Acyl transfer mechanisms of tissue transglutaminase.

Tissue transglutaminase (TG2) is a calcium-dependent enzyme that catalyses several acyl transfer reactions. The most biologically relevant of these involve protein-bound Gln residues as an acyl-donor substrate, and either water or a primary amine as an acyl-acceptor substrate. The former leads to deamidation of Gln to Glu, whereas the latter leads to transamidation, typically resulting in protein cross-linking when the amine substrate is a protein-bound Lys residue. In this review, we present an overview of over fifty years of mechanistic studies that have led to our current understanding of TG2-mediated hydrolysis and transamidation.

Flexible operation of the Cap-and-Trade System for the air pollutants in the Seoul Metropolitan area.

To improve the air quality in the Seoul Metropolitan area (SMA), the Korean government has implemented special measures in the 1990s. As part of these measures, the Cap-and-Trade System (CATS) was introduced and executed in July 1, 2007 for the oxides of nitrogen (NOx) and sulfur (SOx) to provide added flexibility for large sources to meet the required emission reductions. However, the trade via the SMA CATS for the air pollutants has not been active because of the limited buyers and sellers within the system as well as limited tradable species. For more flexible operation of the SMA CATS, following strategies have been suggested and their merits are discussed; (1) to link the SMA CATS with the Korea Voluntary Emission Reduction (KVER) program which is a program to manage greenhouse gases (GHGs), and (2) to extend the system, such as extension of the tradable species, participants, and introducing a project-based certification mechanism for pollutants reduction.

Designing Dual Transglutaminase†2/Histone Deacetylase Inhibitors Effective at Halting Neuronal Death.

In recent years there has been a clear consensus that neurodegenerative conditions can be better treated through concurrent modulation of different targets. Herein we report that combined inhibition of transglutaminase 2 (TG2) and histone deacetylases (HDACs) synergistically protects against toxic stimuli mediated by glutamate. Based on these findings, we designed and synthesized a series of novel dual TG2-HDAC binding agents. Compound 3 [(E)-N-hydroxy-5-(3-(4-(3-oxo-3-(pyridin-3-yl)prop-1-en-1-yl)phenyl)thioureido)pentanamide] emerged as the most interesting of the series, being able to inhibit TG2 and HDACs both in vitro (TG2 IC50 =13.3±1.5 µm, HDAC1 IC50 =3.38±0.14 µm, HDAC6 IC50 =4.10±0.13 µm) and in cell-based assays. Furthermore, compound 3 does not exert any toxic effects in cortical neurons up to 50 µm and protects neurons against toxic insults induced by glutamate (5 mm) with an EC50 value of 3.7±0.5 µm.

Development of new scaffolds as reversible tissue transglutaminase inhibitors, with improved potency or resistance to glutathione addition.

Previous studies within our group have yielded a class of cinnamoyl-based competitive reversible inhibitors for tissue transglutaminase (TG2), with Ki values as low as 1.0 µM (compound CP4d). However, due to the electrophilic nature of their alkene moiety, this class of inhibitors is susceptible to nucleophilic attack by glutathione, a key element in cellular metabolism and toxicity response. To address this issue, we made several modifications to the inhibitor scaffold, ultimately showing that a bis(triazole) scaffold increased resistance to nucleophilic attack, with compound 27d being the most potent (Ki = 10 µM). In the process of reducing reactivity, we also prepared a new class of inhibitors, replacing the alkene of CP4d with an alkyne, leading to a significant increase in potency for compound 22b (Ki = 420 nM).

