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As a new alternative to antibody-drug conjugates, we developed "ligand-targeting" peptide-drug conjugates (PDCs), in which conformationally constrained helix-loop-helix (HLH) peptide M49 targeting human vascular endothelial growth factor-A (VEGF) was used as a drug carrier. The biochemical study showed that HLH peptide M49 made a complex with VEGF in the extracellular environment, and then the M49/VEGF complex interacts with the receptor on the cell surface to induce cellular internalization via the endocytic pathway. Here, we present an X-ray crystal structure of the M49/VEGF complex at 1.5 Å resolution using a protein crystal grown in microgravity. The structure illustrated the "ligand-targeting" cellular uptake mechanism for intracellular drug delivery and the molecular basis on the peptide-VEGF binding mode with tight binding and high target specificity. In addition, mutational studies and thermodynamic analysis provided information on the driving forces of the complex formation. This work would contribute to the design of mid-size molecular-targeting peptides as well as HLH peptides, advancing the research in drug discovery and chemical biology., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Co-authors Masayuki Kamo and Koji Inaka are MARUWA Foods and Biosciences, Inc. Employees., (Copyright © 2024. Published by Elsevier Inc.)

Mid-sized binding peptides have recently emerged as a new therapeutic modality. A helix-loop-helix (HLH) peptide was designed as a scaffold for combinatorial peptide libraries. We screened the HLH peptide libraries against human vascular endothelial growth factor-A (VEGF) to generate a peptide, VS42-LR3, which inhibited VEGF/receptor interaction and suppressed tumor growth in a murine xenograft model of human colorectal cancer. Here, we report the first crystal structure of the HLH peptide in a complex with VEGF at high resolution using space-grown protein crystals. The X-ray structural analysis revealed that the monomeric VS42-LR3 adopted an HLH structure and bound to VEGF at the VEGF receptor-binding site. Interestingly, from the site-directed mutagenesis, thermodynamic analysis, and molecular dynamic simulations, it turned out that the loop region in the non-interacting surface to VEGF affected the structural rigidity of the whole HLH to increase the binding affinity. These findings provide valuable insights for the design of more structurally stable and higher affinity mid-sized binding peptides as well as HLH peptides, that could play a crucial role in advancing molecular-targeting therapies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Co-authors MK and KI are employees of MARUWA Foods and Biosciences, Inc., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The blood concentration of intact proinsulin, but not total proinsulin, has been suggested to be a diagnostic marker for type 2 diabetes mellitus (T2DM), but a sensitive assay specific for rodent intact proinsulin is lacking. Here, a novel enzyme-linked immunosorbent assay (ELISA) for mouse intact proinsulin was developed. The developed ELISA detected mouse intact proinsulin with the working range of 8.3 to 2700pg/ml. Cross-reactivity with mouse split-32,33 proinsulin was approximately 100times lower than the reactivity with mouse intact proinsulin, and no cross-reactivity with mouse insulin was detected. The developed ELISA was sufficiently sensitive to detect low levels of intact proinsulin in normal mouse plasma. The measurement by the developed ELISA revealed that intact proinsulin was elevated in the plasma of type 2 diabetic db/db mice as mice aged, and the ratio of intact proinsulin/insulin in plasma was correlated with levels of glycated hemoglobin A1c as seen in T2DM patients. These results suggest that the plasma level of intact proinsulin, but not total proinsulin, is a sensitive marker for pancreatic dysfunction and the ensuring diabetic disease progression of db/db mice. This ELISA could aid nonclinical evaluation of therapeutic interventions in T2DM., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The measurement of plasma insulin is important for clinical diagnosis of diabetes and for preclinical research of metabolic diseases, especially in rodent models used in drug discovery research for type 2 diabetes. Fasting immunoreactive insulin (F-IRI) concentrations are used to calculate the homeostasis model assessment ratio (HOMA-R), an index of insulin sensitivity. However, even the most sensitive commercially available enzyme-linked immunosorbent assay (ELISA) kits cannot measure the very low F-IRI concentrations in normal rats and mice. Therefore, we sought to develop a new rodent insulin ELISA with greater sensitivity for low F-IRI concentrations. Despite repeated efforts, high-affinity antibodies could not be generated by immunizing mice with mouse insulin (self-antigen). Therefore, we generated two weak monoclonal antibodies (13G4 and 26B2) that were affinity maturated and used to develop a highly sensitive ELISA. The measurement range of the sandwich ELISA with the affinity maturated antibodies (13G4m1 and 26B2m1) was 1.5 to 30,000 pg/ml, and its detection limit was at least 10 times lower than those of commercially available kits. In conclusion, we describe the development of a new ultrasensitive ELISA suitable for measuring very low plasma insulin concentrations in rodents. This ELISA might be very useful in drug discovery research in diabetes., (Copyright © 2015. Published by Elsevier Inc.)

