scholarly journals Kangaroo IGF-II is structurally and functionally similar to the human [Ser29]-IGF-II variant

1999 ◽  
Vol 161 (3) ◽  
pp. 445-453 ◽  
Author(s):  
CA Yandell ◽  
GL Francis ◽  
JF Wheldrake ◽  
Z Upton
Keyword(s):  
Amino Acid ◽  
Human Serum ◽  
Cdna Sequence ◽  
Mammalian Species ◽  
In Vitro Assays ◽  
Igf Binding Proteins ◽  
Western Grey Kangaroo ◽  
Minor Form ◽  
A Minor

Kangaroo IGF-II has been purified from western grey kangaroo (Macropus fuliginosus) serum and characterised in a number of in vitro assays. In addition, the complete cDNA sequence of mature IGF-II has been obtained by reverse-transcription polymerase chain reaction. Comparison of the kangaroo IGF-II cDNA sequence with known IGF-II sequences from other species revealed that it is very similar to the human variant, [Ser29]-hIGF-II. Both the variant and kangaroo IGF-II contain an insert of nine nucleotides that encode the amino acids Leu-Pro-Gly at the junction of the B and C domains of the mature protein. The deduced kangaroo IGF-II protein sequence also contains three other amino acid changes that are not observed in human IGF-II. These amino acid differences share similarities with the changes described in many of the IGF-IIs reported for non-mammalian species. Characterisation of human IGF-II, kangaroo IGF-II, chicken IGF-II and [Ser29]-hIGF-II in a number of in vitro assays revealed that all four proteins are functionally very similar. No significant differences were observed in the ability of the IGF-IIs to bind to the bovine IGF-II/cation-independent mannose 6-phosphate receptor or to stimulate protein synthesis in rat L6 myoblasts. However, differences were observed in their abilities to bind to IGF-binding proteins (IGFBPs) present in human serum. Kangaroo, chicken and [Ser29]-hIGF-II had lower apparent affinities for human IGFBPs than did human IGF-II. Thus, it appears that the major circulating form of IGF-II in the kangaroo and a minor form of IGF-II found in human serum are structurally and functionally very similar. This suggests that the splice site that generates both the variant and major form of human IGF-II must have evolved after the divergence of marsupials from placental mammals.

Decreases in ovine fetal cartilage sulfate uptake and serum sulfate during gestation

1985 ◽  
Vol 249 (1) ◽  
pp. E115-E120
Author(s):  
F. H. Morriss ◽  
R. N. Marshall ◽  
S. S. Crandell ◽  
B. J. Fitzgerald ◽  
L. Riddle
Keyword(s):  
Human Serum ◽  
Costal Cartilage ◽  
Full Term ◽  
Late Gestation ◽  
In Vitro Assays ◽  
Sulfate Uptake ◽  
Fetal Sheep ◽  
Ovine Fetal ◽  
Serum Sulfate

In vitro assays for [35S]sulfate uptake by ovine fetal costal cartilage were used to assess gestational changes in cartilage metabolism. Addition of 20% normal human serum to the incubation medium increased fetal cartilage [35S]sulfate incorporation into glycosaminoglycans. Both basal and human serum-stimulated uptakes of [35S]sulfate by fetal sheep cartilage decreased from midgestation to full term. The incremental response in [35S]sulfate uptake that was stimulated by human serum decreased as gestation proceeded to full-term. Fetal serum sulfate concentration decreased logarithmically during gestation, raising the possibility that cartilage sulfate uptake might become substrate limited as full term is approached. Perfusion of seven late gestation sheep fetuses for 7 days with Na2SO4 to achieve serum sulfate concentrations similar to those observed earlier in gestation resulted in a 33% increase in mean cartilage [35S]sulfate uptake compared with that of control twin fetuses, but uptake was not increased to values that occurred spontaneously earlier in gestation. These results suggest that the decreasing rate of [35S]sulfate uptake by fetal cartilage during the last half of gestation is associated only minimally with decreasing serum sulfate levels and is most consistent with intrinsic change in resting chondrocyte metabolism during gestation.

scholarly journals Purified human serum PON1 does not protect LDL against oxidation in the in vitro assays initiated with copper or AAPH

2004 ◽  
Vol 45 (12) ◽  
pp. 2260-2268 ◽  
Author(s):  
John F. Teiber ◽  
Dragomir I. Draganov ◽  
Bert N. La Du
Keyword(s):  
Human Serum ◽  
In Vitro Assays

scholarly journals Antagonism of Trichoderma harzianum ETS 323 on Botrytis cinerea Mycelium in Culture Conditions

