scholarly journals 5-Fluoromethylornithine, an irreversible and specific inhibitor of l-ornithine:2-oxo-acid aminotransferase

1988 ◽  
Vol 253 (2) ◽  
pp. 481-488 ◽  
Author(s):  
G Daune ◽  
F Gerhart ◽  
N Seiler
Keyword(s):  
Amino Acid ◽  
Acute Treatment ◽  
Specific Inhibitor ◽  
Repeated Administration ◽  
Total Activity ◽  
Toxic Effects ◽  
Irreversible Inhibitor ◽  
Pathological Conditions ◽  
Repeated Doses

5-Fluoromethylornithine (5-FMOrn) is the first specific irreversible inhibitor of L-ornithine:2-oxoacid aminotransferase (OAT) found. Single doses (greater than 10 mg/kg) of this compound inactivate OAT to a residual OAT-like activity. This activity (10-20% of total activity) is resistant to further inactivation by higher or repeated doses of 5-FMOrn, or incubation with the inactivator in vitro. Ornithine concentrations are greatly enhanced in various tissues, and urinary ornithine is dramatically increased, but no other amino acid is affected after acute treatment with 5-FMOrn. Repeated administration decreases carnosine and homocarnosine concentrations in brain. Toxic effects were not observed. The new inactivator is considered as a tool in the establishment of functions of OAT under physiological and pathological conditions.

scholarly journals Triphasic Response of Rat Intracerebral Arterioles to Increasing Concentrations of Vasopressin in vitro

1993 ◽  
Vol 13 (2) ◽  
pp. 304-309 ◽  
Author(s):  
Masakazu Takayasu ◽  
Yasukazu Kajita ◽  
Yoshio Suzuki ◽  
Masato Shibuya ◽  
Kenichiro Sugita ◽  
...  
Keyword(s):  
Subarachnoid Hemorrhage ◽  
Vascular Tone ◽  
Cerebral Arteries ◽  
Specific Inhibitor ◽  
In Vitro Study ◽  
Relaxing Factor ◽  
Pathological Conditions ◽  
Cerebral Arterioles ◽  
Endothelium Derived Relaxing Factor

To determine how vasopressin affects the vascular tone of the smaller cerebral arterioles, we carried out an in vitro study of isolated and cannulated intracerebral arterioles of rats. We found that increasing concentrations of vasopressin induced a triphasic response of vasodilation (10−12–10−11 M), vasoconstriction (10−10–10−8 M), and vasodilation stabilizing to control diameter (10−7–10−6 M) and that the maximum constriction was twice the maximum dilation in these smaller arterioles [21.2 ± 13.1% (mean ± SD) decrease in diameter vs. 11.2 ± 5.7% increase]. Pretreatment of the arterioles with NG-monomethyl-l-arginine (10−4 M), a specific inhibitor of endothelium-derived relaxing factor, abolished the vasopressin-induced vasodilation and significantly increased the vasoconstriction. These results suggest that these arterioles were maintained in a dilated state by an endothelium-derived relaxing factor activated by vasopressin. Both vasodilation and vasoconstriction were found to be mediated through vasopressin V1 receptors in a study of arterioles pretreated with d(CH2)5Tyr(Me)arginine vasopressin (10−6 M), a vasopressin V1 receptor antagonist. These results support the hypothesis that vasopressin may constrict smaller cerebral arterioles while simultaneously dilating larger cerebral arteries. Our results also suggest that vasopressin may aggravate cerebral ischemia in pathological conditions, such as subarachnoid hemorrhage, when the arteriolar response to vasopressin shifts from vasodilation to vasoconstriction due to increased vasopressin levels in plasma and CSF and impaired endothelium-derived relaxation.

scholarly journals Mechanism by Which Mutations at His274 Alter Sensitivity of Influenza A Virus N1 Neuraminidase to Oseltamivir Carboxylate and Zanamivir

2002 ◽  
Vol 46 (12) ◽  
pp. 3809-3816 ◽  
Author(s):  
Michael Z. Wang ◽  
Chun Y. Tai ◽  
Dirk B. Mendel
Keyword(s):  
Drug Resistance ◽  
Amino Acid ◽  
Influenza A ◽  
Specific Inhibitor ◽  
Side Chain ◽  
Amino Acid Side Chain ◽  
Oseltamivir Carboxylate ◽  
Influenza A H1n1 ◽  
Binding Inhibition