Recombinant production of extracellular glycoproteins in stable mammalian cell lines is an ideal technique for obtaining a large quantity of high-quality proteins. In most cases, however, current methodologies do not allow for sufficiently rapid cell line development and protein purification. Here, we describe a 21-residue peptide tag (designated as TARGET tag) and its use for rapid stable cell line development and purification. The ability of the anti-tag antibody P20.1 to withstand repetitive regeneration cycles has enabled the development of a sensitive surface plasmon resonance-based screening format that requires only 20 microl of cell culture supernatants. Direct and semi-quantitative screening at the 96-well culture scale eliminated the need for a second screening, re-cloning, or sorting, thereby minimizing culture pre-production time. Using this system, "high producer" cell lines were established in less than a month, and milligram quantities of target proteins could be purified with a standardized protocol., ((c) 2010 Elsevier B.V. All rights reserved.)

Hyperforin is a pharmacologically active constituent of Hypericum perforatum (St. John's wort). In vitro cultures of this medicinal plant were found to contain hyperforin and three related polyprenylated acylphloroglucinol derivatives. The accumulation of these compounds was coupled to shoot regeneration, with secohyperforin being the major constituent in morphogenic cultures. The structure of secohyperforin was elucidated online by LC-DAD, -MS, and -NMR. In multiple shoot cultures, the ratio of hyperforin to secohyperforin was strongly influenced by the phytohormones N6-benzylaminopurine (BAP) and naphthalene-1-acetic acid (NAA). While increasing concentrations of BAP stimulated the formation of hyperforin, increasing concentrations of NAA elevated the level of secohyperforin. No differential stimulation was observed after elicitor treatment. Hyperforin and secohyperforin are proposed to arise from a branch point in the biosynthetic pathway.

Catalytic antibodies 6D9 and 9C10, which were induced by immunization with a haptenic transition-state analog (TSA), catalyze the hydrolysis of a nonbioactive chloramphenicol monoester derivative to generate a bioactive chloramphenicol. These antibodies stabilize the transition state to catalyze the hydrolysis reaction, strictly according to the theoretical relationship: for 6D9, k(cat)/k(uncat)=895 and K(S)/K(TSA)=900, and for 9C10, k(cat)/k(uncat)=56 and K(S)/K(TSA)=60. To elucidate the molecular basis of the antibody-catalyzed reaction, the crystal structure of 6D9 was determined, and the binding thermodynamics of 6D9 and 9C10 with both the substrate and the TSA were analyzed using isothermal titration calorimetry. The crystal structure of the unliganded 6D9 Fab was determined at 2.25 A resolution and compared with that of the TSA-liganded 6D9 Fab reported previously, showing that the TSA is bound into the hydrophobic pocket of the antigen-combining site in an "induced fit" manner, especially at the L1 and H3 CDR loops. Thermodynamic analyses showed that 6D9 binds the substrate of the TSA with a positive DeltaS, differing from general thermodynamic characteristics of antigen-antibody interactions. This positive DeltaS could be due to the hydrophobic interactions between 6D9 and the substrate or the TSA mediated by Trp H100i. The difference in DeltaG between substrate and TSA-binding to 6D9 was larger than that to 9C10, which is in good correlation with the larger k(cat) value of 6D9. Interestingly, the DeltaDeltaG was mainly because of the DeltaDeltaH. The correlation between k(cat) and DeltaDeltaH is suggestive of "enthalpic strain" leading to destabilization of antibody-substrate complexes. Together with X-ray structural analyses, the thermodynamic analyses suggest that upon binding the substrate, the antibody alters the conformation of the ester moiety in the substrate from the planar Z form to a thermodynamically unstable twisted conformation, followed by conversion into the transition state. Enthalpic strain also contributes to the transition-state stabilization by destabilizing the ground state, and its degree is much larger for the more efficient catalytic antibody, 6D9.

