Identification of rapidly labeled, small molecular weight hydroxyproline-containing peptides in developing mouse limbs in vitro and in vivo

1979 ◽  
Vol 72 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Lewis B. Holmes ◽  
Robert L. Trelstad
1981 ◽  
Author(s):  
Ph Schneider ◽  
M Ruegg ◽  
F Bachmann

Highly purified lew molecular weight urokinase (LMR-UK), moving on SDS-PAGE (reduced and nan-reduced) as a single band of 32 kdalton, was labelled with 125I by the chlora- mine-T method. 106 cpn of this 125I-LMr-UK (94% TCA preci- pitable) were injected into the inferior vena cava of la- par atomized albino rats, which were maintained at 37°C. Blood samples were collected by cardiac puncture 5, 30 and 90 min respectively after the injection. Serun, obtained from these samples, was fractionated on a Sephadex G-100 column, calibrated with proteins of known Mr. Radioactivity was measured in the collected fractions.In the 5 min sample, the radioactivity was distributed in 2 peaks, corresponding to 32 kdalton and to < 70 kdalton respectively. In the 30 min sample, the distribution was characterized by a diminution of the 32 kdalton peak and the appearance of a third peak corresponding to a Mr of < 4 kdalton. In the 90 min sample, the LMr-UK peak had disappeared almost completely. About 40% of the 125I-activity was present in a skewed high Mr peak with a broad maximum in the 85-100 kdalton region; ≥ 60% of the 125I-activity was recovered in late fractions corresponding to < 4 kdalton. In control experiments, pooled rat serum was incubated in vitro with 125I-LMr-UK for 5, 30 and 90 min respectively and samples were fractionated on the same column. The radioactivity distribution shewed only the 32 and > 70 kdalton peaks, but no < 4 kdalton peak.These results suggest that LMr-UK is complexed to a carrier protein, both in vivo and in vitro, but that it is degraded into small fragments in vivo only. Attempts to characterize the nature of these complexes are in progress.


1997 ◽  
Vol 20 (2) ◽  
pp. 81-90 ◽  
Author(s):  
P. Ahrenholz ◽  
R.E. Winkler ◽  
W. Ramlow ◽  
M. Tiess ◽  
W. Müller

Since the introduction of on-line substituate preparation, high substituate rates (Qs) in pre- and postdilution for hemodiafiltration (HDF) procedures can be realized. During postdilution HDF (POD-HDF) and additional convective removal is possible, but in vivo Qs is limited to approx. 1/3Qb (bloodflow). With predilution HDF (PRD-HDF) higher Qs and therefore high convective transport rates by ultrafiltration can be reached. On the other hand the blood concentration is diminished by predilution. Further decrease of the diffusive transport is caused by reduced dialysate flow Qd due to separation of the substituate from the dialysate (Fresenius 4008 On-Line HDF, Gambro AK100 Ultra). The theoretical description of the combined diffusive-convective transport is limited to 1-dimensional models and small UF-rates. Therefore for practical and theoretical purposes the assessment of the efficacy of on-line PRD-HDF and POD-HDF in different molecular weight ranges is desirable. By means of in vitro experiments the effective clearances Keff of hemodialysis (HD, dialyzer: Fresenius F60) for urea, creatinine, vitamin B12 and inulin were compared with measured and theoretical Keff of POD- and PRD-HDF. The theoretical expectation is confirmed that Keff for small molecular weight substances decreases slightly with PRD-HDF and increases for larger molecules. In the case of POD-HDF Keff for small molecular weight substances increases slightly and strongly for larger molecules. In vivo experiments were performed to measure the real substance removal from patient's blood and to figure out the impact of dialysate flow (collection of the used dialysate during the 1. treatment hour and concentration measurements for urea, creatinine, phosphate, ß2-MG). The results show that the substraction of Qs from Qd reduces Keff for urea, creatinine and phosphate but not for ß2-MG. PRD-HDF with Qd = 500 ml/min is significantly less effective for small molecules than HD. There is no significant difference of Keff for urea, creatinine, phosphate during HD and PRD-HDF with Qd = 800 ml/min, but a significant increase of 10-15% for POD-HDF Keff for ß2-MG increases by 75% for PRD-HDF and 95% for POD-HDF compared with HD (Qd = 500 ml/min).


Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2063-2063 ◽  
Author(s):  
Teresa Sellers ◽  
Timothy Hart ◽  
Michael Semanik ◽  
Krishna Murthy

Abstract SB 497115-GR is a small molecular weight Tpo receptor (TpoR) agonist that has properties similar to thrombopoietin (TPO), primarily inducing proliferation and differentiation of megakaryocytes from bone marrow progenitor cells. SB-497115-GR is being developed for the treatment of thrombocytopenias, such as immune thrombocytopenic purpura. In vitro and in vivo studies have demonstrated that SB-497115-GR has very distinct species specificity. SB-497115 or other molecules in this class induced dose dependent STAT activation in platelets from humans and chimpanzees but not in platelets from laboratory animal species commonly used in drug safety studies. In order to demonstrate in vivo activity of SB-497115-GR, a single dose and 5 daily dose pharmacology and safety study in chimpanzees was conducted. To support initiation of clinical trials, a comprehensive package of toxicology studies was conducted including studies up to 14 days duration in rats and dogs. All procedures involving the care and use of animals in these studies were reviewed and approved by the appropriate Institutional Animal Care and Use Committees. Female chimpanzees (1–3/group) were administered vehicle or SB-497115-GR at doses of 0.1 to 10 mg/kg/day by oral gavage. For toxicology studies, SB-497115-GR was administered orally to rats (10/sex/group) by gavage at doses of 3 to 40 mg/kg/day and to dogs (3/sex/group) by capsule at doses of 3 to 30 mg/kg/day for 14 days. SB-497115-GR was well tolerated in chimpanzees, rats and dogs at all doses tested. In chimpanzees, no treatment related increases in platelet counts were observed after administration of single doses of up to 10 mg/kg or 5 daily doses of up to 3 mg/kg/day. However, following 5 daily doses of 10 mg/kg/day SB-497115-GR, there was a 1.3- to 2.4-fold increase in circulating platelet counts in 3 chimpanzees. A similar change in reticulated platelet counts was observed preceding this increase. In contrast, there was no effect of treatment for up to 14 days on platelet counts in rats or dogs. In conclusion, SB-497115-GR, an orally bioavailable small molecular weight agonist of the TpoR, has been shown to increase platelet counts in chimpanzees. These in vivo data confirm the in vitro data demonstrating the unique species-specific effects of this novel Tpo receptor agonist on platelets and were predictive of a pharmacodynamic effect currently being observed in human clinical trials.


1985 ◽  
Vol 109 (1) ◽  
pp. 115-121
Author(s):  
Hannu Rajaniemi ◽  
Jan Sogn ◽  
Paul Holmes ◽  
Björn Källfelt ◽  
Per Olof Janson

Abstract. Pseudopregnant rats were injected with [125I] hCG, anaesthesized 1 h later and after cannulation of the aorta the ovaries were isolated and perfused with Gey & Gey buffer containing 0.2% BSA. The release of radioactivity was monitored for 2 h and analyzed by gel filtration. Five to ten per cent of the radioactivity was released within 2 h and represented small molecular weight peptides and iodotyrosine and [125I]hCG. Analysis of the ovarian radioactivity prior to and after perfusion revealed that virtually all hCG was receptor-bound. Loading the medium with unlabelled hCG displaced [125I]hCG from the receptor but did not enhance its degradation. Histological examination showed that the ovarian tissues were still intact after the 2 h perfusion. Immunohistochemical studies revealed a localization of the hCG at the cell periphery both prior to and after perfusion. These results provide evidence showing that the rate of internalization of receptor-hCG complexes in rat luteal cells is slow in vivo.


Blood ◽  
1981 ◽  
Vol 57 (6) ◽  
pp. 1117-1118 ◽  
Author(s):  
S Pollack ◽  
S Ruocco

Abstract Removal of iron from biologically important ligands by desferrioxamine (DF), in vitro, is exceedingly slow because of a kinetic barrier. This kinetic barrier can be overcome by a variety of a small molecular weight anions, of which nitrilotriacetate (NTA) is a prototype. We explored the effect of NTA on DF iron mobilization in vivo. Iron mobilization was increased and the effect was synergistic.


Author(s):  
Rahul Vijay Kapoore ◽  
Seetharaman Vaidyanathan

Metabolome analyses are a suite of analytical approaches that enable us to capture changes in the metabolome (small molecular weight components, typically less than 1500 Da) in biological systems. Mass spectrometry (MS) has been widely used for this purpose. The key challenge here is to be able to capture changes in a reproducible and reliant manner that is representative of the events that take place in vivo . Typically, the analysis is carried out in vitro , by isolating the system and extracting the metabolome. MS-based approaches enable us to capture metabolomic changes with high sensitivity and resolution. When developing the technique for different biological systems, there are similarities in challenges and differences that are specific to the system under investigation. Here, we review some of the challenges in capturing quantitative changes in the metabolome with MS based approaches, primarily in microbial and mammalian systems. This article is part of the themed issue ‘Quantitative mass spectrometry’.


2005 ◽  
Vol 289 (3) ◽  
pp. E366-E372 ◽  
Author(s):  
Nacide Ercan-Fang ◽  
Miriam R. Taylor ◽  
Judith L. Treadway ◽  
Carolyn B. Levy ◽  
Paul E. Genereux ◽  
...  