ABSTRACT Oseltamivir carboxylate is a potent and specific inhibitor of influenza neuraminidase (NA). An influenza A/H1N1 variant selected in vitro with reduced susceptibility to oseltamivir carboxylate contains a His274Tyr mutation. To understand the mechanism by which a His274Tyr mutation gives rise to drug resistance, we studied a series of NA variant proteins containing various substitutions at position 274. Replacement of His274 with larger side chain residues (Tyr or Phe) reduced the NA sensitivity to oseltamivir carboxylate. In contrast, replacement of His274 with smaller side chain residues (Gly, Asn, Ser, and Gln) resulted in enhanced or unchanged sensitivity to oseltamivir carboxylate. Previous studies have suggested that the slow-binding inhibition of NA by oseltamivir carboxylate is a result of the reorientation of Glu276. Loss of this slow-binding inhibition in the His274Tyr and His274Phe mutant NA but not in His274Asn, His274Gly, His274Ser, or His274Gln supports the conclusion that the conformational change of Glu276 is restricted in the His274Tyr and His274Phe mutant NA upon oseltamivir carboxylate binding. Interestingly, His274Asn, as well as His274Gly, His274Ser, and His274Gln, also displayed reduced sensitivity to zanamivir and its analogue, 4-amino-Neu5Ac2en. Substitution of His274 with Tyr in influenza A/Tokyo/3/67 (H3N2) recombinant NA did not affect the susceptibility to oseltamivir carboxylate. These data indicate that the volume occupied by the amino acid side chain at position 274 can influence the sensitivities of influenza N1 NA but not of N2 NA to both oseltamivir carboxylate and zanamivir.

scholarly journals Effect of Epoxomicin on 3D Neurosphere

2014 ◽  
Author(s):  
Khaled Youssef ◽  
Mohammed Salama ◽  
Mohamed Elhosseny ◽  
Bahaaeldin Awad ◽  
Ahmed Lotfy ◽  
...  
Keyword(s):  
Early Childhood ◽  
Single Cells ◽  
Placental Tissue ◽  
Toxic Effects ◽  
Predictive Toxicology ◽  
Focal Lesions ◽  
Irreversible Inhibitor ◽  
Trypan Blue Exclusion ◽  
Proliferation Medium

Developmental neurotoxicity (DNT) entails the toxic effects imparted by many chemicals on brain during early childhood. Various chemicals would have their maximum effects on brains during early childhood. Some toxicants have confirmed to induce developmental toxic effects on CNS. Most of agents cannot be identified with certainty due to the defective nature of predictive toxicology models used. A novel method that can overcome most of the limitations of conventional techniques is the use of 3D neurospheres system (in-vitro system can recapitulate most of the changes during the period of brain development making it an ideal model for predicting neurotoxic effects). In the present study we verified the possible DNT of epoxomicin which is a potent anti-tumor agent isolated from Actinomycetes that is used as a selective and irreversible inhibitor of the 20S proteasome with anti-inflammatory activity. Methods Rat neural progenitor cells were isolated from rat embryos (E14) extracted from placental tissue. Cortices were aseptically dissected from brains of the fetuses and the tissues were triturated by repeated passage through a fire-polished constricted Pasteur pipette. The dispersed tissues were allowed to settle for 3 min. The supernatant was then transferred to a fresh tube and centrifuged at 1,000 g for 5 min. The pellet was placed in Hank’s balanced salt solution cultured as free-floating neurospheres in proliferation medium. Two doses of epoxomicin (1µM and 10µM) were used in cultured neurospheres for a period of 14 days. For proliferation analysis, spheres were cultured in proliferation medium. After 0,4,5,11 and 14 days, sphere size was determined by software analyses (CellProfiler, Broad Institute, www.cellprofiler.org) Diameter of each neurosphere was measured and exported as excel file to statistical analysis. For viability analysis, trypsin-EDTA solution were added to neurospheres for 3 min to dissociate them into single cells suspension, then viability was evaluated by the Trypan Blue exclusion test. Epoxomicin found to affect differentiation and viability of neurospheres, these effects were positively correlated to doses and progress of time. Results Epoxomicin found to affect proliferation and viability of neuropsheres. Conclusion The study confirms DNT of epoxomicin and suggests possible gross morphologic changes and the decrease in viability, it proposes possible focal lesions on exposure to epoxomicin in early childhood.

