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Biomedical subjects

F Amato

Publications and source records attributed to F Amato.

At least 19 recordsLinked to original sources
PubMed1995

Luteinizing hormone/chorionic gonadotropin bioactivity in the common marmoset (Callithrix jacchus) is due to a chorionic gonadotropin molecule with a structure intermediate between human chorionic gonadotropin and human luteinizing hormone.

Chorionic gonadotropin (CG), a pregnancy-specific heterodimeric hormone found in primates, is responsible for CL rescue with pregnancy maintenance. Of the primates, the human and baboon gene sequences are the only structures so far determined. In order to study the structure and function of CG in other primates, we have isolated and sequenced the coding regions for the two subunits of marmoset CG (mCG) by the reverse transcription/polymerase chain reaction method. Study of multiple clones confirmed a high degree of homology with the human sequences (88% and 80% for the alpha and beta nucleotide sequences, respectively). Marmoset CG alpha has an extra four amino acids compared to hCG alpha, whereas the mCG beta sequence has a 3-bp deletion that maintains the reading frame and C-terminal amino acid sequence. Most of the differences between hCG beta and mCG beta peptides occur in the C-terminal region, which includes the loss of two of the O-linked glycosylation consensus sequences and the presence of an N-linked glycosylation consensus sequence. When mCG alpha and beta were co-expressed in CHO cells, assembly of biologically active hormone was confirmed by induced steroid secretion by MA10 cells. Partially purified mCG beta was used to raise anti-mCG antibodies. To date, an antibody has been obtained that is capable of detecting recombinant mCG beta, recombinant mCG dimer, and mCG dimer secreted by cultured marmoset trophoblast. Marmoset CG alpha and beta were also detectable at the transcriptional level in cultured trophoblast by in situ hybridization. This suggests that the LH/CG bioactivity reported from marmoset placentae and embryos is due to a molecule with structural features common to hLH (glycosylation pattern) and hCG (CG beta C-terminal structure).

Amino Acid Sequence↗
PubMed1993

Molecular heterogeneity of the beta-core fragment of human chorionic gonadotrophin.

We have analysed the structure and composition of the beta-core fragment of human chorionic gonadotrophin (beta C-hCG) from fresh urine specimens obtained from pregnant women and compared our findings with those previously proposed by other groups using different protocols. SDS-PAGE separation of reduced beta C-hCG demonstrated two major bands with apparent molecular weights of M(r) 8900 and M(r) 7500. The molecular weight of the agalacto beta C-hCG was estimated to be M(r) 10,218 from the amino acid analysis after high-performance liquid chromatography (HPLC) separation. Moreover, HPLC separation of its reduced and S-carboxymethylated peptides resulted in three peaks, but only two of them could be sequenced and demonstrated to be the previously reported beta 6-40 (M(r) 5000) and beta 55-92 (M(r) 5300) peptides of the beta hCG subunit. The results showed that 56-78% of beta C-hCG molecules of molecular weight M(r) 12,800 were able to bind Concanavalin A (Con A). While most were lacking all the peripheral monosaccharides and terminated in mannose, some retained other sugar residues on their antennae. Direct carbohydrate analysis showed the following molar content normalized to six mannose molecules: galactose 2.8, glucosamine 5.3, galactosamine 0.3, fucose 1.7 and sialic acid 3.0. Approximately 22-44% of the beta C-hCG molecules did not bind Con A (Con A non-reactive forms), of which 88% were totally deprived of sugar units and had an apparent molecular weight of approximately M(r) 10,000, and 12% were weakly reactive to Con A and reactive to anion exchange (negatively charged forms), being incompletely trimmed of their oligosaccharide chains. Comparison of our results with those of two other groups have indicated that the differences noted among preparations are due to either the source or the methods used to purify and characterize this fragment. In addition, our results showed significant microheterogeneity on the N-linked oligosaccharide moieties with some molecules apparently having no sugar molecules. These results have implications for the origins of beta C-hCG, suggesting secretion of some molecules without sugar chains and in other cases possible metabolism of hCG in the peripheral tissues.

Amino Acids↗
PubMed1992

Comparison of specific immunoassays for detection of the beta-core human chorionic gonadotrophin fragment in body fluids.

