scholarly journals Bioaccumulation and Distribution of Indospicine and Its Foregut Metabolites in Camels Fed Indigofera spicata

Toxins ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 169 ◽  
Author(s):  
Gabriele Netzel ◽  
Eddie Tan ◽  
Mukan Yin ◽  
Cindy Giles ◽  
Ken Yong ◽  
...  
Keyword(s):  
Clearance Rate ◽  
Digestive System ◽  
Plasma Samples ◽  
In Vitro Experiments ◽  
Natural Toxin ◽  
Low Levels ◽  
Almost All ◽  
Indigofera Spicata ◽  
Retrospective Investigation

In vitro experiments have demonstrated that camel foregut-fluid has the capacity to metabolize indospicine, a natural toxin which causes hepatotoxicosis, but such metabolism is in competition with absorption and outflow of indospicine from the different segments of the digestive system. Six young camels were fed Indigofera spicata (337 µg indospicine/kg BW/day) for 32 days, at which time three camels were euthanized. The remaining camels were monitored for a further 100 days after cessation of this indospicine diet. In a retrospective investigation, relative levels of indospicine foregut-metabolism products were examined by UHPLC-MS/MS in plasma, collected during both accumulation and depletion stages of this experiment. The metabolite 2-aminopimelamic acid could be detected at low levels in almost all plasma samples, whereas 2-aminopimelic acid could not be detected. In the euthanized camels, 2-aminopimelamic acid could be found in all tissues except muscle, whereas 2-aminopimelic acid was only found in the kidney, pancreas, and liver tissues. The clearance rate for these metabolites was considerably greater than for indospicine, which was still present in plasma of the remaining camels 100 days after cessation of Indigofera consumption.

scholarly journals Evidence of Thrombin-Independent Generation of Activated Protein C

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2568-2568
Author(s):  
Heiko Rühl ◽  
Janine Rossa ◽  
Christina Berens ◽  
Anna Winterhagen ◽  
Johannes Oldenburg ◽  
...  
Keyword(s):  
Protein C ◽  
De Novo ◽  
Activated Protein C ◽  
Autologous Serum ◽  
Thrombin Inhibitor ◽  
Thrombin Time ◽  
Plasma Samples ◽  
In Vitro Experiments ◽  
Healthy Human

Abstract Introduction: In a recent study, we performed autologous serum infusions to evaluate the elimination kinetics of hemostasis-related biomarkers in healthy human subjects. In order to monitor a serum-induced activation of coagulation, we measured free thrombin in the infused serum and in plasma samples taken during and after infusion, but did not detect any de novo thrombin formation [PLoS One. 2015; 10(12): e0145012]. To study if the low levels of free thrombin in the infused serum induce generation of activated protein C (APC) we additionally measured APC in samples drawn after autologous serum infusion and in vitro in a purified system. Methods: Autologous serum was infused (50 mL/30 min) into 19 healthy volunteers. Four of them were simultaneously receiving infusions of the thrombin inhibitor argatroban (1 µg/kg/min), initiated 1 h before and ceased 1 h after starting the infusion of serum. Thrombin and APC were measured in serum and in plasma samples drawn before and in 15-min intervals during the infusion of serum, using a highly-sensitive oligonucleotide-based enzyme capture assay (OECA) platform. In in vitro experiments, APC formation was induced by addition of purified thrombin or serum to buffer containing protein C and thrombomodulin in excess, and CaCl2 at physiological concentrations. The formation of APC was subsequently measured by OECA. Results: In the autologous serum median (interquartile range) concentrations of thrombin and APC were 6.68 (4.63 - 8.73) ng/mL and 9.17 (7.63 - 13.91) ng/mL, thus doses of 0.12 (0.07 - 0.15) ng/mL of thrombin and 0.16 (0.14 - 0.22) ng/mL of APC were infused per mL of the subjects' plasma volume. In the plasma of probands, that did not receive argatroban, peak thrombin levels of 0.04 (0.00 - 0.08) ng/mL were measured, indicating a rapid inactivation of thrombin by endogenous inhibitors present in the plasma. However, with 1.41 (0.76 - 2.97) ng/mL peak APC levels exceeded the infused APC doses by a multiple. This was also true for the plasma samples from the probands that received argatroban, in which peak levels of APC of 0.94 (0.79 - 1.22) ng/mL were measured despite thrombin inhibition indicated by prolongation of the aPTT of 42.9 (40.1 - 44.4) s and thrombin time of 78.3 (69.3 - 87.2) s. In the in vitro experiments addition of argatroban at the concentrations achieved in the probands completely abolished APC generation up to a thrombin concentration of 5 ng/ml. Addition of human serum as a source for thrombin in the same purified system consistently induced generation of greater amounts of APC than expected on the basis of the amount of thrombin present in the serum samples. Conclusions: The data obtained provide evidence for a thrombin-independent mechanism of APC formation. Further in vitro studies with endothelial cells are required to identify the components that are involved in this alternative way of APC generation. Disclosures Rühl: CSL Behring: Research Funding; Bayer: Consultancy, Honoraria. Müller:Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Impairment of platelet aggregation in hemolytic uremic syndrome: evidence for platelet "exhaustion"