scholarly journals Graphene Oxide Exacerbates Dextran Sodium Sulfate-Induced Colitis via ROS/AMPK/p53 Signaling to Mediate Apoptosis

2021 ◽  
Author(s):  
Siliang Liu ◽  
Angao Xu ◽  
Yanfei Gao ◽  
Yue Xie ◽  
Zhipeng Liu ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Sodium Sulfate ◽  
Specific Inhibitor ◽  
Lactic Dehydrogenase ◽  
Dextran Sodium Sulfate ◽  
Toxic Effects ◽  
Intestinal Epithelial ◽  
P53 Pathway ◽  
Compound C

Abstract Background: Graphene oxide (GO), a novel carbon-based nanomaterial, has promising applications in biomedicine. However, it induces potential cytotoxic effects on the gastrointestinal (GI) tract cells, and these effects have been largely uncharacterized. The present study aimed to explore the toxic effects of GO on the intestinal tract especially under pre-existing inflammatory conditions, such as inflammatory bowel disease (IBD), and elucidate underlying mechanisms.Results: Our findings indicated that oral gavage of GO worsened acute colitis induced by 2.5% dextran sodium sulfate in mice. In vitro, toxic effects including decreased cell viability, as well as the elevated release of lactic dehydrogenase (LDH) in FHC cell line, an ideal model of intestinal epithelial cells (IECs), were observed. Moreover, GO treatment triggered apoptosis in FHC cells through the activation of reactive oxygen species (ROS)/AMP-activated protein kinase (AMPK)/p53 pathway, as evidenced by the upregulation of cytochrome c (Cytc), Bax, and cleaved caspase-3 (c-cas3) and the downregulation of Bcl-2. Interestingly, pretreatment with an antioxidant, N-acetyl-L-cysteine, and a specific inhibitor of AMPK activation, Compound C (Com.C), effectively inhibited GO-induced apoptosis in FHC cells.Conclusions: Our data demonstrate that GO-induced IECs apoptosis via ROS/AMPK/p53 pathway activation accounts for the exacerbation of colitis in mice. These findings provide a new insight into the pathogenesis of IBD induced by environmental factors. Furthermore, our findings enhance our understanding of GO as a potential environmental toxin, which helps delineate the risk of exposure to patients with disturbed intestinal epithelial barrier/inflammatory disorders such as IBD.

Effects of D-amino acid substituents on degradation of LHRH analogues by proximal tubule

1987 ◽  
Vol 252 (3) ◽  
pp. E320-E326 ◽  
Author(s):  
G. Flouret ◽  
T. Majewski ◽  
D. R. Peterson ◽  
A. J. Kenny ◽  
F. A. Carone
Keyword(s):  
Amino Acid ◽  
Specific Inhibitor ◽  
Multiple Bonds ◽  
Lhrh Analogues ◽  
Angiotensin I Converting Enzyme ◽  
Endopeptidase 24.11 ◽  
Renal Brush Border Membranes ◽  
Hydrolysis Of