We have validated two new methods, one radioimmunoassay (RIA) and one immunoradiometric assay (IRMA), for the detection of beta-core hCG fragment (beta C-hCG) in body fluids. In addition, we have compared their performance with two other assays designed for beta C-hCG quantification. The RIA uses a rabbit polyclonal antibody raised against pure beta C-hCG which has a high affinity constant, is sensitive to 5 pmol/l, and has significant cross-reaction only with the free beta LH subunit. The IRMA, designed in a liquid phase, uses the same polyclonal antibody associated with a 125I-labelled mouse monoclonal antibody (32H2) raised against beta hCG, is sensitive to 1.5 pmol/l, and does not cross-react significantly with any related glycoprotein. Comparison between these two assays and two others previously published was made by measuring beta C-hCG in urine from healthy pregnant women (n = 47) and gave correlation coefficients higher than r = 0.960 with any combination. Analysis of beta C-hCG in urine of non-pregnant subjects (n = 238) showed measurable beta C-hCG in 8.8% (levels ranged from 5 to 34 pmol/l) with the IRMA and 88.3% with the RIA (n = 30; ranging from 28.4 to 228 pmol/l) (P = 0.05). We concluded that, despite different affinities of the antibody involved and different cross-reactivities with related glycoproteins, the four assays we examined may be equally employed to detect beta C-hCG in pregnancy urine. However, the IRMA appears to be more appropriate for beta C-hCG analysis in non-pregnant individuals, specifically in postmenopausal women because of the high cross-reactivity of the RIA with free beta LH or beta fragments of other glycoproteins. These studies have significance for our understanding of the physiology of beta C-hCG in cancer, pregnancy and after the menopause.

Antibodies↗
PubMed1992

Distribution of the beta-core human chorionic gonadotrophin fragment in human body fluids.

The origins of a fragment of the human chorionic gonadotrophin (hCG) molecule, beta-core (beta C-hCG) were studied by analysis of beta C-hCG concentrations in biological fluids. In addition, the ability of the placenta to produce the fragment and the metabolism of hCG to beta C-hCG by human granulosa cells was determined in tissue culture. Finally the conversion of exogenous hCG to beta C-hCG was studied in vivo. The fragment was present in pregnancy urine as well as that from premenopausal and postmenopausal subjects. The highest concentrations were found in pregnant women. Ratios of beta C-hCG to intact hCG were higher in pregnancy urine when radioimmunoassay (RIA) was used compared with immunoradiometric assay (IRMA) (0.67 and 0.37 respectively). Concentrations of beta C-hCG were higher in postmenopausal urine than in premenopausal specimens. A significant amount of a high molecular weight beta C-hCG immunoreactive material was found in serum samples after size separation, and the molar ratio of beta C-hCG/hCG was estimated as 0.019. Amniotic fluid also contained small quantities of two forms of immunoreactive beta C-hCG and the ratio of 0.01 for authentic beta C-hCG/hCG increased to 0.026 when the high molecular weight form was considered. Cultured trophoblastic tissue released material with beta C-hCG immunoreactivity in the medium and chromatographic separation revealed that the majority of this material was of higher molecular weight compared with the authentic beta C-hCG form. beta C-hCG was the principal glycoprotein found in follicular fluid after hyperstimulated folliculogenesis and intramuscular injection of 5000 IU hCG. We also demonstrated that 26% of follicular fluid samples (n = 50) were positive for beta C-hCG; levels ranged from 5.2 to 23.0 pmol/l (13.1 +/- 5.7); S.D.) when a specific IRMA was used. The RIA could detect beta C-hCG in 48 samples (96%), levels ranging from 7.0 to 28.5 pmol/l (19.4 +/- 5.2). Moreover, granulosa cells cultured in the presence of hCG were able to degrade the intact molecule to both high molecular weight and authentic immunoreactive forms of beta C-hCG. After gel filtration, material of molecular weight over a wide range and immunoreactive for beta C-hCG was present in human seminal plasma. Assaying 74 samples of this fluid by IRMA, beta C-hCG was detected in 42 (56.7%), levels ranging between 5.5 and 59.5 pmol/l (24.9 +/- 15.2).(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1992

Urinary concentrations of beta core fragment of hCG throughout pregnancy.