2012 ◽  
Vol 102 (11) ◽  
pp. 1054-1063 ◽  
Author(s):  
Chi-Hua Cheng ◽  
Chia-Ann Yang ◽  
Kou-Cheng Peng
Keyword(s):  
Amino Acid ◽  
Botrytis Cinerea ◽  
Trichoderma Harzianum ◽  
Hyphal Growth ◽  
Extracellular Proteins ◽  
In Vitro Assays ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Trichoderma Spp

Previous studies have shown that the extracellular proteins of Trichoderma harzianum ETS 323 grown in the presence of deactivated Botrytis cinerea in culture include a putative l-amino acid oxidase and have suggested the involvement of this enzyme in the antagonistic mechanism. Here, we hypothesized that the mycoparasitic process of Trichoderma spp. against B. cinerea involves two steps; that is, an initial hyphal coiling stage and a subsequent hyphal coiling stage, with different coiling rates. The two-step antagonism of T. harzianum ETS 323 against B. cinerea during the mycoparasitic process in culture was evaluated using a biexponential equation. In addition, an l-amino acid oxidase (Th-l-AAO) was identified from T. harzianum ETS 323. The secretion of Th-l-AAO was increased when T. harzianum ETS 323 was grown with deactivated hyphae of B. cinerea. Moreover, in vitro assays indicated that Th-l-AAO effectively inhibited B. cinerea hyphal growth, caused cytosolic vacuolization in the hyphae, and led to hyphal lysis. Th-l-AAO also showed disease control against the development of B. cinerea on postharvest apple fruit and tobacco leaves. Furthermore, an apoptosis-like response, including the generation of reactive oxygen species, was observed in B. cinerea after treatment with Th-l-AAO, suggesting that Th-l-AAO triggers programmed cell death in B. cinerea. This may be associated with the two-step antagonism of T. harzianum ETS 323 against B. cinerea.

scholarly journals Pyridoxal-5′-phosphate as an oxygenase cofactor: Discovery of a carboxamide-forming, α-amino acid monooxygenase-decarboxylase

2018 ◽  
Vol 115 (5) ◽  
pp. 974-979 ◽  
Author(s):  
Ying Huang ◽  
Xiaodong Liu ◽  
Zheng Cui ◽  
Daniel Wiegmann ◽  
Giuliana Niro ◽  
...  
Keyword(s):  
Amino Acid ◽  
Flavin Adenine Dinucleotide ◽  
Sequence Similarity ◽  
In Vitro Assays ◽  
Complete Conversion ◽  
Mechanistic Studies ◽  
Biochemical Characteristics ◽  
Biochemical Studies ◽  
Isomeric Product

Capuramycins are antimycobacterial antibiotics that consist of a modified nucleoside named uridine-5′-carboxamide (CarU). Previous biochemical studies have revealed that CarU is derived from UMP, which is first converted to uridine-5′-aldehyde in a reaction catalyzed by the dioxygenase CapA and subsequently to 5′-C-glycyluridine (GlyU), an unusual β–hydroxy-α-amino acid, in a reaction catalyzed by the pyridoxal-5′-phosphate (PLP)-dependent transaldolase CapH. The remaining steps that are necessary to furnish CarU include decarboxylation, O atom insertion, and oxidation. We demonstrate that Cap15, which has sequence similarity to proteins annotated as bacterial, PLP-dependent l-seryl-tRNA(Sec) selenium transferases, is the sole catalyst responsible for complete conversion of GlyU to CarU. Using a complementary panel of in vitro assays, Cap15 is shown to be dependent upon substrates O2 and (5′S,6′R)-GlyU, the latter of which was unexpected given that (5′S,6′S)-GlyU is the isomeric product of the transaldolase CapH. The two products of Cap15 are identified as the carboxamide-containing CarU and CO2. While known enzymes that catalyze this type of chemistry, namely α-amino acid 2-monooxygenase, utilize flavin adenine dinucleotide as the redox cofactor, Cap15 remarkably requires only PLP. Furthermore, Cap15 does not produce hydrogen peroxide and is shown to directly incorporate a single O atom from O2 into the product CarU and thus is an authentic PLP-dependent monooxygenase. In addition to these unusual discoveries, Cap15 activity is revealed to be dependent upon the inclusion of phosphate. The biochemical characteristics along with initiatory mechanistic studies of Cap15 are reported, which has allowed us to assign Cap15 as a PLP-dependent (5′S,6′R)-GlyU:O2 monooxygenase-decarboxylase.

scholarly journals Inhibitory Effects of Amino-Acid Fluids on Drug Binding to Site II of Human Serum Albumin in Vitro