Less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, the luteinizing hormone-releasing hormone, LHRH, is degraded in renal proximal tubules (PT) in vivo (rat) and in vitro (rabbit) to less than Glu-His (2), less than Glu-His-Trp (3), and less than Glu-His-Trp-Ser (4). LHRH may be cleaved by endopeptidases simultaneously at multiple bonds, or initially at Ser4-Tyr5 followed by carboxypeptidase hydrolysis of 4 to 3 and then 2. To distinguish between these mechanisms, [3H]LHRH analogues were incubated with rabbit renal brush-border membranes (BBM), microinfused into PT in vivo or in vitro, and products were analyzed by HPLC. [D-Ser4]LHRH was not cleaved at D Ser4-Tyr5 but yielded less than Glu-His-Trp-D-Ser-Tyr-Gly as the major metabolite plus 2 and 3. [D-Trp6]LHRH was cleaved by BBM and PT to 2 and 3, but not to 4. [D-Ser4, D-Trp6]LHRH was not cleaved by BBM, but was degraded to 2 by PT in vivo. Thus, D-amino acid substituents altered the expected cleavage pattern of these analogues. [3H]LHRH was cleaved by BBM or by endopeptidase-24.11 from porcine PT to metabolites 2, 4, small amounts of 3, and less than Glu-His-Trp-Ser-Tyr-Gly, but cleavage was strongly inhibited by the specific inhibitor phosphoramidon. Thus, normally LHRH may be cleaved in PT by endopeptidase-24.11 to 2 and 4, and by angiotensin I-converting enzyme to 3, its known cleavage site.

scholarly journals Glycine, the smallest amino acid, confers neuroprotection against d-galactose-induced neurodegeneration and memory impairment by regulating c-Jun N-terminal kinase in the mouse brain

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Rahat Ullah ◽  
Myeung Hoon Jo ◽  
Muhammad Riaz ◽  
Sayed Ibrar Alam ◽  
Kamran Saeed ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Amino Acid ◽  
Protein Expression ◽  
Mouse Brain ◽  
Memory Impairment ◽  
Specific Inhibitor ◽  
Synaptic Dysfunction ◽  
Ht22 Cells

Abstract Background Glycine is the smallest nonessential amino acid and has previously unrecognized neurotherapeutic effects. In this study, we examined the mechanism underlying the neuroprotective effect of glycine (Gly) against neuroapoptosis, neuroinflammation, synaptic dysfunction, and memory impairment resulting from d-galactose-induced elevation of reactive oxygen species (ROS) during the onset of neurodegeneration in the brains of C57BL/6N mice. Methods After in vivo administration of d-galactose (d-gal; 100 mg/kg/day; intraperitoneally (i/p); for 60 days) alone or in combination with glycine (1 g/kg/day in saline solution; subcutaneously; for 60 days), all of the mice were sacrificed for further biochemical (ROS/lipid peroxidation (LPO) assay, Western blotting, and immunohistochemistry) after behavioral analyses. An in vitro study, in which mouse hippocampal neuronal HT22 cells were treated with or without a JNK-specific inhibitor (SP600125), and molecular docking analysis were used to confirm the underlying molecular mechanism and explore the related signaling pathway prior to molecular and histological analyses. Results Our findings indicated that glycine (an amino acid) inhibited d-gal-induced oxidative stress and significantly upregulated the expression and immunoreactivity of antioxidant proteins (Nrf2 and HO-1) that had been suppressed in the mouse brain. Both the in vitro and in vivo results indicated that d-gal induced oxidative stress-mediated neurodegeneration primarily by upregulating phospho-c-Jun N-terminal kinase (p-JNK) levels. However, d-gal + Gly cotreatment reversed the neurotoxic effects of d-gal by downregulating p-JNK levels, which had been elevated by d-gal. We also found that Gly reversed d-gal-induced neuroapoptosis by significantly reducing the protein expression levels of proapoptotic markers (Bax, cytochrome c, cleaved caspase-3, and cleaved PARP-1) and increasing the protein expression level of the antiapoptotic protein Bcl-2. Both the molecular docking approach and the in vitro study (in which the neuronal HT22 cells were treated with or without a p-JNK-specific inhibitor (SP600125)) further verified our in vivo findings that Gly bound to the p-JNK protein and inhibited its function and the JNK-mediated apoptotic pathway in the mouse brain and HT22 cells. Moreover, the addition of Gly alleviated d-gal-mediated neuroinflammation by inhibiting gliosis via attenuation of astrocytosis (GFAP) and microgliosis (Iba-1) in addition to reducing the protein expression levels of various inflammatory cytokines (IL-1βeta and TNFα). Finally, the addition of Gly reversed d-gal-induced synaptic dysfunction by upregulating the expression of memory-related presynaptic protein markers (synaptophysin (SYP), syntaxin (Syn), and a postsynaptic density protein (PSD95)) and markedly improved behavioral measures of cognitive deficits in d-gal-treated mice. Conclusion Our findings demonstrate that Gly-mediated deactivation of the JNK signaling pathway underlies the neuroprotective effect of Gly, which reverses d-gal-induced oxidative stress, apoptotic neurodegeneration, neuroinflammation, synaptic dysfunction, and memory impairment. Therefore, we suggest that Gly (an amino acid) is a safe and promising neurotherapeutic candidate that might be used for age-related neurodegenerative diseases.