The catalytic antibody 6D9, which was raised against a transition-state analogue (TSA), catalyzes the hydrolysis of a non-bioactive chloramphenicol monoester to generate chloramphenicol. It has been shown that 6D9 utilizes the binding affinity in the catalysis; the differential affinity of the TSA relative to the substrate is equal to the rate enhancement. To reveal the recognition mechanism of 6D9 for the TSA and the substrate, we performed NMR analysis of the Fv fragment of 6D9 (6D9-Fv), together with site-directed mutagenesis and stopped-flow kinetic analyses. Among six 6D9-Fv mutants, Y58(H)A and W100i(H)A displayed significant reductions in their affinities to the TSA, while their substrate-binding affinities were identical with that of the wild-type 6D9-Fv. The stopped-flow kinetic studies revealed that the TSA binding to 6D9-Fv occurred by an induced-fit mechanism. In contrast, no induced-fit type of TSA-binding mechanism was observed for Y58(H)A and W100i(H)A. From NMR experiments, we identified the residues with chemical shifts that were perturbed by the ligand-binding. The residues affected by the TSA binding were located on the TSA-binding site determined by the X-ray study, and on the regions far from the binding site. On the other hand, the residues affected by the substrate binding were localized on the TSA-binding site. As for W100i(H)A, no residue other than those in the binding site was affected by the ligand binding. On the basis of these results and the crystal structure, we concluded that the TSA binding induced a conformational change involving the formation of aromatic-aromatic interactions and a hydrogen bond. These interactions can account for the differential affinity for the TSA relative to the substrate. W100i(H) probably plays an important role in inducing the conformational changes. The present NMR studies have enabled us to visualize the concept of transition-state stabilization in enzymatic catalysis, in which the transition-state contacts are better than those of the substrate.

MyoD, a master regulatory gene for myogenesis, converts mesoderm derived cells to the skeletal muscle phenotype MyoD gene transfer into skin fibroblasts has been attempted in an effort to diagnose genetic muscle diseases. Although the gene transduction efficiency of adenoviral gene delivery systems is higher than that of various other systems, the rate of myo-conversion is insignificant. Since high adenovirus doses are cytotoxic and exogenous MyoD expression is insufficient for skin fibroblasts to re-differentiate into muscle cells, we constructed the novel adeno-MyoD vector, Ad.CAGMyoD using the recombinant CAG promoter. Even at a lower multiplicities of infection most skin fibroblasts infected with Ad.CAGMyoD could convert into myotubes without vector-induced cytotoxicity. The converted cells expressed muscle-specific desmin and full-length dystrophin, both of which were detected by Western blotting. Genetic and immunohistochemical analyses using skin fibroblasts and our vector system are reliable and useful for the clinical diagnosis of genetic muscle diseases.

A biosynthetic gene cluster containing five genes, alt1-5, was cloned from Alternaria solani, a causal fungus of early blight disease to tomato and potato. Homology searching indicated that the alt1, 2, and 3 genes code for cytochrome P450s and the alt4 gene for a FAD-dependent oxygenase/oxidase. The alt5 gene encodes a polyketide synthase (PKS), named PKSN, that was found to be an iterative type I complex reduced-type PKS with a C-methyltransferase domain. To identify the PKSN function, the alt5 gene was introduced into the fungal host Aspergillus oryzae under an alpha-amylase promoter. The transformant produced a polyketide compound, named alternapyrone, whose structure is shown to be 3,5-dimethyl-4-hydroxy-6-(1,3,5,7,11,13-hexamethyl-3,5,11-pentadecatrienyl)-pyran-2-one. Labeling experiments confirmed that alternapyrone is a decaketide with octa-methylation from methionine on every C(2) unit except the third unit.