OBJECTIVE: We sought to determine a reference range for urinary immunoreactive beta core fragment of hCG (beta C-hCG) in pregnancy, the ratio between beta C-hCG and intact hCG, and the earliest detectable rise of beta C-hCG in urine. METHODS: Urine was obtained from 741 pregnant women between 6-41 weeks' gestation, as well as from women undergoing donor insemination with timed ovulation peaks. RESULTS: The beta core fragment of hCG reached a maximum between 8-15 weeks, with a decrease between 20-29 weeks. The molar ratio of beta C-hCG to intact hCG was always greater than 1. CONCLUSION: In pregnancy, beta C-hCG concentrations increase in the urine in parallel to intact hCG but at a higher molar ratio, suggesting either placental production of beta C-hCG or enhanced metabolism of hCG to beta C-hCG in peripheral organs.

Chorionic Gonadotropin↗
PubMed1991

Stability of immunoreactive beta-core fragment of hCG.

The beta core fragment of hCG (beta C-hCG) accounts for a large proportion of hCG immunoreactivity in the urine of pregnant women. It is often increased in the urine of patients with gynecologic tumors and may become an important diagnostic tool in early pregnancy and cancers. Despite the importance of beta C-hCG, little is known about its stability in urine under conditions of differing pH and temperature. This study examined the effect of repeated freeze-thaw cycles; storage at room temperature, 4C, and -20 C over several months; and the effect of alteration of urine pH. The two specific immunoassays for beta C-hCG used do not significantly cross-react with intact hCG or the free alpha subunit. Despite different cross-reactivities of the antibodies to the free beta subunit and higher immunoactivity when the radioimmunoassay was used, there was excellent correlation between the two assays in pregnancy urine. This suggests that there is little free beta subunit in urine from pregnant women. In addition, this study evaluated the stability of intact hCG and beta-hCG under identical conditions. No alterations in their immunoactivities were found under most conditions of storage. It is concluded that, for clinical purposes, beta C-hCG as well as intact hCG and free beta subunit are very stable molecules.

Chorionic Gonadotropin↗
PubMed1990

Epidemiological study of urinary tract stones in a northern Italian city.

An epidemiological study of stone disease in a Northern Italian city was carried out by means of a postal questionnaire mailed to 6000 individuals (2.5% of the entire population). It was found that the incidence of stone disease was comparable to that of industrialised Western Europe. There was a relationship between stone disease and gout and stone disease and a positive family history. The frequency of uric acid stones was high (26.5%). Stone-formers showed no alimentary differences from non-stone formers apart from the use of spices and herbs. Stone-formers used less water from public aqueducts and more uncarbonated mineral water, but only 19% of these drank at least 2 litres a day.

Adult↗
PubMed1989

[Use of atracurium in continuous intravenous infusion].

In this study the use of atracurium was evaluated for use in continuous endovenous infusion by controlling infusion levels using the simple technique of TOF monitoring during general surgical operations. In conclusion, continuous infusion of blocking agents is the preferential technique for controlling and maintaining a neuromuscular block; moreover, the use of besylate of atracurium is advised to ensure a reduced rate of individual variation.

Adult↗
PubMed1989

[Comparative evaluation of several muscle relaxants].

The use of different muscle relaxants have been studied in two homogeneous groups of patients, in the first group atracurium and in the second succinylcholine-pancuronium. The utility of atracurium in short-medium abdominal surgery has been demonstrated.

Abdomen↗
PubMed1988

Acute effect of indapamide on urine calcium excretion in nephrolithiasis and human essential hypertension.

The effects of indapamide (2.5 mg once a day) on urinary composition are reported in 20 patients (10 with recurrent calcium nephrolithiasis and 10 with essential hypertension) compared with 20 controls. Indapamide was well absorbed in every patient (mean plasma level at the steady state was 111 +/- 41 ng/ml) and its antihypertensive action was more pronounced in hypertensive than in normotensive patients. It lowered calcium excretion in 18/20 patients (mean fall on the 7th day of treatment: 53%) and raised the Mg/Ca ratio in 20/20 patients (mean increase on the 7th day: 167%). The effect on Ca2+ and Mg2+ excretion was not associated with a strong diuretic effect. During intravenous calcium loading (0.375 mmol/kg body weight) 6 normal subjects after a single oral dose of indapamide excreted less calcium, suggesting a direct renal hypocalciuric action by the drug. Indapamide could represent an alternative drug to thiazide diuretics in diseases with dangerous renal calcium losses, but long-term studies are needed.

Adult↗
PubMed1987

[Renal colic: behavior of urinary parameters in the assessment of lithogenic risk].

In patients with renal colic we studied lithogenic urinary risk factors before and after the stone passage. We showed abnormalities in water, electrolytes and other substances excretion due to retention and metabolic disorders. The effects more pronounced is on urinary sodium, calcium, magnesium and ammonium. Citrate behaviour suggests a transient intracellular acidosis.