Metalloprotease inhibitor profiles of human ADAM8 in vitro and in cell-based assays

2019 ◽  
Vol 400 (6) ◽  
pp. 801-810 ◽  
Author(s):  
Uwe Schlomann ◽  
Kristina Dorzweiler ◽  
Elisa Nuti ◽  
Tiziano Tuccinardi ◽  
Armando Rossello ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Specific Inhibitor ◽  
Hek293 Cells ◽  
Ectodomain Shedding ◽  
Pathological Conditions ◽  
Catalytic Sites ◽  
Affinity Receptor ◽  
Metalloprotease Inhibitor

AbstractADAM8 as a membrane-anchored metalloproteinase-disintegrin is upregulated under pathological conditions such as inflammation and cancer. As active sheddase, ADAM8 can cleave several membrane proteins, among them the low-affinity receptor FcεRII CD23. Hydroxamate-based inhibitors are routinely used to define relevant proteinases involved in ectodomain shedding of membrane proteins. However, for ADAM proteinases, common hydroxamates have variable profiles in their inhibition properties, commonly known for ADAM proteinases 9, 10 and 17. Here, we determined the inhibitor profile of human ADAM8 for eight ADAM/MMP inhibitors byin vitroassays using recombinant ADAM8 as well as thein vivoinhibition in cell-based assays using HEK293 cells to monitor the release of soluble CD23 by ADAM8. ADAM8 activity is inhibited by BB94 (Batimastat), GW280264, FC387 and FC143 (two ADAM17 inhibitors), made weaker by GM6001, TAPI2 and BB2516 (Marimastat), while no inhibition was observed for GI254023, an ADAM10 specific inhibitor. Modeling of inhibitor FC143 bound to the catalytic sites of ADAM8 and ADAM17 reveals similar geometries in the pharmacophoric regions of both proteinases, which is different in ADAM10 due to replacement in the S1 position of T300 (ADAM8) and T347 (ADAM17) by V327 (ADAM10). We conclude that ADAM8 inhibitors require maximum selectivity over ADAM17 to achieve specific ADAM8 inhibition.

Effects of chloracetophenone and di- iso propyl fluorophosphonate on amphibian eggs

1951 ◽  
Vol 138 (893) ◽  
pp. 575-599 ◽  
Keyword(s):  
Hydrolysis Product ◽  
Specific Inhibitor ◽  
Subsequent Treatment ◽  
Irreversible Inhibitor ◽  
Animal Pole ◽  
Developmental Arrest ◽  
Vegetal Pole ◽  
Micro Organisms ◽  
Giant Nuclei

Chloracetophenone, a specific and powerful —SH reactant and an irreversible inhibitor of —SH enzymes, is highly toxic to frog and newt eggs and to frog tadpoles. The toxic action on tadpoles cannot be reversed by subsequent treatment with cysteine, but can be nullified by simultaneous presence of cysteine. Frog and newt eggs exposed to minute concentrations of chloracetophenone comparable with those required for inhibition of —SH enzymes in vitro exhibit abnormalities in development that have been called the ‘blocked —SH syndrome’. Some items of the syndrome are: ( a ) the development in the frog of heteroploid pigmented ectodermal and epidermal cells, the unpigmented cells being unaffected, and, in the newt, the development of giant nuclei, presumed to be heteroploid: ( b ) cleavage of animal pole while vegetal pole is inhibited; ( c ) a developmental arrest at gastrulation under various dosage conditions. The effects are not due to a hydrolysis product or an altered pH in the solution. Cilia continue to beat in cells damaged by CAP. Frog eggs exposed to di- iso propyl fluorophosphonate, a powerful and specific inhibitor of esterases, and not an —SH reactant, do not develop giant or heteroploid cells or a ‘blocked —SH syndrome’, and show a developmental arrest just before gastrulation. —SH reagents are considered to constitute an important class of c -mitotically active substances. It is concluded that their c -mitotic action is, in fact, due to blocking of —SH groups, probably of enzymes. Cell division is prevented by cytoplasmic inhibition, either with or without spindle inhibition, the result being different types of heteroploidy and polynuclearity. The conclusions concerning mitosis apply also to plants, and have relations with growth processes in micro-organisms. The mode of action of —SH poisons is discussed.