Plants interact with their environment by producing a diverse array of secondary metabolites. A majority of these compounds are phenylpropanoids and flavonoids which are valued for their medicinal and agricultural properties. The phenylpropanoid biosynthesis pathway proceeds with the basic C6-C3 carbon skeleton of phenylalanine, and involves a wide range of enzymes viz., phenylalanine ammonia lyase, coumarate hydroxylase, coumarate ligase, chalcone synthase, chalcone reductase and chalcone isomerase. Recently, bacteria have also been shown to contain homodimeric polyketide synthases belonging to the plant chalcone synthase superfamily linking the capabilities of plants and bacteria in the biosynthesis of flavonoids. We report here the presence of genes encoding the core enzymes of the phenylpropanoid pathway in an industrially useful fungus, Aspergillus oryzae. Although the assignment of enzyme function must be confirmed by further biochemical evidences, this work has allowed us to anticipate the phenylpropanoid metabolism profile in a filamentous fungus for the first time and paves way for research on identifying novel fungal flavonoid-like metabolites.

Identification of genes encoding type III polyketide synthase (PKS) superfamily members in the industrially useful filamentous fungus, Aspergillus oryzae, revealed that their distribution is not specific to plants or bacteria. Among other Aspergilli (Aspergillus nidulans and Aspergillus fumigatus), A. oryzae was unique in possessing four chalcone synthase (CHS)-like genes (csyA, csyB, csyC, and csyD). Expression of csyA, csyB, and csyD genes was confirmed by RT-PCR. Comparative genome analyses revealed single putative type III PKS in Neurospora crassa and Fusarium graminearum, two each in Magnaporthe grisea and Podospora anserina, and three in Phenarocheate chrysosporium, with a phylogenic distinction from bacteria and plants. Conservation of catalytic residues in the CHSs across species implicated enzymatically active nature of these newly discovered homologs.

The catalytic antibody, 6D9, was subjected to directed evolution in the phage-display system using two structurally related transition-state analogs (TSAs) for panning. One analog, TSA 3, was originally used for immunization, and the other, TSA 4, a derivative of TSA 3, was designed to optimize the differential affinity for the transition state relative to the ground state so as to provide variants with improved reaction rates. We previously reported that by panning with TSA 4, we could obtain variants with highly improved catalytic rate enhancement (k(cat)/k(uncat)), and Tyr (L27e) seemed to play a key role in stabilizing the transition-state structure [Nat. Biotechnol. 19 (2001) 563]. Here, we examined in detail a large number of the variants selected by these haptens, in order to elucidate the mechanism of the directed evolution driven by them. ELISA with 3- and 4-bovine serum albumin (BSA) showed that variants selected by these TSAs exhibited distinct binding patterns. All the variants whose rate enhancement was greater than five-fold of that of 6D9 had Tyr (L27e) and were obtained from the library panned with TSA 4, but not from the library panned with TSA 3. Kinetic studies showed that TSA 4 could efficiently select variants with increased differential binding affinity for the transition state relative to the ground state, and these variants exhibited improved rate enhancements. This study verified the difference of in vitro evolution driven by the two structurally related TSAs and stresses the importance of designing an appropriate hapten for panning.