Hydroxy-substituted trans-cinnamoyl derivatives as multifunctional tools in the context of Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial pathology that requires multifaceted agents able to address its peculiar nature. In recent years, a plethora of proteins and biochemical pathways has been proposed as possible targets to counteract neurotoxicity. Although the complex scenario is not completely elucidated, close relationships are emerging among some of these actors. In particular, increasing evidence has shown that aggregation of amyloid beta (Aß), glycogen synthase kinase 3ß (GSK-3ß) and oxidative stress are strictly interconnected and their concomitant modulation may have a positive and synergic effect in contrasting AD-related impairments. We designed compound 3 which demonstrated the ability to inhibit both GSK-3ß (IC50 = 24.36 ± 0.01 µM) and Aß42 self-aggregation (IC50 = 9.0 ± 1.4 µM), to chelate copper (II) and to act as exceptionally strong radical scavenger (kinh = 6.8 ± 0.5 · 105 M-1s-1) even in phosphate buffer at pH 7.4 (kinh = 3.2 ± 0.5 · 105 M-1s-1). Importantly, compound 3 showed high-predicted blood-brain barrier permeability, did not exert any significant cytotoxic effects in immature cortical neurons up to 50 µM and showed neuroprotective properties at micromolar concentration against toxic insult induced by glutamate.

Transglutaminase inhibitors: a patent review.

INTRODUCTION: Transglutaminases (TGases) are a class of enzymes that play multifunctional roles. Their protein-crosslinking activity has been linked to fibrosis and Huntington's disease, their glutamine deamidation activity has been related to celiac disease and their GTP-binding activity has been implicated in cancer. All of these physiological disorders have prompted the development of inhibitors, which has accelerated dramatically over the past decade. AREAS COVERED: This review presents an overview of TGase inhibitors published in the patent literature, from the first compounds developed in the late 1980's, to the current date. This article is focussed on the chemical structure of new inhibitors and their probable mechanism of action. EXPERT OPINION: Comparison of effective TGase inhibitors reveals common structural features that may guide future design. Many of these elements are embodied in the first TGase inhibitor to recently enter into clinical trials.

Inhibition by tectorigenin and tectoridin of prostaglandin E2 production and cyclooxygenase-2 induction in rat peritoneal macrophages.

Tectorigenin and tectoridin, isolated from the rhizomes of Korean Belamcanda chinensis (Iridaceae) which are used as Chinese traditional medicine for the treatment of inflammation, suppressed prostaglandin E2 production by rat peritoneal macrophages stimulated by the protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), or the endomembrane Ca2+-ATPase inhibitor, thapsigargin. Tectorigenin inhibited prostaglandin E2 production more potently than tectoridin. Neither compound inhibited the release of radioactivity from [3H]arachidonic acid-labeled macrophages stimulated by TPA or thapsigargin. In addition, activities of isolated cyclooxygenase (COX)-1 and COX-2 were not inhibited by the two compounds. Western blot analysis revealed that the induction of COX-2 by TPA or thapsigargin was inhibited by the two compounds in parallel with the inhibition of prostaglandin E2 production. These findings suggest that one of the mechanisms of the anti-inflammatory activities of the rhizomes of Belamcanda chinensis is the inhibition of prostaglandin E2 production by tectorigenin and tectoridin due to the inhibition of the induction of COX-2 in the inflammatory cells.

Inhibitors of tissue transglutaminase.

Tissue transglutaminase (TG2) catalyzes the cross-linking of proteins by the formation of isopeptide bonds between glutamine (Gln) and lysine (Lys) side chains. Although TG2 is essential for the stabilization of the extracellular matrix, its unregulated activity has been implicated in celiac disease, fibrosis, and cancer metastasis, among other disorders. Given the importance and range of TG2-related pathologies, recent work has focused on the development of potent and selective inhibitors against TG2. In this review, we present the latest and most noteworthy irreversible and reversible inhibitors of TG2, and offer perspectives for the design of future inhibitors, in the hope that lead compounds with therapeutic potential may soon be discovered.

Peroxiredoxin is ubiquitously expressed in rat skin: isotype-specific expression in the epidermis and hair follicle.