Blood ◽  
1982 ◽  
Vol 60 (3) ◽  
pp. 564-570 ◽  
Author(s):  
JS Fong ◽  
BS Kaplan
Keyword(s):  
Renal Failure ◽  
Acute Renal Failure ◽  
Hemolytic Uremic Syndrome ◽  
Acute Illness ◽  
Uremic Toxin ◽  
In Vitro Experiments ◽  
Microangiopathic Hemolytic Anemia ◽  
Uremic Syndrome ◽  
Low Levels

Thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure are the hallmarks of hemolytic-uremic syndrome (HUS). This report presents the results on platelet studies from 10 consecutive HUS patients in childhood. During their acute illness, they all displayed a characteristic pattern of impaired platelet function: no aggregating responses to epinephrine, some to ADP, and moderate to collagen. In addition, platelet contents of beta-thromboglobulin (beta TG) were markedly reduced. As these patients improved clinically, their platelet- aggregating responses also normalized despite their uremic state. Incubation of platelets with uremic plasma or guanidino-succinic acid, a uremic toxin, had minor effects on platelet-aggregating activity. Since low levels of platelet beta TG suggest that these platelets were in an exhausted state, in vitro experiments were performed to exhaust normal platelets by incubation at 37 degrees C. A proportional impairment of platelet-aggregating responses and decreasing levels of platelet beta TG were noted. Furthermore, the pattern of impairment was similar to that found in the platelet-aggregating activities of HUS patients. Thus, “exhaustion,” in addition to azotemia and thrombocytopenia, are factors that contribute to the functional impairment of platelets in these patients. Further studies to reveal mechanisms that lead to platelet exhaustion in HUS are of fundamental importance in the understanding of this illness.

scholarly journals Impairment of platelet aggregation in hemolytic uremic syndrome: evidence for platelet "exhaustion"

Blood ◽  
1982 ◽  
Vol 60 (3) ◽  
pp. 564-570 ◽  
Author(s):  
JS Fong ◽  
BS Kaplan
Keyword(s):  
Renal Failure ◽  
Acute Renal Failure ◽  
Hemolytic Uremic Syndrome ◽  
Acute Illness ◽  
Uremic Toxin ◽  
In Vitro Experiments ◽  
Microangiopathic Hemolytic Anemia ◽  
Uremic Syndrome ◽  
Low Levels

