system composed of the Hsp70-chaperone HscA and the J-protein cochaperone HscB synergistically interacts with IscU and facilitates cluster transfer from IscU to recipient apo-proteins. Here, we report that the otherwise essential roles of HscA and HscB can be bypassed in vivo by a number of single amino acid substitutions in IscU. CD spectroscopic studies of the variant IscU proteins capable of this bypass activity revealed dynamic interconversion between two conformations: the denatured (D) and the structured (S) state in the absence and presence of Zn2+ , respectively, which was far more prominent than interconversion observed in wild-type IscU. Furthermore, we found that neither the S-shifted (more structured) variants of IscU nor the perpetually denatured variants could perform their in vivo role regardless of whether the chaperone system was present or not. The present study thus provides for the first time evidence that an in vivo D-state of IscU exists and implies that conformational interconversion between the S- and D-states of the scaffolding protein is a fundamental requirement for the assembly and transfer of the Fe-S cluster.

Healed Edentulous Sites: Suitability for Dental Implant Placement, Need for Secondary Procedures, and Contemporary Implant Designs.

PURPOSE: The purpose of this retrospective computed tomography study was to evaluate bone availability for dental implant placement, frequency of bone augmentation procedures, frequency of anatomical structures that compromise implant placement, and frequency of implant dimensions, and to determine which edentulous sites would benefit from the use of a sloped implant versus a traditional flat design. MATERIALS AND METHODS: Recorded parameters included the width of the ridge, the buccal and lingual/palatal alveolar bone height in reference to different anatomical landmarks, determination of implant placement, selection of an implant with a flat or sloped top, and need for a secondary bone augmentation procedure. RESULTS: One thousand three hundred seventy edentulous sites were evaluated in 216 patients. Implants could be placed in 60.6% of the total sites, where the coronal portion would be sloped in 59% of sites and conventionally flat in 41%; 39.4% of sites were not adequate for implant placement, where 56.5% needed additional guided bone regeneration procedures and 43.5% required sinus augmentation procedures. The inferior alveolar canal was the most frequent anatomical structure limiting size and/or placement. CONCLUSION: The study indicates that implants can be placed in slightly over half of edentulous sites without a secondary grafting procedure. The possibility of dental implant placement varied according to the volume and morphology of alveolar bone and related anatomical structures. This decreased from anterior to posterior in both arches. The sloped implant design was beneficial. In addition, the sloped implant design resulted in the placement of a longer implant.

Recent Advances in the Understanding of the Reaction Chemistries of the Heme Catabolizing Enzymes HO and BVR Based on High Resolution Protein Structures.

In mammals, catabolism of the heme group is indispensable for life. Heme is first cleaved by the enzyme Heme Oxygenase (HO) to the linear tetrapyrrole Biliverdin IXα (BV), and BV is then converted into bilirubin by Biliverdin Reductase (BVR). HO utilizes three Oxygen molecules (O2) and seven electrons supplied by NADPH-cytochrome P450 oxidoreductase (CPR) to open the heme ring and BVR reduces BV through the use of NAD(P)H. Structural studies of HOs, including substrate-bound, reaction intermediate-bound, and several specific inhibitor-bound forms, reveal details explaining substrate binding to HO and mechanisms underlying-specific HO reaction progression. Cryo-trapped structures and a time-resolved spectroscopic study examining photolysis of the bond between the distal ligand and heme iron demonstrate how CO, produced during the HO reaction, dissociates from the reaction site with a corresponding conformational change in HO. The complex structure containing HO and CPR provides details of how electrons are transferred to the heme-HO complex. Although the tertiary structure of BVR and its complex with NAD+ was determined more than 10 years ago, the catalytic residues and the reaction mechanism of BVR remain unknown. A recent crystallographic study examining cyanobacterial BVR in complex with NADP+ and substrate BV provided some clarification regarding these issues. Two BV molecules are bound to BVR in a stacked manner, and one BV may assist in the reductive catalysis of the other BV. In this review, recent advances illustrated by biochemical, spectroscopic, and crystallographic studies detailing the chemistry underlying the molecular mechanism of HO and BVR reactions are presented.