Peroxiredoxins are a family of peroxidases that are ubiquitously and abundantly expressed in mammalian tissues; however, comparatively less is known about their expression in the skin. In this study, we examined the expression of three isotypes of peroxiredoxins (I-III) in rat skin. Western blot analyses showed strong expression of peroxiredoxins I-III in the epidermis and dermis of intact skin. Additionally, they were expressed in cultured rat keratinocytes and fibroblasts. Confocal image analyses revealed that peroxiredoxin II was present in the cytoplasm as a diffuse, reticulated pattern. In immunohistochemical staining of rat skin, peroxiredoxin expression was mainly localized to the epidermis, hair follicles, and sebaceous glands. In the epidermis, peroxiredoxins I and II were expressed in all layers with a gradient of increasing expression to the granular layer. In contrast, peroxiredoxin III was expressed in all layers with a gradient of expression decreasing to the granular layer. In the hair follicle, peroxiredoxins I-III were mainly expressed in the outer root sheath, except peroxiredoxin II, which was strongly expressed in the inner root sheath. In situ hybridization showed that mRNA expression was commensurate with the level of protein. Ultraviolet B radiation increased peroxiredoxin II expression in rat skin within 15 min after irradiation. From this study we conclude that peroxiredoxin isoforms are ubiquitously expressed in rat skin, and expression of at least peroxiredoxin II can be regulated by ultraviolet irradiation as a peroxidase in the skin. J Invest Dermatol 115:1108-1114 2000

Expression of differentiation markers during fetal skin development in humans: immunohistochemical studies on the precursor proteins forming the cornified cell envelope.

The cornified cell envelope is formed during the terminal differentiation of epidermis through cross-linking of specific proteins by transglutaminases. The specific arrangement of individual protein in the cornified cell envelope and participation of individual protein in the cornified cell envelope at different regions of skin, i.e., palm, foreskin, lips, etc. are not clearly understood. In order to understand the pattern and expression schedule of each individual precursor protein during the differentiation and formation of cornified cell envelope, the expression of precursor proteins in developing human fetal skins from the first to the third trimester were examined by immunohistochemical studies. Involucrin was found in the periderm and intermediate layer from 14 wk estimated gestational age, while loricrin and small proline-rich protein 1 were found in the periderm from 16 wk estimated gestational age. Filaggrin and trichohyalin that are absent in the adult cornified cell envelope were found in the granular and horny layers from 24 wk estimated gestational age. The precursor proteins except trichohyalin did not change their patterns after the onset of initial expression during development. Trichohyalin was transiently expressed in the granular and horny layers of the epidermis from 24 wk estimated gestational age with peak expression at 27 wk estimated gestational age, but was not detected in adult skin. In hair follicles, trichohyalin expression was stable without change from 20 wk estimated gestational age. These findings suggest that fetal skin may have different sets of barriers from the second trimester; the immature cornified cell envelope is formed in the early second trimester and the mature cornified cell envelope is formed in the late second or early third trimester when filaggrin and trichohyalin appear.

Age-related changes of water content in the rat skin.

The water content of the skin is greatly influenced by ground substances, which may be responsible for wrinkling and laxity of the skin accompanying the cutaneous aging. Therefore, water content in the skin is presumed to be a critical determinant in cutaneous aging. This study was aimed at clarifying the change in water content and the content of glycosaminoglycans (GAG) of rat skin in relation to aging. Sprague-Dawley rats were divided into 6 groups: 1-, 3-, 6-, 12-, 18- and 24-month-old groups. Two-to-three grams of skin tissue samples were taken from the back, and a half of sample was dried at 160 degrees C for 30 min with electronic moisture balance, and water content was assessed as decreased weight by heating. To measure change of GAG of the rat skin, another half of samples were extracted with 0.1 M sodium phosphate buffer (pH 7.4 NaPB) and 2 M guanidine-HCl/Tris buffer (pH 7.4). The resultant insoluble pellet was dried at 50 degrees C in a drying over for 72 h after two washings and the dry weight was recorded. The amount of sulfated GAG in the skin extracts was measured by alcian blue precipitation assay, and the amount of uronic acid (UA) was assayed in the skin tissue extracts and the dried skin using the carbazole reaction. The water content of the rat skin decreased with age, and a similar decreasing pattern in the amount of sulfated GAG and UA of the rat skin tissue was observed with aging. One hundred times of UA was obtained in dry rat skin tissue, as compared with that of the skin extracts. In conclusion, there occurs a significant decrease of water content in the aged rat skin, which may be related to the change of GAG with intrinsic aging of the skin.

