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Biomedical subjects

G Stefano

Publications and source records attributed to G Stefano.

16 recordsLinked to original sources
PubMed2002

Endogenous morphine modulates acute thermonociception in mice.

The endogenous synthesis of morphine has been clearly demonstrated throughout the phylogenesis of the nervous system of mammals and lower animals. Endogenous morphine, serving as either a neurotransmitter or neurohormone, has been demonstrated in the nervous system of both vertebrates and invertebrates. As one of the effects of exogenous morphine is the modulation of pain perception, we investigated the effects that the depletion of endogenous morphine had on nociceptive transmission. The immunoneutralization of endogenous morphine from brain extracellular spaces was obtained through the intracerebroventricular administration of affinity purified anti-morphine IgG to mice, which then underwent the hot plate test. Endogenous morphine immunoneutralization decreased thermal response latency and attenuated the anti-nociceptive effect of the mu selective agonist DAMGO in hot plate test suggesting that endogenous morphine is involved in pain modulation.

Acute Disease↗
PubMed2000

Biochemical evidence of specific trypsin-chymotrypsin inhibitors in the rhynchobdellid leech, Theromyzon tessulatum.

The presence of two specific trypsin-chymotrypsin inhibitors from head parts of the rhynchobdellid leech Theromyzon tessulatum is reported. Two proteins, anti-trypsin chymotrypsin A (ATCA; 14636.6 +/- 131 Da) and anti-trypsin-chymotrypsin B (ATCB; 14368 +/- 95 Da) were purified by size exclusion and anion-exchange chromatography followed by reversed-phase HPLC. Based on amino-acid composition, N-terminal sequence determination (MELCELGQSCSRD-NPQPSNM), matrix assisted laser desorption-time of flight measurement (MALDI-TOF), trypsin mapping comparison, inhibition constant determination (Ki), and influence on amidolytic activity of different serine proteases, it is demonstrated that ATCA and ATCB are novel and highly potent serine-protease inhibitors of trypsin and chymotrypsin (ATCA: 350fM towards trypsin and chymotrypsin; ATCB: 400 and 75 fM towards trypsin and chymotrypsin, respectively). It is further surmised that ATCA and ATCB are linked, in that ATCB would lead to the formation of ATCA after loss of few amino acid residues.

Amino Acid Sequence↗
PubMed1997

Prodynorphin in invertebrates.

We have characterized a prodynorphin-like molecule in an invertebrate, specifically in the rhynchobdellid leech Theromyzon tessulatum. The 14270 Da protein was purified by gel permeation chromatography, anti-leucine-enkephalin-affinity column separation followed by reverse-phase HPLC. Its complete characterization was performed by Edman degradation, enzymatic treatments, and matrix assisted laser-desorption time of flight mass spectrometry. This 119 amino-acid protein exhibits 28.8% sequence identity with rat prodynorphin, 22.9% with human prodynorphin, and 21.8% with the pig molecule. Within the leech precursor, alpha-Neo-endorphin, dynorphin-A, and dynorphin B-like peptides are present at the C-terminus as in vertebrate prodynorphin. These biological active peptides exhibit 100%, 50%, and 76.6% sequence identity with their counterparts in mammals, respectively. The amount of leucine-enkephalin is identical to that found in vertebrates. Leech prodynorphin is distinguished from that found in mammals in that the N-terminus is shorter. This report constitutes the first complete biochemical characterization of a prodynorphin in invertebrates.

Amino Acid Sequence↗
PubMed1997

Production and physiological actions of anandamide in the vasculature of the rat kidney.

