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Biomedical subjects

O Toffoletto

Publications and source records attributed to O Toffoletto.

14 recordsLinked to original sources
PubMed2001

In vitro cytotoxicity of the LDE: daunorubicin complex in acute myelogenous leukemia blast cells.

Acute myelogenous leukemia (AML) blast cells show high-affinity degradation of low-density lipoprotein (LDL), suggesting an increased expression of cellular LDL receptors. LDE is a lipid microemulsion easily synthesized in vitro which is known to mimic the metabolic pathway of LDL. We used LDE as a carrier for daunorubicin and assayed the cytotoxicity of the complex using AML blast cells since RT-PCR analysis showed that AML cells express LDL receptor mRNA. The LDE:daunorubicin complex killed 46.7% of blast cells and 20.2% of normal bone marrow cells (P<0.001; Student t-test). Moreover, this complex destroyed AML blast cells as efficiently as free daunorubicin. Thus, LDE might be a suitable carrier of chemotherapeutic agents targeting these drugs to neoplastic cells and protecting normal tissues.

Adolescent↗
PubMed1998

Plasma kinetics of chylomicron-like emulsion in renal transplant patients receiving cyclosporin-based immunosuppression.

BACKGROUND: Atherosclerotic cardiovascular disease is prevalent among renal transplant patients. Increase in serum total cholesterol, low-density lipoprotein, and very low-density lipoprotein is common in those patients. Alterations in chylomicron metabolism, however, are also related to atherogenesis and were not studied in renal transplant. HYPOTHESIS: The aim of this study was to evaluate chylomicron metabolism in renal transplant recipients receiving cyclosporin-based immunosuppression. We determined the plasma kinetics of triglyceride-rich emulsions labeled with [3H]triolein and [14C]cholesteryl oleate that are known to mimic the chylomicron metabolism when injected into the blood stream. METHODS: Fourteen renal transplant recipients with normal renal function (10 men, 4 women, aged 40 +/- 6.1 years) and 17 age- and gender-matched healthy controls received bolus injections of the chylomicron-like emulsion. Plasma samples were then taken at regular intervals during 60 min. Disappearance curves of the labels and the respective fractional clearance rates (FCR) were calculated in order to measure lipolysis and chylomicron remnant removal from the plasma. RESULTS: Fasting serum lipid levels did not differ in the two groups. The difference between Median FCR of [3H]triolein emulsion in renal transplant patients and that obtained in the controls (0.07 vs. 0.11 min-1, NS) was not statistically significant. Median FCR of [14C]cholesteryl oleate also did not differ between the groups (patients: 0.044; controls: 0.046, NS). CONCLUSION: These results indicate that neither chylomicron lipolysis nor remnant removal are affected in stable renal transplant patients treated with cyclosporin-based immunosuppression.

Adult↗
PubMed1997

Plasma kinetic behavior in hyperlipidemic subjects of a lipidic microemulsion that binds to low density lipoprotein receptors.

It was previously reported that a protein-free microemulsion (LDE) with structure roughly resembling that of the lipid portion of low density lipoprotein (LDL) was presumably taken up by LDL receptors when injected into the bloodstream. In contact with plasma, LDE acquires apolipoproteins (apo) including apo E that would be the ligand for receptor binding. Currently, apo were associated to LDE by incubation with high density lipoprotein (HDL). LDE-apo uptake by mononuclear cells showed a saturation kinetics, with an apparent K(m) of 13.1 ng protein/mL. LDE-apo is able to displace LDL uptake by mononuclear cells with a Ki of 11.5 ng protein/mL. LDE without apo is, however, unable to displace LDL. The uptake of 14C-HDL is not dislocated by increasing amounts of LDE-apo, indicating that HDL and LDE-apo do not bind to the same receptor sites. In human hyperlipidemias, LDE labeled with 14C-cholesteryl ester behaved kinetically as expected for native LDL. LDE plasma disappearance curve obtained from eight hypercholesterolemic patients was markedly slower than that from 10 control normolipidemic subjects [fractional clearance rate (FCR) = 0.02 +/- 0.01 and 0.12 +/- 0.04 h-1, respectively; P < 0.0001]. On the other hand, in four severely hypertriglyceridemic patients, LDE FCR was not significantly different from the controls (0.07 +/- 0.03 h-1). These results suggest that LDE can be a useful device to study lipoprotein metabolism.

Adult↗
PubMed1996

Plasma kinetics of a chylomicron-like emulsion in patients with coronary artery disease.