Ciguatoxins (CTXs) are a family of toxins that contaminate a variety of fish and cause ciguatera seafood poisoning. The limited availability of CTXs from natural sources has hampered preparation of antibodies and, thus, the development of immunoassays for these toxins. In the current studies, we utilized synthetic fragments as haptens to prepare antibodies against CTX3C, a congener of CTXs, thereby avoiding the problem of its scarcity. Synthetic ABC-ring fragment (ABC) of CTX3C was conjugated with keyhole limpet hemocyanin (KLH) and immunized on mice. Phage-displayed antibodies were first screened based on affinity to a soluble biotin-linked ABC-ring fragment that was captured on streptavidin-linked magnetic beads. The beads were then eluted with the ABCD-ring fragment (ABCD), and recovered phages were amplified. This elution with a synthetic fragment allowed the preparation of antibodies to ABCD as well as to ABC. Three antibodies with affinities of K(d) approximately 10(-5) M for ABCD were selected and prepared as soluble recombinant Fabs (rFabs). One rFab, 1C49, bound to CTX3C itself, although the binding affinity for CTX3C was weaker than for ABCD.

Objectives: The clinical implications of autoantibodies (Abs) were investigated as upstream indicators of paroxysmal atrial fibrillation in patients with congestive heart failure., Methods: Circulating Abs against myosin (M-Abs) detected by immunofluorescence, Abs against beta 1-adrenergic receptors (Beta 1-Abs) detected by enzyme-linked immunosorbent assay (ELISA), and Abs against NA-K-ATPase (NKA-Abs) detected by ELISA were screened in 95 congestive heart failure patients with < or = 45% left ventricular ejection fraction (coronary artery disease, n = 48; dilated cardiomyopathy, n = 47) and 48 age-matched control patients with hypertension. No patient received antiarrhythmic therapy. All patients were enrolled with angiotensin converting enzyme inhibitors in the chronic stable state. Relationship of the presence of paroxysmal atrial fibrillation to other clinical variables were assessed by 48-hour Holter monitoring., Results: No control patient had Abs. However, M-Abs, Beta 1-Abs, and NKA-Abs were detected in 22%, 26% and 16% of patients with congestive heart failure (coronary artery disease; 8%, 10%, and 4%, dilated cardiomyopathy; 36%, 43%, and 28%, respectively). Paroxysmal atrial fibrillation was more frequent in patients with dilated cardiomyopathy than in those with coronary artery disease (47% vs 15%, p < 0.01). Multivariate analysis suggested that NKA-Abs was an independent risk factor for the occurrence of paroxysmal atrial fibrillation (p < 0.01), although there were no differences in other clinical factors: age, sex, New York Heart Association functional class, concomitant medication, left ventricular ejection fraction, left atrial diameter, severity of mitral regurgitation, serum potassium, plasma norepinephrine, and atrial natriuretic peptide concentration., Conclusions: Autoantibodies against sarcolemmal Na-K-ATPase were closely related to the occurrence of paroxysmal atrial fibrillation in patients with congestive heart failure, so an autoimmune process may be an upstream factor in atrial fibrillation.

Prodrug activation by catalytic antibodies (abzymes) conjugated with anti-tumor antibodies, called antibody-directed abzyme prodrug therapy (ADAPT), has been proposed as a strategy for site-specific drug delivery systems for anti-tumor drugs. The delivery of abzymes is achieved by making a bi-specific antibody with a monovalent catalytic antibody and a monovalent binding antibody. To achieve ADAPT, we focused on specific requirements for prodrugs and catalytic antibodies, the stability of the prodrugs against natural enzymes, and the applicability of abzymes for a wide range of prodrugs. Attention was paid to the design of a pro-moiety rather than a parent drug. As a common pro-moiety, we chose vitamin B(6), because the bulky vitamin B(6) esters are relatively stable against hydrolytic enzymes in serum. We have generated catalytic antibodies by immunization of a vitamin B(6) phosphonate transition state analog. The elicited antibodies were found to hydrolyze several anti-cancer and anti-inflammatory prodrugs with the vitamin B(6) pro-moiety. Finally, we evaluated antibody-catalyzed prodrug activation by examining the growth inhibition of human cervical cancer (HeLa) cells with the vitamin B(6) ester of butyric acid. These results suggest that the pro-moiety of vitamin B(6) ester is stable enough to resist natural enzymes in serum and is removed by the tailored catalytic antibodies. The combination of catalytic antibodies and prodrugs masked with vitamin B(6) would allow hydrophobic and highly toxic drugs to be used.