Abstract Thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure are the hallmarks of hemolytic-uremic syndrome (HUS). This report presents the results on platelet studies from 10 consecutive HUS patients in childhood. During their acute illness, they all displayed a characteristic pattern of impaired platelet function: no aggregating responses to epinephrine, some to ADP, and moderate to collagen. In addition, platelet contents of beta-thromboglobulin (beta TG) were markedly reduced. As these patients improved clinically, their platelet- aggregating responses also normalized despite their uremic state. Incubation of platelets with uremic plasma or guanidino-succinic acid, a uremic toxin, had minor effects on platelet-aggregating activity. Since low levels of platelet beta TG suggest that these platelets were in an exhausted state, in vitro experiments were performed to exhaust normal platelets by incubation at 37 degrees C. A proportional impairment of platelet-aggregating responses and decreasing levels of platelet beta TG were noted. Furthermore, the pattern of impairment was similar to that found in the platelet-aggregating activities of HUS patients. Thus, “exhaustion,” in addition to azotemia and thrombocytopenia, are factors that contribute to the functional impairment of platelets in these patients. Further studies to reveal mechanisms that lead to platelet exhaustion in HUS are of fundamental importance in the understanding of this illness.

Neurofilaments and Paired Helical Filaments

1985 ◽  
Vol 43 ◽  
pp. 740-743
Author(s):  
J. Metuzals
Keyword(s):  
Animal Model ◽  
Posttranslational Modifications ◽  
Posttranslational Modification ◽  
Giant Axon ◽  
Paired Helical Filaments ◽  
Squid Giant Axon ◽  
In Vitro Experiments ◽  
Thin Sections ◽  
Structural Relationships

It has been demonstrated that the neurofibrillary tangles in biopsies of Alzheimer patients, composed of typical paired helical filaments (PHF), consist also of typical neurofilaments (NF) and 15nm wide filaments. Close structural relationships, and even continuity between NF and PHF, have been observed. In this paper, such relationships are investigated from the standpoint that the PHF are formed through posttranslational modifications of NF. To investigate the validity of the posttranslational modification hypothesis of PHF formation, we have identified in thin sections from frontal lobe biopsies of Alzheimer patients all existing conformations of NF and PHF and ordered these conformations in a hypothetical sequence. However, only experiments with animal model preparations will prove or disprove the validity of the interpretations of static structural observations made on patients. For this purpose, the results of in vitro experiments with the squid giant axon preparations are compared with those obtained from human patients. This approach is essential in discovering etiological factors of Alzheimer's disease and its early diagnosis.

Fibrinogen-Fibrin-Related Antigen Pattern in Human Blood

1974 ◽  
Vol 32 (02/03) ◽  
pp. 266-276
Author(s):  
Carl D. Jacobsen ◽  
John C. Hoak ◽  
Kenneth K. WU ◽  
Glenna L. Fry
Keyword(s):  
Human Blood ◽  
Plasma Samples

SummaryIn serum from patients with DIC at least 3 different FR-antigenic components could be found. It was difficult to demonstrate these components in the corresponding plasma samples. It is possible that a portion of these antigens formed as a result of in vitro clotting despite the presence of proteolytic inhibitors. These results suggest that the interpretation of “increased split products in serum” may be more complex than current concepts indicate.

In Vitro Effects of Urokinase — Prevention by Different Inhibitors

1990 ◽  
Vol 64 (03) ◽  
pp. 402-406 ◽  
Author(s):  
M D Oethinger ◽  
E Seifried
Keyword(s):  
In Vitro Study ◽  
Thrombin Time ◽  
Plasma Samples ◽  
Temperature Dependent ◽  
Chloromethyl Ketone ◽  
Control Value ◽  
Dose Time ◽  
In Vitro Effects ◽  
Full Protection