The endogenous cannabinoid receptor agonist anandamide is present in central and peripheral tissues. As the kidney contains both the amidase that degrades anandamide and transcripts for anandamide receptors, we characterized the molecular components of the anandamide signaling system and the vascular effects of exogenous anandamide in the kidney. We show that anandamide is present in kidney homogenates, cultured renal endothelial cells (EC), and mesangial cells; these cells also contain anandamide amidase. Reverse-transcriptase PCR shows that EC contain transcripts for cannabinoid type 1 (CB1) receptors, while mesangial cells have mRNA for both CB1 and CB2 receptors. EC exhibit specific, high-affinity binding of anandamide (Kd = 27.4 nM). Anandamide (1 microM) vasodilates juxtamedullary afferent arterioles perfused in vitro; the vasodilation can be blocked by nitric oxide (NO) synthase inhibition with L-NAME (0.1 mM) or CB1 receptor antagonism with SR 141716A (1 microM), but not by indomethacin (10 microM). Anandamide (10 nM) stimulates CB1-receptor-mediated NO release from perfused renal arterial segments; a similar effect was seen in EC. Finally, anandamide (1 microM) produces a NO-mediated inhibition of KCl-stimulated [3H]norepinephrine release from sympathetic nerves on isolated renal arterial segments. Hence, an anandamide signaling system is present in the kidney, where it exerts significant vasorelaxant and neuromodulatory effects.

Amidohydrolases↗
PubMed1997

A renin-like enzyme in the leech Theromyzon tessulatum.

We report on the biochemical isolation and characterization of a 32 kDa aspartyl protease from the leech Theromyzon tessulatum. Following a three step purification (gel permeation chromatography, pepstatin A-sepharose affinity column separation followed by reversed-phase HPLC) a renin-like enzyme was purified to homogeneity. The first 124 amino acid residues of the N-terminal part of the purified S-pyridylethylated leech renin exhibits a 26.5-35.5% sequence identity with that of mammals. The 20-81 region of leech renin exhibits a 80% sequence homology with the 175-232 region in mammals. This highly conserved region, which is also found in all aspartic proteases, possesses the aspartyl catalytic residue (D11TGSS). Leech renin hydrolyses at neutral pH and at 37 degrees C the Leu10-Leu11 bond of synthetic porcine angiotensinogen tetradecapeptide yielding the angiotensin I and the Leu11-Val12-Tyr13-Ser14 peptides, with a specific activity of 115 microg AI/min/mg (K[M] 22 microM; K[cat], 2.7). This hydrolysis is inhibited by pepstatin A (IC50: 4.6 microM). Moreover, this enzyme is found on a multiple hormone precursor of 19 kDa which exhibits a specific activity of 850 pmol AI/min/mg of renin. This is the first biochemical characterization of a renin-like enzyme in invertebrates and non-mammalian vertebrates.

Amino Acid Sequence↗
PubMed1997

Presence and biochemical properties of a molluscan invertebrate angiotensin-converting enzyme.

A soluble 65582.9 Da (in MALDI-TOF) angiotensin converting (ACE)-like enzyme has been purified by a captopril-Sepharose affinity column chromatography from the mollusk Mytilus edulis. This glycosylated peptidyl dipeptidase, with an N-terminal sequence of LDPELSPGCFVANQAGGQLF, hydrolyses the Phe8-His9 bond (at pH 8.4 and 37 degrees C) of angiotensin I with a high catalytic activity i.e. Km: 168 microM and Kcat/Km: 262 s-1 mM-1. The hydrolysis of angiotensin I is inhibited by the specific ACE inhibitors captopril and lisonopril (Ic50 = 50 nM). This activity is increased by Cl- (optimal Cl- concentration 400 mM) and by Zn2+. This zinc metallopeptidase also attacks peptides having a Gly-His, Gly-Phe or a Phe-His bond in their sequence e.g. leucine-enkephalin (Kcat/Km: 1200 s-1 mM-1 or bradykinin (Kcat/Km: 2500 s-1 mM-1). Mytilus ACE displays properties of the C-domain of human ACE, indicating a high degree of conservation during evolution. These results are consistent with an ACE activity implicated in metabolism of several neuropeptides in mollusks.

Amino Acid Sequence↗
PubMed1997

Paget's disease.

Explore the source record for details and available documents.

Alleles↗
PubMed1995

Opioid peptides in the nervous system of Aplysia: a combined biochemical, immunocytochemical, and electrophysiological study.