Background: Based on the homology with fatty acid synthases and bacterial polyketide synthases (PKSs), thioesterase domains have been assigned at the C-terminus regions of fungal iterative type I PKSs. We previously overexpressed Aspergillus nidulans wA PKS gene in a heterologous fungal host and identified it to encode a heptaketide naphthopyrone synthase. In addition, expression of C-terminus-modified WA PKS gave heptaketide isocoumarins suggesting that the C-terminus region of WA PKS is involved in the cyclization of the second aromatic ring of naphthopyrone. To unravel the actual function of the C-terminus region, we carried out functional analysis of WA PKS mutants by C-terminus deletion and site-directed mutagenesis., Results: Only the 32 amino acid deletion from the C-terminus of WA PKS caused product change to heptaketide isocoumarins from heptaketide naphthopyrone, YWA1 1, a product of intact WA PKS. Further C-terminus deletion mutant of WA PKS up to Ser(1967), an active site residue of so far called thioesterase, still produced isocoumarins. Site-directed mutagenesis of amino acid residues in this C-terminus region showed that even a single mutation of S1967A or H2129Q caused production of isocoumarin instead of naphthopyrone. Furthermore, the role of tandem acyl carrier proteins (ACPs), a typical feature of fungal aromatic PKSs, was examined by site-directed mutagenesis and the results indicated that both ACPs can function as ACP independently., Conclusions: Claisen-type cyclization is assumed to be involved in formation of aromatic compounds by some fungal type I PKSs. These PKSs have a quite identical architecture of active site domain organization, beta-ketoacyl synthase, acyltransferase, tandem ACPs and thioesterase (TE) domains. Since the C-terminus region of WA PKS of this type was determined to be involved in Claisen-type cyclization of the second ring of naphthopyrone, we propose that the so far called TE of these PKSs work not just as TE but as Claisen cyclase.

We developed a muscle-specific gene delivery system based on two-step gene transfer. The first step involved adenovirus-mediated transfer of the ecotropic retrovirus receptor (EcoRec) gene driven by the muscle-specific desmin promoter. Both human primary myoblasts and fibroblasts were efficiently transduced with this adenovirus vector. However, expression of EcoRec was detected only in myoblasts. In the second step, EcoRec-expressing myoblasts could be stably transduced with the ecotropic retroviral vector with the beta-galactosidase gene. Approximately 15% of myoblasts were transduced by this two-step strategy. When the transduced myoblasts were differentiated into myotubes, extensive cell-cell fusion occurred, and the apparent number of beta-galactosidase-positive cells increased to 28%. These results indicate that our two-step gene delivery system could be used for targeted and stable gene transfer into muscle cells., (Copyright 2000 Academic Press.)

The three-dimensional structure of a catalytic antibody, 6D9, has been solved as a complex with a transition state analog. The structure was determined from two different crystal forms, and was refined at a resolution of 1.8 A. The antibody 6D9, which was induced by immunization with the phosphonate transition state analog 3, hydrolyzes a prodrug of chloramphenicol monoester 1 to generate the parent drug 2. The kinetic studies have shown that the antibody is catalytic by virtue of the theoretical relationship between the affinity for the transition state and the catalytic efficiency (kcat/kuncat=KS/KTSA). The crystal structure makes it possible to visualize the theoretical relationship. A side-chain (Nepsilon) of HisL27D is placed in a key position to make a hydrogen bond to the phosphonate oxygen of the transition state analog with a distance of 2.72 A, suggesting a hydrogen bond to the oxyanion developing in the transition state of the hydrolysis. There are no catalytic residues, other than the histidine, around the phosphonate moiety. In addition, in the antibody-hapten complex, the hapten bears a folded conformation and the two stacked aromatic rings are buried deep in the antigen-combining site through aromatic-aromatic interaction with TrpH100I and TyrH58. The conformation of the bound hapten suggests that the antibody binds the substrate to change the conformation of the ester moiety to a thermodynamically unstable E-form, thereby making it easy for the substrate to reach the transition-state during catalysis. These observations reveal that the catalytic mechanism is explained purely on the basis of the stabilization of the transition state. The refined high resolution structures reported here are envisaged to have an impact on the understanding of other hydrolytic antibodies, since their haptens share some unique features with the hapten used in this study., (Copyright 1998 Academic Press.)