2005 ◽  
Vol 28 (3) ◽  
pp. 549-552 ◽  
Author(s):  
Keishi Yamasaki ◽  
Noriyuki Kuga ◽  
Norito Takamura ◽  
Yumiko Furuya ◽  
Muneaki Hidaka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Drug Binding ◽  
Inhibitory Effects

scholarly journals The penicillin binding protein 1A of Helicobacter pylori, its amoxicillin binding site and access routes

Gut Pathogens ◽  
2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Bahareh Attaran ◽  
Najmeh Salehi ◽  
Bahareh Ghadiri ◽  
Maryam Esmaeili ◽  
Shadi Kalateh ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
3D Structure ◽  
Resistance Mechanisms ◽  
Drug Binding ◽  
In Vitro Assays ◽  
Amino Acid Residues ◽  
Clinical Strains ◽  
H Pylori

Abstract Background Amoxicillin-resistant H. pylori strains are increasing worldwide. To explore the potential resistance mechanisms involved, the 3D structure modeling and access tunnel prediction for penicillin-binding proteins (PBP1A) was performed, based on the Streptococcus pneumoniae, PBP 3D structure. Molecular covalent docking was used to determine the interactions between amoxicillin (AMX) and PBP1A. Results The AMX-Ser368 covalent complex interacts with the binding site residues (Gly367, Ala369, ILE370, Lys371, Tyr416, Ser433, Thr541, Thr556, Gly557, Thr558, and Asn560) of PBP1A, non-covalently. Six tunnel-like structures, accessing the PBP1A binding site, were characterized, using the CAVER algorithm. Tunnel-1 was the ultimate access route, leading to the drug catalytic binding residue (Ser368). This tunnel comprises of eighteen amino acid residues, 8 of which are shared with the drug binding site. Subsequently, to screen the presence of PBP1A mutations, in the binding site and tunnel residues, in our clinical strains, in vitro assays were performed. H. pylori strains, isolated under gastroscopy, underwent AMX susceptibility testing by E-test. Of the 100 clinical strains tested, 4 were AMX-resistant. The transpeptidase domain of the pbp1a gene of these resistant, plus 10 randomly selected AMX-susceptible strains, were amplified and sequenced. Of the amino acids lining the tunnel-1 and binding site residues, three (Ser414Arg, Val469Met and Thr556Ser) substitutions, were detected in 2 of the 4 resistant and none of the sequenced susceptible strains, respectively. Conclusions We hypothesize that mutations in amino acid residues lining the binding site and/or tunnel-1, resulting in conformational/spatial changes, may block drug binding to PBP1A and cause AMX resistance.

scholarly journals Feedback Regulation of N Fixation in Frankia-Alnus Symbiosis Through Amino Acids Profiling in Field and Greenhouse Nodules

2020 ◽  
Vol 33 (3) ◽  
pp. 499-508
Author(s):  
Anne-Emmanuelle Hay ◽  
Aude Herrera-Belaroussi ◽  
Marjolaine Rey ◽  
Pascale Fournier ◽  
Philippe Normand ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nitrogen Fixation ◽  
Amino Acid ◽  
Feedback Regulation ◽  
Metabolic Model ◽  
In Vitro Assays ◽  
N Fixation ◽  
Abundance Pattern ◽  
High Level

Symbiosis established between actinorhizal plants and Frankia spp., which are nitrogen-fixing actinobacteria, promotes nodule organogenesis, the site of metabolic exchange. The present study aimed to identify amino acid markers involved in Frankia-Alnus interactions by comparing nodules and associated roots from field and greenhouse samples. Our results revealed a high level of citrulline in all samples, followed by arginine (Arg), aspartate (Asp), glutamate (Glu), γ-amino-n-butyric acid (GABA), and alanine (Ala). Interestingly, the field metabolome approach highlighted more contrasted amino acid patterns between nodules and roots compared with greenhouse samples. Indeed, 12 amino acids had a mean relative abundance significantly different between field nodule and root samples, against only four amino acids in greenhouse samples, underlining the importance of developing “ecometabolome” approaches. In order to monitor the effects on Frankia cells (respiration and nitrogen fixation activities) of amino acid with an abundance pattern evocative of a role in symbiosis, in-vitro assays were performed by supplementing them in nitrogen-free cultures. Amino acids had three types of effects: i) those used by Frankia as nitrogen source (Glu, Gln, Asp), ii) amino acids stimulating both nitrogen fixation and respiration (e.g., Cit, GABA, Ala, valine, Asn), and iii) amino acids triggering a toxic effect (Arg, histidine). In this paper, a N-metabolic model was proposed to discuss how the host plant and bacteria modulate amino acids contents in nodules, leading to a fine regulation sustaining high bacterial nitrogen fixation.