Comparative review of diagnostic accuracy in tubal pregnancy: a 14-year survey of 1040 cases.

This paper summarizes our experience from 1972-1985 with the changing tools used in the diagnosis of ectopic pregnancy. The study was divided into three time periods based on the diagnostic methods used in each. In the first period (1972-1975), diagnosis was by culdocentesis and the urine pregnancy test. In the second period (1976-1978), diagnostic laparoscopy was added. Finally, in the third period (1979-1985), serum beta-human chorionic gonadotropin (beta-hCG) assay and pelvic ultrasonography were added. A rising trend in diagnostic accuracy was observed from the first period to the third. The predictability and the negative predictive value of diagnostic tests in the third period were 96.3 and 94.9%, respectively--significantly higher than in the first and second periods. The incidence of unruptured tubal pregnancy increased significantly, from 27.6% in the first and the second periods to 42.5% in the third period. Laparoscopy, ultrasonography, and serum beta-hCG assay each revealed a positive rate of more than 90% in the diagnosis of tubal pregnancy. Laparoscopy had the highest positive predictive value as a single test in the third period. Among suspected cases, however, laparoscopy was found to have a low accuracy in discriminating between tubal pregnancy and infection. The combination of serum beta-hCG testing and ultrasonography produced a 99% positive predictive value, and revealed a higher degree of accuracy in the diagnosis of tubal pregnancy than any other single test or combination of tests.

Caffeine inhibits exercise-induced increase in tryptophan hydroxylase expression in dorsal and median raphe of Sprague-Dawley rats.

Effect of caffeine on the expression of tryptophan hydroxylase (TPH), rate limiting enzyme of serotonin synthesis, in dorsal and median raphe was investigated via immunohistochemistry. In exercise groups, Sprague-Dawley rats were put on treadmill running for 30 min per day for 6 consecutive days. On the seventh day, animals of control-with-caffeine group were injected subcutaneously with 4 mg/kg caffeine, while control-without-caffeine group were injected with 0.9% NaCl, sacrificed 2 h later. Exercise-with-caffeine group and exercise-without-caffeine group were injected with caffeine and NaCl, respectively; all-out time was determined 1 h after injection, and then sacrificed. Caffeine increased all-out time in exercised rats, and inhibited the exercise-induced elevation in TPH expression. The suppressive effect of caffeine on TPH expression in exercised rats can be suggested as one possible ergogenic mechanism of caffeine.

T-cell functions in ovarian cancer patients treated with viral oncolysates: I. Increased helper activity to immunoglobulins production.

We investigated the humoral and cellular responses of ovarian cancer patients treated with viral oncolysates (VO) tumor vaccines. All patients treated with VO developed tumor cell surface-reacting antibodies and anti-hemagglutinin antibodies. Furthermore, PBMC from these patients developed cellular proliferative responses to the cellular oncolysates (CO). The in vitro proliferating population consisted mainly of CD4+ cells, as demonstrated by depletion experiments using the corresponding monoclonal antibodies and complement and by examination of the phenotype of the cells stimulated in culture with VO or cellular oncolysates. Furthermore, in the patients treated with VO we observed an increased helper activity to immunoglobulin production by PBMC collected post-VO treatment in the PWM-driven differentiation system. The helper activity was much higher in patients treated with IL-2 plus VO than IL-2 alone.

Pyridindolols K1 and K2, new alkaloids from Streptomyces sp. K93-0711.

Two new alkaloids with the beta-carboline skeleton, pyridindolols K1 (C18H18N2O5) and K2 (C16H16N2O4), were isolated from the culture both of Streptomyces sp. K93-0711. The structure of pyridindolols were established by spectroscopic investigations and chemical transformations. Pyridindolol K2 inhibited the adhesion of HL-60 cells to LPS-activated HUVEC monolayer (IC50 = 75 micrograms/ml).