SummaryThe present in vitro study investigated dose-, time- and temperature-dependent effects of two-chain urokinase plasminogen activato(u-PA, urokinase) on normal citrated plasma. When 10 μg/ml u-PA wereadded to pooled normal plasma and incubated for 30 min at an ambient temperature (25° C), α2-antiplas-min decreased to 8% of the control value. Incubation on ice yielded a decrease to 45% of control,whereas α2-antiplasmin was fully consumed at 37° C. Fibrinogen and plasminogen fell to 46% and 39%, respectively, after a 30 min incubation at 25° C. Thrombin time prolonged to 190% of control.Various inhibitors were studied with respect to their suitability and efficacy to prevent these in vitro effects. Aprotinin exhibited a good protective effect on fibrinogen at concentrations exceeding 500 KlU/ml plasma. Its use, however, was limited due to interferences with some haemostatic assays. We could demonstrate that L-Glutamyl-L-Glycyl-L-Arginyl chloromethyl ketone (GGACK) and a specific polyclonal anti-u-PA-antibody (anti-u-PA-IgG) effectively inhibited urokinase-induced plasmin generation without interfering with haemostatic assays. The anti-u-PA-antibody afforded full protection ofα2-antiplasmin at therapeutic levels of u-PA.It is concluded that u-PA in plasma samples from patients during thrombolytic therapy may induce in vitro effects which should be prevented by the use of a suitable inhibitor such as GGACK or specific anti-u-PA-antibody.

Effect of Aspirin on the Thrombelastogram of Human Blood

1973 ◽  
Vol 30 (03) ◽  
pp. 494-498 ◽  
Author(s):  
G de Gaetano ◽  
J Vermylen
Keyword(s):  
Oral Dose ◽  
Single Oral Dose ◽  
Platelet Function ◽  
Human Blood ◽  
Platelet Rich Plasma ◽  
Plasma Samples ◽  
Release Reaction ◽  
Platelet Release

SummaryThrombelastograms of both native blood and re-calcified platelet-rich plasma samples taken from subjects given a single oral dose of aspirin (1 gram) were not significantly different from the pretreatment recordings. Aspirin also did not modify the thrombelastogram when preincubated in vitro with platelet-rich plasma at concentrations inhibiting the platelet “release reaction” by collagen. Thrombelastography therefore cannot evaluate the effect of aspirin on platelet function.

Enhanced Increases in Cytosolic Ca2+ in ADP-Stimulated Platelets from Patients with Delta-Storage Pool Deficiency – A Possible Indicator of Interactions between Granule-Bound ADP and the Membrane ADP Receptor

1997 ◽  
Vol 77 (02) ◽  
pp. 376-382 ◽  
Author(s):  
Bruce Lages ◽  
Harvey J Weiss
Keyword(s):  
Platelet Rich Plasma ◽  
Limited Range ◽  
Dense Granule ◽  
Receptor Desensitization ◽  
Fura 2 ◽  
Storage Pool ◽  
Low Levels ◽  
The Right

SummaryThe possible involvement of secreted platelet substances in agonist- induced [Ca2+]i increases was investigated by comparing these increases in aspirin-treated, fura-2-loaded normal platelets and platelets from patients with storage pool deficiencies (SPD). In the presence and absence of extracellular calcium, the [Ca2+]i response induced by 10 µM ADP, but not those induced by 0.1 unit/ml thrombin, 3.3 µM U46619, or 20 µM serotonin, was significantly greater in SPD platelets than in normal platelets, and was increased to the greatest extent in SPD patients with Hermansky-Pudlak syndrome (HPS), in whom the dense granule deficiencies are the most severe. Pre-incubation of SPD-HPS and normal platelets with 0.005-5 µM ADP produced a dose-dependent inhibition of the [Ca2+]i response induced by 10 µ M ADP, but did not alter the [Ca2+]i increases induced by thrombin or U46619. Within a limited range of ADP concentrations, the dose-inhibition curve of the [Ca2+]i response to 10 µM ADP was significantly shifted to the right in SPD-HPS platelets, indicating that pre-incubation with greater amounts of ADP were required to achieve the same extent of inhibition as in normal platelets. These results are consistent with a hypothesis that the smaller ADP-induced [Ca2+]i increases seen in normal platelets may result from prior interactions of dense granule ADP, released via leakage or low levels of activation, with membrane ADP receptors, causing receptor desensitization. Addition of apyrase to platelet-rich plasma prior to fura-2 loading increased the ADP-induced [Ca2+]i response in both normal and SPD-HPS platelets, suggesting that some release of ADP derived from both dense granule and non-granular sources occurs during in vitro fura-2 loading and platelet washing procedures. However, this [Ca2+]i response was also greater in SPD-HPS platelets when blood was collected with minimal manipulation directly into anticoagulant containing apyrase, raising the possibility that release of dense granule ADP resulting in receptor desensitization may also occur in vivo. Thus, in addition to enhancing platelet activation, dense granule ADP could also act to limit the ADP-mediated reactivity of platelets exposed in vivo to low levels of stimulation.