Specific molecular interactions involved in catalysis by antibody 6D9 were investigated by site-directed mutagenesis. The catalytic antibody 6D9, which was generated against a transition state analog (III), hydrolyzes a non-bioactive chloramphenicol monoester derivative (I) to produce chloramphenicol (II). Construction of a three-dimensional molecular model of 6D9 and sequence comparison within a panel of related antibodies suggested candidates for catalytic residues, His (L27d), Tyr (L32), Tyr (H58) and Arg (H100b); these were targeted for the site-directed mutagenesis study. The Y-H58-F and R-H100b-A mutants possessed catalytic activities comparable to that of the wild-type, and the Y-H58-H and Y-L32-F mutant displayed an approximately fivefold decrease in k(cat)/Km. In the transition state analysis, the plots of logK(TSA) versus log(k(cat)/Km) for the mutants are linear, with a slope of approximately 1.0, indicating that the entire hapten-binding energy in the mutants is also utilized to bind the transition state and to accelerate the catalysis. In addition, a dramatic change in the catalytic activity was observed when the histidine residue (27d) in the CDR1 light chain was replaced with alanine. The H-L27d-A mutant had no detectable catalytic activity. This mutation led to a large, 40-fold reduction in transition state binding, with no change in substrate binding. Coupled with the previous kinetic studies and chemical modifications of the intact 6D9 antibody, this mutagenesis study has demonstrated that His L27d plays an essential role in stabilization of the transition state, the mechanism of catalysis by the 6D9 antibody.

Serotonin 5-HT2C receptor-mediated intracellular Ca2+ mobilization was investigated in 5-HT2C receptors expressed in Chinese hamster ovary (CHO) cells; and fura-2/AM was used to investigate the regulation of 5-HT2C receptor function. CHO cells, transfected with a cDNA clone for the 5-HT2C receptor, expressed 287 fmol/mg of the receptor protein as determined by mianserin-sensitive [3H]-mesulergine binding (kd = 0.49 nM). The addition of 5-HT mobilized intracellular Ca2+ in a dose-dependent fashion, ranging from basal level of 99 +/- 1.8 nM up to 246 +/- 21.2 nM, with an EC50 value for 5-HT of .015 microM. Exposure to 5-HT, a 5-HT receptor agonist, mCPP [1-(3-chlorophenyl)piperazine dihydrochloride], a 5-HT2C agonist, and DOI [1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane], a 5-HT2C and 5-HT2 agonist, resulted in increased intracellular Ca2+ levels. Mianserin, mesulergine, ritanserin, and ketanserin each blocked 5-HT-mediated intracellular Ca2+ mobilization more effectively than spiperone. Mianserin and amoxapine inhibited 5-HT-mediated intracellular Ca2+ mobilization completely; amitriptyline, nortriptyline, and imipramine reduced it about 50%. These results suggest that antagonism in CHO cells transfected with human 5-HT2C receptors is a component of the serotonergic properties of a number of established antidepressants.