Macrosphelide, a novel inhibitor of cell-cell adhesion molecule. II. Physiochemical properties and structural elucidation.

New anticell-adherence compounds, macrosphelides A and B, were isolated from the fermentation broth of Microsphaeropis sp. FO-5050, and their structures were elucidated by spectroscopic methods and chemical transformations. Macrosphelides A (M.W. 342, C16H22O8) and B (M.W. 340, C16H20O8) with three esters in their molecules were classified as 16-membered macrocyclic compounds. Macrosphelide B was found to be a corresponding oxidative product of macrosphelide A at the C-14 position.

Macrosphelide, a novel inhibitor of cell-cell adhesion molecule. I. Taxonomy, fermentation, isolation and biological activities.

Potent anti-adherent activity was detected in fermentation extracts of microbial strain FO-5050. Active compounds designated macrosphelide A and B were isolated and the structure was determined to be 16-membered macrolide antibiotics possessing three ester bonds in the ring structure. Macrosphelide A dose-dependently inhibited the adhesion of HL-60 cells to LPS-activated HUVEC monolayer (IC50, 3.5 microM); macrophelide B also inhibited HL-60 adhesion but to a lesser extent (IL50, 36 microM). Macrosphelide A did not show any antimicrobial and cytocidal activities at the concentration of 1000 micrograms/ml and 100 micrograms/ml, respectively.

Chlovalicin, a new cytocidal antibiotic produced by Sporothrix sp. FO-4649. I. Taxonomy, fermentation, isolation and biological activities.

Selective growth inhibition against IL-6 dependent cells was detected in fermentation extracts of a fungal strain FO-4649 which was characterized as Sporothrix species. An active metabolite (1) termed chlovalicin was isolated together with ovalicin and two other ovalicin derivatives (compounds 3 and 4). Chlovalicin, a ovalicin derivative with a chlorinated methylene moiety at the C-1 position of the cyclohexane ring, dose-dependently inhibited the growth of IL-6 dependent MH60 cells (IC50, 7.5 microM) in the presence of 0.2 U/ml IL-6 and, to a lesser extent, the growth of B16 melanoma cells (IC50, 38 microM). Among the other three compounds, only ovalicin showed inhibitory activity (IC50, 27 microM) against MH60 cells. These four compounds did not show any antimicrobial activity at a concentration of 1000 micrograms/ml.

Chlovalicin, a new cytocidal antibiotic produced by Sporothrix sp. FO-4649. II. Physicochemical properties and structural elucidation.

A new growth inhibitor of IL-6 responsive MH60 cells, chlovalicin (MW; 332, C16H25O5Cl), was found in cultures of Sporothrix sp. FO-4649, together with a known sesquiterpene, ovalicin. The structure of chlovalicin was elucidated by spectroscopic methods. Chlovalicin possesses a chlorinated methylene moiety at the C-1 position, and it corresponds to halogenated products derived from the epoxide ring attached to the C-1 position of ovalicin. The absolute configuration of chlovalicin was clarified as 1S, 2R, 3S, 1'S, 2'R by chemical transformation from ovalicin.

Madindoline, a novel inhibitor of IL-6 activity from Streptomyces sp. K93-0711. I. Taxonomy, fermentation, isolation and biological activities.

Selective growth inhibition against IL-6-dependent cells was detected in fermentation extracts of a microbial strain, K93-0711, which was characterized as Streptomyces species. Active metabolite, termed madindoline A and B, were isolated, and the structure was determined to be 3a-hydroxy-indoline with diketocyclopentene at the N position. Madindoline A and B displayed dose-dependent inhibition of MH60 cells, an IL-6-dependent cell line, in presence of 0.1 U/ml IL-6. The IC50 for madindoline A and B against this cell line was 8 microM and 30 microM, respectively. These compounds did not inhibit the growth of cell lines which are not IL-6 dependent and the growth inhibition of the MH60 cell line was reversed by addition of excess, 0.4 U/ml, of IL-6 to the culture media. These compounds did not show any antimicrobial activity at a concentration of 1,000 micrograms/ml.