The Involvement of Platelets and the Coronary Vasculature in Collagen-Induced Sudden Death in Rabbits

1985 ◽  
Vol 53 (01) ◽  
pp. 070-074 ◽  
Author(s):  
G Mallarkey ◽  
G M Smith
Keyword(s):  
Blood Pressure ◽  
Heart Rate ◽  
Platelet Count ◽  
Sudden Death ◽  
Myocardial Ischaemia ◽  
Plasma Levels ◽  
Sodium Citrate ◽  
Plasma Samples ◽  
Calcium Entry Blockers

SummaryThe mechanism of collagen-induced sudden death in rabbits was studied by measuring blood pressure (BP), heart rate, ECG, the continuous platelet count and the plasma levels of thromboxane B2 (TxB2) and 6-keto prostaglandin Fia (6-keto PGF1α). Death was preceded by myocardial ischaemia and a sharp fall in BP which occurred before any fall in platelet count was observed. The calcium entry blockers (CEBs), verapamil, nifedipine and PY 108-068 protected the rabbits from sudden death without any significant effect on the decrease in the platelet count or increase in plasma TxB2 levels. 6-keto PGF1α could not be detected in any plasma samples. Indomethacin and tri-sodium citrate also protected the rabbits but significantly reduced the fall in platelet count and plasma TxB2. In vitro studies on isolated aortae indicated that verapamil non-specifically inhibited vasoconstriction induced by KC1, adrenaline and U46619 (a thromboxane agonist). It is concluded that CEBs physiologically antagonize the vasoconstricting actions of platelet-derived substances and that it is coronary vasoconstriction that is primarily the cause of death.

IN VITRO METHODS FOR THE STUDY OF THE ADENOHYPOPHYSIAL FUNCTIONS TO SECRETE GONADOTROPHIN

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S27-S40 ◽  
Author(s):  
T. Kobayashi ◽  
T. Kigawa ◽  
M. Mizuno ◽  
T. Watanabe
Keyword(s):  
Cell Culture ◽  
Organ Culture ◽  
Cultured Cells ◽  
Cell Sheet ◽  
Short Term ◽  
Hypothalamic Extract ◽  
Numerous Cell ◽  
Single Cell Culture ◽  
Almost All

ABSTRACT There are several in vitro methods to analyse the function of the adenohypophysis or the mechanisms of its regulation. The present paper deals with single cell culture, organ culture and short term incubation techniques by which the morphology and gonadotrophin-secreting function of the adenohypophysis were studied. In trypsin-dispersed cell culture, the adenohypophysial cells showed extensive propagation to form numerous cell colonies and finally develop into a confluent monolayer cell sheet covering completely the surface of culture vessels. Almost all of the cultured cells, however, became chromophobic, at least at the end of the first week of cultivation, when gonadotrophin was detectable neither in the culture medium nor in the cells themselves. After the addition of the hypothalamic extract, gonadotrophin became detectable again, and basophilic or PAS-positive granules also reappeared within the cells, suggesting that the gonadotrophs were stimulated by the extract to produce gonadotrophin. In organ culture and short term incubation, the incorporation of [3H] leucine into the adenohypophysial cells in relation to the addition of hypothalamic extract was examined. It was obvious that the ability to incorporate [3H] leucine into the gonadotrophs in vitro was highly dependent upon the presence of the hypothalamic extract.

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