1. We have used biochemical, immunocytochemical, and electrophysiological techniques to evaluate the role of opioid peptides in the central nervous system of the marine mollusc, Aplysia californica. 2. Binding studies using 3H-D-Ala2, met-enkephalinamide (3H-DAMA) showed a single class of high-affinity binding sites with a Kd of 1.3 nM and a binding density of 45 pmol/g. 3. HPLC extracts of ganglia revealed multiple peaks with immunoreactivity for either leu (LEU-IR)- or met-enkephalin (MET-IR), but the amounts were not uniformly distributed in all ganglia. 4. LEU-IR and MET-IR neurons were demonstrated immunocytochemically in all ganglia, but MET-IR neurons were more frequent and were concentrated in pedal and pleural ganglia. While absorption control studies abolished MET-IR, LEU-IR was only partially abolished in the neuropil. 5. In electrophysiological studies, both depolarizing and hyperpolarizing responses were found to D-Ala2-leu-enkephalin (DALEU) and D-Ala2-met enkephalin (DAMET) on some and different neurons. 6. HPLC fractions from regions with retention times corresponding to authentic leu- or met-enkephalin showed physiologic responses similar to those of DALEU and DAMET, respectively. 7. These studies suggest that a variety of endogeneous opioid peptides play physiologically important roles in the nervous system of Aplysia, including but not necessarily limited to leu- and met-enkephalin.

Analgesics, Opioid↗
PubMed1993

Evidence of immunocyte stimulatory molecule(s) in plasma of patients undergoing cardiopulmonary bypass.

Previous studies have demonstrated that cardiopulmonary bypass (CPB) induces a diffuse inflammatory response as well as cellular immunosuppression. To better characterize and localize this immune dysfunction, we examined the effect of CPB-exposed plasma on granulocytes and macrophages obtained from normal blood. Pre- and intra-CPB blood samples were obtained from seven patients admitted for elective coronary artery bypass graft surgery. Plasma from the pre-CPB samples served as controls. Evaluation of the behavior of the granulocytes and macrophages was based on their ability to respond by way of chemokinesis or chemotaxis versus a stimulus (D-Ala2-Met5-enkephalin [DAME], interleukin[IL]-1 alpha). Non-CPB exposed granulocytes and macrophages were found to be capable of chemotaxis when stimulated by DAME and by IL-1 alpha. Subjected to CPB, these cells were significantly more activated than controls (p < 0.005). They behaved in a chemokinetic fashion when incubated with cell-free CPB plasma and did not respond to either DAME or IL-1 alpha in a chemotaxic situation when compared with controls (p < 0.005). Plasma subjected to CPB as stimulus provoked a chemotaxic response in non-CPB exposed cells. Taken together, these results indicate that at least one type of potent signal molecule was present, and possibly generated, in plasma subjected to CPB.

Cardiopulmonary Bypass↗
PubMed1992

Distinct receptors for Leu- and Met-enkephalin on the metacerebral giant cell of Aplysia.

1. The effects of D-Ala2-Leu-enkephalin (DALEU), D-Ala2-Met-enkephalin (DAMET), and FMRFamide on the metacerebral cell (MCC) of Aplysia were determined in current- and voltage-clamp experiments. 2. Distinct receptors exist on this neuron for the three substances. 3. DALEU elicited a depolarizing response due to an inward current but not accompanied by a significant change in membrane conductance. 4. In contrast, DAMET elicited a hyperpolarizing response due to an outward current, also not associated with a significant change in membrane conductance. 5. Both the DALEU and the DAMET responses increased with hyperpolarization, decreased with depolarization, but did not reverse at potentials less than -30 mV. Neither response was sensitive to naloxone. 6. FMRFamide induced a voltage-dependent outward current that reversed at about -76 mV. This neuron was responsive to much lower concentrations of FMRFamide than either of the enkephalins, and the response to FMRFamide appears to be a conductance increase to K+. 7. These results suggest that the MCC neuron has distinct receptors for Leu- and Met-enkephalin that activate unusual responses of opposite polarity, as well as more usual inhibitory responses to FMRFamide.