Crystal structure of phytochromobilin synthase in complex with biliverdin IXα, a key enzyme in the biosynthesis of phytochrome.

Phytochromobilin (PΦB) is a red/far-red light sensory pigment in plant phytochrome. PΦB synthase is a ferredoxin-dependent bilin reductase (FDBR) that catalyzes the site-specific reduction of bilins, which are sensory and photosynthesis pigments, and produces PΦB from biliverdin, a heme-derived linear tetrapyrrole pigment. Here, we determined the crystal structure of tomato PΦB synthase in complex with biliverdin at 1.95 Å resolution. The overall structure of tomato PΦB synthase was similar to those of other FDBRs, except for the addition of a long C-terminal loop and short helices. The structure further revealed that the C-terminal loop is part of the biliverdin-binding pocket and that two basic residues in the C-terminal loop form salt bridges with the propionate groups of biliverdin. This suggested that the C-terminal loop is involved in the interaction with ferredoxin and biliverdin. The configuration of biliverdin bound to tomato PΦB synthase differed from that of biliverdin bound to other FDBRs, and its orientation in PΦB synthase was inverted relative to its orientation in the other FDBRs. Structural and enzymatic analyses disclosed that two aspartic acid residues, Asp-123 and Asp-263, form hydrogen bonds with water molecules and are essential for the site-specific A-ring reduction of biliverdin. On the basis of these observations and enzymatic assays with a V121A PΦB synthase variant, we propose the following mechanistic product release mechanism: PΦB synthase-catalyzed stereospecific reduction produces 2(R)-PΦB, which when bound to PΦB synthase collides with the side chain of Val-121, releasing 2(R)-PΦB from the synthase.

Super-activator variants of the cyanobacterial transcriptional regulator ChlR essential for tetrapyrrole biosynthesis under low oxygen conditions.

ChlR is a MarR-type transcriptional regulator that activates the transcription of the chlAII-ho2-hemN operon in response to low oxygen conditions in the cyanobacterium Synechocystis sp. PCC 6803. Upon exposure to low oxygen conditions, ChlR activates transcription of the operon that encodes enzymes critical to tetrapyrrole biosynthesis under low oxygen conditions. We previously identified a super-activator variant, D35H, of ChlR that constitutively activates transcription of the operon. To gain insight into the low-oxygen induced activation of ChlR, we obtained eight additional super-activator variants of ChlR including D35H from pseudorevertants of a chlAI-disrupted mutant. Most substitutions were located in the N-terminal region of ChlR. Mapping of the substituted amino acid residues provided valuable structural insights that uncovered the activation mechanism of ChlR.

Identification of IscU residues critical for de novo iron-sulfur cluster assembly.

IscU is a central component of the ISC machinery and serves as a scaffold for the de novo assembly of iron-sulfur (Fe-S) clusters prior to their delivery to target apo-Fe-S proteins. However, the molecular mechanism is not yet fully understood. In this study, we have conducted mutational analysis of E. coli IscU using the recently developed genetic complementation system of a mutant that can survive without Fe-S clusters. The Fe-S cluster ligands (C37, C63, H105, C106) and the proximal D39 and K103 residues are essential for in vivo function of IscU and could not be substituted with any other amino acids. Furthermore, we found that substitution of Y3, a strictly conserved residue among IscU homologs, abolished in vivo functions. Surprisingly, a second-site suppressor mutation in IscS (A349V) reverted the defect caused by IscU Y3 substitutions. Biochemical analysis revealed that IscU Y3 was crucial for functional interaction with IscS and sulfur transfer between the two proteins. Our findings suggest that the critical role of IscU Y3 is linked to the conformational dynamics of the flexible loop of IscS, which is required for the ingenious sulfur transfer to IscU.

Bilin-metabolizing enzymes: site-specific reductions catalyzed by two different type of enzymes.