scholarly journals Regenerative Medicine: where their origin makes species meet

2019 ◽  
Vol 37 (3) ◽  
pp. 6-7
Author(s):  
Jos Malda
Keyword(s):  
Articular Cartilage ◽  
Ex Vivo ◽  
Mammalian Species ◽  
Cartilage Tissue ◽  
In Vitro Assays ◽  
Cartilage Defects ◽  
In Vivo Models ◽  
Societal Pressure

The articular cartilage of joints serves diverse functions, including absorbing shock, transmitting force, and enabling low-friction joint motion. Regeneration of articular cartilage defects remains, however, a significant challenge in both human and veterinary orthopaedic practice. Ex vivo and in vivo models play a crucial role in translating novel potential regenerative treatments from bench to bedside. However, in view of the predictive power of these models and the One Medicine concept that proclaim that there should be no dividing lines between human and animal medicine to learn, it is important to understand the similarities as well as differences in the cartilage tissue between species. To this aim, osteochondral cores of the femoral condyles were studied in 58 different mammalian species ranging from mouse to elephant. Interestingly, while biochemical composition remained relatively constant, cartilage thickness and cellularity were similar underscoring the importance of the equine species as a model for human orthopaedic interventions. Nevertheless, political ambition and societal pressure are now asking for a drastically reduction of animal experimentation and have further spiked the development of more predictive in vitro and ex vivo models. In addition to a range of more sophisticated in vitro assays, this has now also provided an ex vivo osteochondral defect model that can be generated based on equine donor tissue. Although such models better represent the situation in the native tissue and can be used to assess (osteo)chondral repair strategies, they still lack some important aspects of the in vivo (patho)physiological (inflammatory) environment, as well as the exposure to mechanical loading. This illustrates the challenges that we still face in translating these novel approaches from bench to bedside.

scholarly journals Use of In Vitro Assays To Determine Effects of Human Serum on Biological Characteristics of Acanthamoeba castellanii

2006 ◽  
Vol 44 (7) ◽  
pp. 2595-2600 ◽  
Author(s):  
J. Sissons ◽  
S. Alsam ◽  
M. Stins ◽  
A. O. Rivas ◽  
J. L. Morales ◽  
...  
Keyword(s):  
Human Serum ◽  
Acanthamoeba Castellanii ◽  
Biological Characteristics ◽  
In Vitro Assays

scholarly journals Cytotoxicity, metabolism, and isozyme mapping of the synthetic cannabinoids JWH-200, A-796260, and 5F-EMB-PINACA studied by means of in vitro systems

2021 ◽  
Author(s):  
Tanja M. Gampfer ◽  
Lea Wagmann ◽  
Anouar Belkacemi ◽  
Veit Flockerzi ◽  
Markus R. Meyer
Keyword(s):  
Phase I ◽  
Synthetic Cannabinoids ◽  
Liver Microsomes ◽  
In Vitro Assays ◽  
New Psychoactive Substances ◽  
Minor Role ◽  
Screening Assay ◽  
Mediated Effects ◽  
A Minor

AbstractIntake of synthetic cannabinoids (SC), one of the largest classes of new psychoactive substances, was reported to be associated with acute liver damage but information about their hepatotoxic potential is limited. The current study aimed to analyze the hepatotoxicity including the metabolism-related impact of JWH-200, A-796260, and 5F-EMB-PINACA in HepG2 cells allowing a tentative assessment of different SC subclasses. A formerly adopted high-content screening assay (HCSA) was optimized using a fully automated epifluorescence microscope. Metabolism-mediated effects in the HCSA were additionally investigated using the broad CYP inhibitor 1-aminobenzotriazole. Furthermore, phase I metabolites and isozymes involved were identified by in vitro assays and liquid chromatography–high-resolution tandem mass spectrometry. A strong cytotoxic potential was observed for the naphthoylindole SC JWH-200 and the tetramethylcyclopropanoylindole compound A-796260, whereas the indazole carboxamide SC 5F-EMB-PINACA showed moderate effects. Numerous metabolites, which can serve as analytical targets in urine screening procedures, were identified in pooled human liver microsomes. Most abundant metabolites of JWH-200 were formed by N-dealkylation, oxidative morpholine cleavage, and oxidative morpholine opening. In case of A-796260, most abundant metabolites included an oxidative morpholine cleavage, oxidative morpholine opening, hydroxylation, and dihydroxylation followed by dehydrogenation. Most abundant 5F-EMB-PINACA metabolites were generated by ester hydrolysis plus additional steps such as oxidative defluorination and hydroxylation. To conclude, the data showed that a hepatotoxicity of the investigated SC cannot be excluded, that metabolism seems to play a minor role in the observed effects, and that the extensive phase I metabolism is mediated by several isozymes making interaction unlikely.

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