Action Potentials↗
PubMed1990

Granulocyte activation after coronary angioplasty in humans.

To determine whether percutaneous transluminal coronary angioplasty (PTCA) would lead to neutrophil activation with subsequent discharge of proteolytic enzymes, like elastase, and oxygen free radicals, like superoxide anion, blood samples were taken from the coronary sinus and aorta in 14 patients with stable angina and one-vessel disease who underwent PTCA. Neutrophils were separated by means of the Ficoll-Hypaque system and were stimulated to detect release of elastase and generation of superoxide anion. Plasma levels of elastase were also measured by an immunoenzymatic method. PTCA was successful in all patients. Plasma elastase levels increased significantly at the end of the procedure compared with pre-PTCA values both in the coronary sinus (from 129.2 +/- 16.6 to 286.6 +/- 39.7 micrograms/l, p less than 0.005) and in the aorta (from 117.4 +/- 13.6 to 258.1 +/- 41.3 micrograms/l, p less than 0.005). On the other hand, superoxide anion released in the supernatants after neutrophil stimulation by phorbol-myristate-acetate decreased after PTCA in the coronary sinus (before PTCA, 60.1 +/- 7.1; after PTCA, 40.7 +/- 6.8 nmol 1 x 10(7) granulocytes/ml/15 min, p less than 0.05), whereas a mild but not significant decrease was observed in the aorta (from 58.3 +/- 10.9 to 55.3 +/- 8.6 nmol 1 x 10(7) granulocytes/ml/15 min, p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1984

Cyclic AMP in normal and malignant hyperpyrexia susceptible individuals following exercise.

Plasma cAMP concentrations were compared in normal and malignant hyperpyrexia susceptible individuals. Samples were taken before, at the peak, and at 60 and 120 min after a standardized physical stress test. It was found that, at the peak of the test, cAMP concentrations were significantly higher in malignant hyperpyrexia susceptible individuals. In addition, the return to control values was significantly prolonged in this group. The results support the hypothesis of abnormal cAMP metabolism in malignant hyperpyrexia susceptible individuals.

Adolescent↗
PubMedDate not supplied

Stop-flow in mediastinum and thorax for resistant lymphoma.

BACKGROUND/AIMS: Management of patients with heavily pretreated malignant lymphoma failing frontline treatment and salvage high-dose chemotherapy and autologous peripheral stem cell rescue, is problematic. A pilot study was conducted to evaluate isolated thoracic perfusion of drugs by means of stopflow technique. METHODOLOGY: Six patients were enrolled in the study; diagnoses included 4 advanced Hodgkin's disease, 1 primary mediastinal B-cell lymphoma, and 1 anaplastic large cell lymphoma. Patients were aged 18-37 years; 4 presented with bulky mediastinum. They had never achieved a complete response since all had progressed from front-line treatment, and 3 had even failed salvage high-dose chemotherapy with autologous peripheral stem cell rescue. Cisplatin (100 mg/m2) and melphalan (35 mg/m2) were used. Carmustine (100 mg/m2) were added to these 2 drugs and cytarabine (2000 mg/m2) in patients not previously treated by carmustine, etoposide, cytarabine, and melphalan. Epidoxorubicin (70 mg/m2) was added in patients who previously received a suboptimal dosage of antracycline. Drugs were delivered monthly via aortic perfusion performed by means of Aigner's stop-flow technique. RESULTS: Overall 13 cycles of perfusional chemotherapy were administered with a median number of 2 cycles. During the procedures there were no technical, hemodynamic, or vascular complications, and no deaths occurred during surgery. After 1 month, 6 (100%) objective responses after isolated thoracic perfusion were recorded, 3 (50%) of which were complete. Tolerance to therapy was excellent. Hematological toxicity was mild and transfusional support was needed only in one course. At the last follow-up, 2 patients are alive (1 complete response and 1 very good partial response, maintained). CONCLUSIONS: This new therapeutical approach seems very active in recurrent/refractory malignant lymphoma and may play an important role in this setting.

Adolescent↗