We retrospectively evaluated the ability of equations with in vivo population binding parameters of our previous study (Method 1) or an average unbound fraction of 0.25 of Pynnönen (Method 2) to predict the unbound serum carbamazepine (CBZ) concentration in 50 serum samples from 28 polytherapy pediatric patients with epilepsy. In 12 serum samples from 10 patients, the ability of equations for unbound serum carbamazepine-10,11-epoxide (CBZ-E) concentration prediction was also determined in predictive performance with in vivo population binding parameters of our previous study (Method A) or an average unbound fraction of 0.5 of Pynnönen (Method B). Mean prediction error, mean absolute prediction error (MAE), and root mean squared error (RMSE) were calculated for each method, and these values served as a measure of prediction bias and precision. Method 1 shows a bias to underpredict unbound serum CBZ. The MAE and RMSE were lower in Method 2 (MAE = 0.696 microM, RMSE = 0.912 microM) than in Method 1 (MAE = 0.946 microM, RMSE = 1.138 microM). Method 2 is superior to Method 1 in accuracy and precision. The effects of antiepileptic co-medications on predictive performance of Method 1 are relatively larger in a co-medicated group of serum samples with valproic acid (n = 33, MAE = 0.994 microM, RMSE = 1.211 microM) than in a group of serum samples without valproic acid co-medication (n = 17, MAE = 0.853 microM, RMSE = 0.979 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

To determine whether non-rheumatic (NR) aortic regurgitation (AR) has the same clinical and postoperative courses as rheumatic (R) AR, we performed a retrospective study using pre- and postoperative M-mode echocardiograms in 23 patients who underwent aortic valve replacement (AVR) under myocardial protection with hypothermic cardioplegia. The etiology of AR was diagnosed by two-dimensional echocardiography. The NR-AR group consisted of nine patients including four with aortic valve prolapse (AP) and five with bicuspid valve (BV), and the R-AR group included 14 patients. Patients with preoperative end-diastolic dimensions (EDD) of less than 6.0 cm were excluded from this study. The indication for AVR was NYHA functional class III or severer. The severity of preoperative NYHA functional class was similar among these three groups. During the 18-month follow-up period (range 2-32 months), there were no post-operative deaths nor congestive heart failure. Ages at surgery ranged from 17 to 54 years; 10 (71%) of 14 patients with R-AR were 40 years old or older, while seven (78%) of nine with NR-AR were under 39 years old (p less than 0.05). The pre-operative left ventricular end-diastolic pressure (LVEDP) in patients with BV-AR was highest among these three groups (R-AR: 14.5 +/- 3.9 mmHg, AP-AR: 9.5 +/- 4.1 mmHg, BV-AR: 22.0 +/- 2.7 mmHg, p less than 0.05). There was no significant difference in pre-operative M-mode echocardiographic results, except for the end-systolic dimension (ESD) between R-AR (5.20 +/- 0.55 cm) and BV-AR (4.78 +/- 0.18 cm) (p less than 0.05). The EDD one month after AVR was still abnormal (greater than or equal to 5.4 cm) in seven of the 14 patients with R-AR, and three of the four patients with AP-AR but none of the patients with BV-ARs (p less than 0.05 vs AP-AR). All patients with pre-operative ESD of less than 5.2 cm had normal EDD one month after AVR. In conclusion, the clinical course of NR-AR is different from that of R-AR. Furthermore, AP-AR regresses more differently after AVR than does BV-AR. Therefore, it is important to consider the etiology of chronic AR in determining the timing of surgery.

Major categorical diagnosis by International Classification of Diseases and type-specific diagnosis for cardiovascular diseases in death certificates were compared to the diagnosis made at autopsy in 864 consecutive autopsy cases aged 20 or over, among the Japanese residents in Hisayama town. Cerebral stroke was correctly diagnosed in 84%, malignant neoplasms in 78% and cardiac disease in 66%. Cerebral stroke and cardiac disease tended to be overdiagnosed, while malignant neoplasms were underdiagnosed. The validation of certified diagnosis was less reliable in the aged population, and in type-specific diagnosis of cardiovascular diseases. Cerebral hemorrhage with false negative or false positive diagnoses was usually classified into type unspecified stroke or different categories of cerebral stroke, while those misdiagnosed as cases of cerebral infarction frequently had no significant lesions in the autopsied brain. Finally, the relationship between the validation of diagnosis on the death certificates and the secular trend in cardiovascular disease in the Japanese vital statistics was discussed.