Adult↗
PubMed1985

On the origin of dehydroepiandrosterone sulphate in the blood of fetal sheep.

Total sulphoconjugated and unconjugated dehydroepiandrosterone (DHA) and total oestrone were measured in plasma of intact sheep fetuses, fetuses hypophysectomized at 104-112 days and fetuses bilaterally adrenalectomized at 98-101 days. At 120-127 days, the mean concentrations of total DHA and oestrone in intact fetuses (n = 13) were 29.7 +/- 4.2 (S.E.M.) nmol/l and 14.3 +/- 2.8 nmol/l respectively. At term, the values for total DHA and oestrone in hypophysectomized fetuses (n = 13) of 18.0 +/- 1.9 nmol/l and 9.1 +/- 2.0 nmol/l were significantly (P less than 0.05) lower than the intact group whereas in the adrenalectomized fetuses (n = 8) total DHA (80.8 +/- 13.0 nmol/l) was higher (P less than 0.05) and total oestrone values were similar to the intact animals. Intrafetal infusion of cortisol at term (1 mg/h for 84 h) raised levels of total oestrone in intact (n = 6; 12.3 +/- 2.9 vs 31.6 +/- 8.5 nmol/l) and adrenalectomized (n = 4; 14.2 +/- 2.6 vs 190.6 +/- 53.0 nmol/l) fetuses and of total DHA in hypophysectomized fetuses (n = 7; 16.0 +/- 1.9 vs 31.6 +/- 8.5 nmol/l) while infusion of ACTH (1-24) (5 micrograms/h) was without significant effect in any group. It is concluded that the ovine fetal adrenal in late pregnancy makes no significant contribution either to the high circulating concentrations of DHA sulphate or to the substrates for placental oestrogen synthesis.

Adrenal Glands↗
PubMed1984

N-acetyl-5-methoxy kynurenamine, a brain metabolite of melatonin, is a potent inhibitor of prostaglandin biosynthesis.

Although the hypothesis that melatonin may act by inhibiting prostaglandin synthesis arose in part from the similar structures of melatonin and indomethacin, melatonin does not share the in vitro efficacy of indomethacin in inhibiting prostaglandin synthesis. One possibility is that a metabolite of melatonin formed within the target cell might inhibit prostaglandin synthesis and in this study we have tested this by examining the action of the two oxidised metabolites, N-formyl-N-acetyl-5-methoxy kynurenamine formed by the action of indole 2,3-dioxygenase and N-acetyl-5-methoxy kynurenamine , formed in the brain by the action of formamidase . This latter compound which has a structure resembling the fenamate inhibitors of PG biosynthesis had a marked and time dependant inhibiting effect in synthesis using bovine and ovine seminal vesicle microsome preparations and measuring the products by using (1-14C) arachidonic acid and by using a specific antiserum for PGE methyl oxime.

Animals↗
PubMed1983

Blood flow in the ovary and adjacent structures of the non-pregnant sheep.

Blood flow of the ovary, vascular pedicle and oviduct was measured in anaesthetized non-cycling and cycling ewes by timed collection of ovarian venous blood. The degree of arterio-venous shunting across the ovary and pedicle was estimated both in vivo and in vitro by perfusing the tissues with 15 +/- 5 micrometers radioactive microspheres. The mean ovarian blood flow in non-cycling animals was 1.9 ml/min, which was 51% of blood flow in the ovarian vein. In cycling animals ovarian blood flow at midcycle was 2.9 ml/min (66% of ovarian venous flow) in non-luteal ovaries and 4.3 ml/min (79% of venous flow) in luteal ovaries. The degree of arterio-venous shunting was low in all stages of the cycle (1.0-2.6% across ovary + pedicle). The degree of shunting was also found to be very small in vitro (0.007-1.38%) in both non-luteal and luteal ovaries. A considerable number of microspheres was entrapped in the vascular pedicle of the ovary indicating the presence of an extensive capillary bed. There was an inverse relationship between blood flow in the ovary and flow in the vascular pedicle. Alterations in distribution of blood flow between the ovary and adjacent structures supplied by the ovarian artery may be of functional significance in allowing rapid changes in ovarian blood flow. The results of the present study indicate that changes in ovarian blood flow during the oestrus cycle are not caused by an action on arterio-venous shunt vessels.

Animals↗