In mammals, the green heme metabolite biliverdin is converted to a yellow anti-oxidant by NAD(P)H-dependent biliverdin reductase (BVR), whereas in O2-dependent photosynthetic organisms it is converted to photosynthetic or light-sensing pigments by ferredoxin-dependent bilin reductases (FDBRs). In NADP+-bound and biliverdin-bound BVR-A, two biliverdins are stacked at the binding cleft; one is positioned to accept hydride from NADPH, and the other appears to donate a proton to the first biliverdin through a neighboring arginine residue. During the FDBR-catalyzed reaction, electrons and protons are supplied to bilins from ferredoxin and from FDBRs and waters bound within FDBRs, respectively. Thus, the protonation sites of bilin and catalytic residues are important for the analysis of site-specific reduction. The neutron structure of FDBR sheds light on this issue.

Crystal structure of a NADPH-cytochrome P450 oxidoreductase (CYPOR) and heme oxygenase 1 fusion protein implies a conformational change in CYPOR upon NADPH/NADP+ binding.

Heme oxygenase-1 (HMOX1) catalyzes heme degradation utilizing reducing equivalents supplied from NADPH-cytochrome P450 reductase (CYPOR). Recently, we determined the complex structure of NADP+ -bound open-conformation stabilized CYPOR and heme-HMOX1, but the resolution was limited to 4.3 Å. Here, we determined the crystal structure of the fusion protein of open-conformation stabilized CYPOR and heme-HMOX1 at 3.25 Å resolution. Unexpectedly, no NADP+ was bound to this fusion protein in the crystal. Structural comparison of the NADP+ -bound complex and the NADP+ -free fusion protein suggests that NADP+ binding regulates the conformational change in the FAD-binding domain of CYPOR. As a result of this change, the FMN-binding domain of CYPOR approaches heme-bound HMOX1 upon NADP+ binding to enhance the electron-transfer efficiency from FMN to heme.

Alveolar Ridge Augmentation with Three-Dimensional Printed Hydroxyapatite Devices: A Preclinical Study.

The objective of this study was to evaluate the effectiveness of precise three-dimensional hydroxyapatite printed micro- and macrochannel devices for alveolar ridge augmentation in a canine model. All grafts induced minimal inflammatory and fibrotic reactions. Examination of undecalcified sections revealed that both types of grafts demonstrated bone ingrowth. The majority of the bone growth into the block graft was into the channels, though a portion grew directly into the construct in the form of small bony spicules. In conclusion, bone ingrowth was readily demonstrated in the middle of the implanted printed devices.

Synthesis and evaluation of the inhibitory activity of the four stereoisomers of the potent and selective human γ-glutamyl transpeptidase inhibitor GGsTop.

2-Amino-4-{[3-(carboxymethyl)phenoxy](methoxy)phosphoryl}butanoic acid (GGsTop) is a potent, highly selective, nontoxic, and irreversible inhibitor of γ-glutamyl transpeptidase (GGT). GGsTop has been widely used in academic and medicinal research, and also as an active ingredient (Nahlsgen) in commercial anti-aging cosmetics. GGsTop consists of four stereoisomers due to the presence of two stereogenic centers, i.e., the α-carbon atom of the glutamate mimic (l/d) and the phosphorus atom (RP/SP). In this study, each stereoisomer of GGsTop was synthesized stereoselectively and their inhibitory activity against human GGT was evaluated. The l- and d-configurations of each stereoisomer were determined by a combination of a chiral pool synthesis and chiral HPLC analysis. The synthesis of the four stereoisomers of GGsTop used chiral synthetic precursors that were separated by chiral HPLC on a preparative scale. With respect to the configuration of the α-carbon atom of the glutamate mimic, the l-isomer (kon=174M-1s-1) was ca. 8-fold more potent than the d-isomer (kon=21.5M-1s-1). In contrast, the configuration of the phosphorus atom is critical for GGT inhibitory activity. Based on a molecular modeling approach, the absolute configuration of the phosphorus atom of the active GGsTop isomers was postulated to be SP. The SP-isomers inhibited human GGT (kon=21.5-174M-1s-1), while the RP-isomers were inactive even at concentrations of 0.1mM.