Phosphorylase is regulated by a number of small-molecular-weight effectors that bind to three sites on the enzyme. Recently, a fourth site referred to as the indole-inhibitor site has been identified. Synthetic compounds bind to the site and inhibit activity. However, the effects of these compounds in the presence of other endogenous effectors are unknown. We have determined the effects of four indole derivative glycogen phosphorylase inhibitors (GPI) on recombinant human liver glycogen phosphorylase a activity. The GPIs tested were all potent inhibitors. However, the endogenous inhibitors (glucose, ADP, ATP, fructose 1-phosphate, glucose 6-phosphate, UDP-glucose) and the activator (AMP) markedly reduced the inhibitory effect of GPIs. Consistent with these in vitro findings, the IC50 for the inhibition of glycogenolysis in cells and the liver drug concentration associated with glucose-lowering activity in diabetic ob/ ob mice in vivo were also significantly higher than those determined in in vitro enzyme assays. The inhibitory effect of indole-site effectors is modulated by endogenous small-molecular-weight effectors of phosphorylase a activity. However, at higher concentrations (10–30 μM), the GPI effect was dominant and resulted in inhibition of phosphorylase a activity irrespective of the presence or absence of the other modulators of the enzyme.


Blood ◽  
1981 ◽  
Vol 57 (6) ◽  
pp. 1117-1118
Author(s):  
S Pollack ◽  
S Ruocco

Removal of iron from biologically important ligands by desferrioxamine (DF), in vitro, is exceedingly slow because of a kinetic barrier. This kinetic barrier can be overcome by a variety of a small molecular weight anions, of which nitrilotriacetate (NTA) is a prototype. We explored the effect of NTA on DF iron mobilization in vivo. Iron mobilization was increased and the effect was synergistic.


1994 ◽  
Vol 72 (06) ◽  
pp. 942-946 ◽  
Author(s):  
Raffaele Landolfi ◽  
Erica De Candia ◽  
Bianca Rocca ◽  
Giovanni Ciabattoni ◽  
Armando Antinori ◽  
...  

SummarySeveral “in vitro” and “in vivo” studies indicate that heparin administration may affect platelet function. In this study we investigated the effects of prophylactic heparin on thromboxane (Tx)A2 biosynthesis “in vivo”, as assessed by the urinary excretion of major enzymatic metabolites 11-dehydro-TxB2 and 2,3-dinor-TxB2. Twenty-four patients who were candidates for cholecystectomy because of uncomplicated lithiasis were randomly assigned to receive placebo, unfractionated heparin, low molecular weight heparin or unfractionaed heparin plus 100 mg aspirin. Measurements of daily excretion of Tx metabolites were performed before and during the treatment. In the groups assigned to placebo and to low molecular weight heparin there was no statistically significant modification of Tx metabolite excretion while patients receiving unfractionated heparin had a significant increase of both metabolites (11-dehydro-TxB2: 3844 ± 1388 vs 2092 ±777, p <0.05; 2,3-dinor-TxB2: 2737 ± 808 vs 1535 ± 771 pg/mg creatinine, p <0.05). In patients randomized to receive low-dose aspirin plus unfractionated heparin the excretion of the two metabolites was largely suppressed thus suggesting that platelets are the primary source of enhanced thromboxane biosynthesis associated with heparin administration. These data indicate that unfractionated heparin causes platelet activation “in vivo” and suggest that the use of low molecular weight heparin may avoid this complication.


1964 ◽  
Vol 12 (01) ◽  
pp. 232-261 ◽  
Author(s):  
S Sasaki ◽  
T Takemoto ◽  
S Oka

SummaryTo demonstrate whether the intravascular precipitation of fibrinogen is responsible for the toxicity of heparinoid, the relation between the toxicity of heparinoid in vivo and the precipitation of fibrinogen in vitro was investigated, using dextran sulfate of various molecular weights and various heparinoids.1. There are close relationships between the molecular weight of dextran sulfate, its toxicity, and the quantity of fibrinogen precipitated.2. The close relationship between the toxicity and the precipitation of fibrinogen found for dextran sulfate holds good for other heparinoids regardless of their molecular structures.3. Histological findings suggest strongly that the pathological changes produced with dextran sulfate are caused primarily by the intravascular precipitates with occlusion of the capillaries.From these facts, it is concluded that the precipitates of fibrinogen with heparinoid may be the cause or at least the major cause of the toxicity of heparinoid.4. The most suitable molecular weight of dextran sulfate for clinical use was found to be 5,300 ~ 6,700, from the maximum value of the product (LD50 · Anticoagulant activity). This product (LD50 · Anticoagulant activity) can be employed generally to assess the comparative merits of various heparinoids.5. Clinical use of the dextran sulfate prepared on this basis gave satisfactory results. No severe reaction was observed. However, two delayed reactions, alopecia and thrombocytopenia, were observed. These two reactions seem to come from the cause other than intravascular precipitation.


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