Role of tyrosine kinases in gastrin induction of ornithine decarboxylase in colonic mucosa

1990 ◽  
Vol 259 (4) ◽  
pp. G626-G630 ◽  
Author(s):  
A. P. Majumdar
Keyword(s):  
Ornithine Decarboxylase ◽  
Tyrosine Kinases ◽  
Colonic Mucosa ◽  
Specific Inhibitor ◽  
Irreversible Inhibitor ◽  
Mucosal Explants ◽  
Tyrosyl Phosphorylation ◽  
Stimulation Of

An organ culture system was utilized to evaluate the role of tyrosine kinases (Tyr-k) and tyrosine-specific phosphorylation of proteins in gastrin regulation of ornithine decarboxylase (ODC) activity in colonic mucosa. Exposure of colonic mucosal explants to gastrin (50-100 ng G-17 I/ml) resulted in a profound stimulation of both Tyr-k and ODC activities compared with the corresponding basal levels. Whereas the maximal stimulation (ranging between 70 and 150%) of Tyr-k occurred within 10-15 min of exposure to gastrin, ODC activity was significantly stimulated (180%) 2 h after exposure to the hormone, and at 4 h it was found to be 750% above the corresponding basal level. Difluoromethylornithine (DFMO; 2 mM), an irreversible inhibitor of ODC, completely abolished the gastrin-mediated stimulation of ODC but not Tyr-k activity. On the other hand, genistein (100 micrograms/ml), a specific inhibitor of Tyr-k, caused a total suppression of the gastrin-induced stimulation of both Tyr-k and ODC. Gastrin also stimulated tyrosyl phosphorylation of a colonic mucosal membrane protein with molecular mass of 57 kDa, and genistein greatly attenuated this effect. We conclude that gastrin stimulates colonic mucosal ODC in vitro, and Tyr-k may be required for the regulation of this process.

Branched-chain amino acid metabolism in rat muscle: abnormal regulation in acidosis

1987 ◽  
Vol 252 (6) ◽  
pp. E712-E718 ◽  
Author(s):  
R. C. May ◽  
Y. Hara ◽  
R. A. Kelly ◽  
K. P. Block ◽  
M. G. Buse ◽  
...  
Keyword(s):  
Metabolic Acidosis ◽  
Amino Acid ◽  
Acid Metabolism ◽  
Muscle Protein ◽  
Total Activity ◽  
Keto Acid ◽  
Acid Dehydrogenase ◽  
Branched Chain Amino Acid ◽  
Pathological Conditions ◽  
Branched Chain

Branched-chain amino acid (BCAA) metabolism is frequently abnormal in pathological conditions accompanied by chronic metabolic acidosis. To study how metabolic acidosis affects BCAA metabolism in muscle, rats were gavage fed a 14% protein diet with or without 4 mmol NH4Cl X 100 g body wt-1 X day-1. Epitrochlearis muscles were incubated with L-[1-14C]-valine and L-[1-14C]leucine, and rates of decarboxylation, net transamination, and incorporation into muscle protein were measured. Plasma and muscle BCAA levels were lower (P less than 0.05) in acidotic rats. Rates of valine and leucine decarboxylation and net transamination were higher (P less than 0.05) in muscles from acidotic rats; these differences were associated with a 79% increase in the total activity of branched-chain alpha-keto acid dehydrogenase and a 146% increase in the activated form of the enzyme. We conclude that acidosis affects the regulation of BCAA metabolism by enhancing flux through the transaminase and by directly stimulating oxidative catabolism through activation of branched-chain alpha-keto acid dehydrogenase.

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