Chylomicron catabolism in the bloodstream consists of lipolysis by lipoprotein lipase and uptake of remnants by the liver. In rats, triglyceride-rich emulsions can mimic chylomicron metabolism. To further validate this model in man, the emulsion was injected intravenously into fasting and into subjects previously fed a test fatty meal. The plasma kinetic curves of the emulsion 3H-triglyceride and 14C-cholesteryl ester were determined. The fractional clearance rate (FCR) of both labels was markedly reduced in the fed subjects (triglycerides: fed = 0.018 +/- 0.007; fasting = 0.105 +/- 0.013 min-1, P < 0.001; cholesteryl ester: fed = 0.016 +/- 0.001; fasting = 0.040 +/- 0.006 min-1; P < 0.05) indicating that the emulsion and chylomicrons generated from the testinal lipid absorption compete for the same catabolic processes, confirming the validity of the method. The emulsion was injected into 11 patients with CAD and into 11 controls. All had plasma cholesterol < 240 and triglycerides < 250 mg/dl. FCR of triglycerides was 5-fold smaller in CAD compared to controls (0.028 +/- 0.004 and 0.141 +/- 0.069 min-1, respectively, P < 0.01). FCR of cholesteryl ester was 4-fold smaller in CAD than in controls (0.015 +/- 0.004 and 0.056 +/- 0.067 min-1 respectively, P < 0.05). These results indicate that both chylomicron lipolysis and remnant removal are diminished in CAD.

Adult↗
PubMed1995

Malignant hypertension is accompanied by marked alterations in chylomicron metabolism.

Malignant hypertension (MH) is a severe complication of untreated arterial hypertension that damages the vascular system. It is often accompanied by disturbances in lipid metabolism that could contribute to its pathophysiology. We examined chylomicron metabolism in MH patients using a triglyceride-rich emulsion known to mimic natural chylomicrons when injected into the bloodstream. The emulsion was labeled with [3H]triolein and [14C]cholesteryl oleate and injected intravenously into 15 normolipidemic MH patients aged 29 to 56 years (8 men) for comparison with 17 healthy control subjects. Consecutive plasma samples were taken at regular intervals during 1 hour for determination of the disappearance curves of the labels. The fractional clearance rate of the [3H]triolein emulsion in MH patients was twice as small as that of control subjects (0.061 +/- 0.012 and 0.141 +/- 0.074 min-1, respectively). On the other hand, [14C]cholesteryl oleate fractional clearance rate was not statistically different in MH patients and control subjects (0.032 +/- 0.004 and 0.056 +/- 0.014 min-1, respectively). These results indicate that in MH, lipolysis (measured by the fractional clearance rate of [3H]triolein) is pronounced diminished, whereas the removal of the remnant particles (measured by the fractional clearance rate of [14C]cholesteryl oleate) is not importantly affected. In conclusion, there is an alteration in the circulatory transport of dietary lipids that may be an important component in the vascular disease associated with MH.

Adult↗
PubMed1994

Structural requirements of bioactive peptides for interaction with endopeptidase 22.19.

A series of biologically active peptides and related compounds (opioid peptides, neurotensin, and bradykinin) were used as substrates or competitive inhibitors to study the structural requirements for peptide interaction with endopeptidase 22.19. The kinetics of hydrolysis of these peptides indicated that, in contrast to other proteases, the substrate specificity of endopeptidase 22.19 is not determined by the amino acids flanking the sensitive bonds of the substrates. The competition between bioactive peptide analogues and the quenched fluorescence substrate of endopeptidase 22.19 indicated that their length and their flexibility may be the dominant factors to explain their binding specificities. These peculiar features of endopeptidase 22.19 may be of importance to understand the physiological processes of conversion and inactivation of biologically active peptides.

Amino Acid Sequence↗
PubMed1991

Targeting of frog prodermorphin to the regulated secretory pathway by fusion to proenkephalin.

We have investigated the sorting and processing of the amphibian precursor prepro-dermorphin in mammalian cells. Dermorphin, a D-alanine-containing peptide with potent opioid activity, has been isolated from the skin of the frog Phyllomedusa sauvagei. The maturation of this peptide from the precursor involves several posttranslational steps. Recombinant vaccinia viruses were used to infect AtT-20, PC12, and HeLa cells to study the sorting and processing of prepro-dermorphin. While this precursor was not processed in any of the examined cell lines, AtT-20 cells were able to process approximately 40% of a chimeric precursor consisting of the first 241 amino acids of prepro-enkephalin fused to a carboxy-terminal part of pro-dermorphin. By immunogold-EM, we could show that the chimeric protein, but not pro-dermorphin, was sorted to dense-core secretion granules. The processing products could be released upon stimulation by 8-Br-cAMP. We conclude that the pro-enkephalin part of the fusion protein contains the information for targeting to the regulated pathway of secretion, while this sorting information is missing in pro-dermorphin. This indicates that sorting mechanisms may differ between amphibian and mammalian cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Enkephalin is liberated from metorphamide and dynorphin A1-8 by endo-oligopeptidase A, but not by metalloendopeptidase EC 3.4.24.15.

It has been previously reported that both the cysteinyl-endo-oligopeptidase A and the metalloendopeptidase EC 3.4.24.15 are able to generate enkephalin from a number of enkephalin-containing peptides, including dynorphin A1-8. The present study shows that only endo-oligopeptidase A is able to generate [Leu5]enkephalin and [Met5]enkephalin from dynorphin A1-8 and from metorphamide respectively. It is also shown that endo-oligopeptidase A neither hydrolyses the specific EC 3.4.24.15 substrate alpha-N-benzoyl-Gly-Ala-Ala-Phe p-aminobenzoate, nor is inhibited by the specific EC 3.4.24.15 inhibitor N-[1(RS)-carboxy-2-phenylethyl]-alpha-Ala-Ala-Phe p-aminobenzoate.

Chemical Precipitation↗
PubMed1988

Liberation of enkephalins from enkephalin-containing peptides by brain endo-oligopeptidase A.

Endo-oligopeptidase A, highly purified from the cytosol fraction of bovine brain by immunoaffinity chromatography, has been characterised as a thiol endopeptidase. This enzyme, known to hydrolyse the Phe5-Ser6 bond of bradykinin and the Arg8-Arg9 bond of neurotensin has been shown to produce, by a single cleavage, [Leu]enkephalin or [Met]enkephalin from small enkephalin-containing peptides. Enkephalin formation could be inhibited in a concentration dependent manner by the alternative substrate bradykinin. The optimal substrate size was found to be 8-13 amino acids, with enkephalin the only product released from precursors in which this sequence is immediately followed by a pair of basic residues. However, the specificity constants (kcat/Km) obtained for endo-oligopeptidase A hydrolysis of bradykinin, neurotensin and dynorphin B are of the same order. Taken together, these results indicate that the substrate amino acid sequence is not the only factor determining the cleavage site of this enzyme. Finally, endo-oligopeptidase A and metalloendopeptidase EC 3.4.24.15 are two different enzymes. The latter is not able to liberate enkephalins from metorphamide and dynorphin.

Amino Acid Sequence↗
PubMed1987

Brain endo-oligopeptidase A, a putative enkephalin converting enzyme.

Endo-oligopeptidase A, highly purified from the cytosol fraction of bovine brain by immunoaffinity chromatography, has been characterized as a thiol endopeptidase. This enzyme, known to hydrolyze the Phe5-Ser6 bond of bradykinin and the Arg8-Arg9 bond of neurotensin, has been shown to produce, by a single cleavage, Leu5-enkephalin or Met5-enkephalin from small enkephalin-containing peptides. Enkephalin formation could be inhibited in a concentration-dependent manner by the alternative substrate bradykinin. The optimal substrate size was found to be eight to 13 amino acids, with enkephalin the only product released from precursors in which this sequence is immediately followed by a pair of basic residues. However, the specificity constants (kcat/Km) obtained for endo-oligopeptidase A hydrolysis of bradykinin, neurotensin, and dynorphin B are of the same order, a result indicating that the substrate amino acid sequence is not the only factor determining the cleavage site of this enzyme.

Animals↗
PubMed1986

A processing enzyme for prodynorphin derived peptides.

Endo-oligopeptidase A known to hydrolyse the Phe5-Ser6 bond of bradykinin and the Arg8-Arg9 bond of neurotensin has been shown to produce, by a single cleavage, leucine5-enkephalin from small prodynorphin derived enkephalin-containing peptides. The specificity constants (kcat/km) obtained for the hydrolysis of bradykinin, neurotensin and dynorphin B are of the same order, suggesting that the substrate amino acid sequence is not the only factor determining the cleavage site of this enzyme.

Amino Acid Sequence↗