Biomedical subjects

M S Moukhtar

Publications and source records attributed to M S Moukhtar.

At least 19 recordsLinked to original sources

PubMed1995

Sequence of a novel mRNA coding for a C-terminal-truncated form of human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase.

We amplified, using the polymerase chain reaction (PCR) and NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (type-I 15-PGDH)-specific primers, RNA extracted from the HL-60 cell line. Two bands, differing in size by approx. 160 bp, were detected with ethidium bromide staining after electrophoresis of amplification products and hybridization with a 15-PGDH-specific probe. Sequencing these DNA bands revealed that the largest corresponded to the 15-PGDH cloned from human placenta [Ensor et al., J. Biol. Chem. 265 (1990) 14888-14891]. The smaller sequence coded for a predicted C-terminal-truncated form of 15-PGDH. This subtype of the type-I 15-PGDH mRNA was also found using RT-PCR in human liver, placenta and a cell line derived from a human medullary thyroid carcinoma (TT cells). Hybridization studies using specific probes indicated that this new mRNA form probably corresponded to the 3.4-kb mRNA, one of the two 15-PGDH mRNAs previously detected in Northern blot analysis.

Amino Acid Sequence↗

PubMed1995

Calcitonin secretion, C cell differentiation and proliferation during the spontaneous development of murine medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC), a C cell neoplasm, synthesizes large amounts of calcitonin (CT), its biological marker. However, in some cases with a poor prognosis, MTC is associated with low basal CT levels owing to a decrease in the thyroid CT content. Using a murine model of human MTC, we studied the relationships between CT biosynthesis, C cell proliferation, and the circulating CT level during MTC progression. Cell proliferation was revealed by autoradiography of radioactive thymidine incorporation in dividing nuclei, after CT or CT mRNA detection by immunocytochemistry (ICC) or in situ hybridization (ISH). All rat thyroids showed a severe hyperplasia of C cells containing significant amounts of CT and CT mRNA, and a very low mitotic index. Tumours were found in 68% of the thyroids. In the strongly immunoreactive small nodules (ICC+), many labelled nuclei were observed. Subsequently some nodular cells, still containing detectable CT mRNA (ISH+), were not detected by immunocytochemistry (ICC-) owing to a dramatic decrease in secretory granules. Their mitotic index increased, and a rise of the basal CT plasma level was noted. These ISH+, ICC- tumour MTC cells represent a modified aggressive tumour C cell population exhibiting an increased ability to proliferate and were detected by the rise in the basal circulating CT level.

Animals↗

PubMed1992

Calcitonin gene-related peptide: an autocrine growth factor with regulatory activity in vitro.

We show that an autocrine system for calcitonin gene-related peptide (CGRP) exists in F9 teratocarcinoma cells. Synthesis of CGRP by F9 cells was demonstrated by measuring the peptide concentration in cells and medium and by determining specific mRNA in cells. During six days of culture, CGRP secretion did not vary significantly in the medium, while intracellular CGRP and CGRP mRNA levels increased. F9 cells contained a CGRP-sensitive adenylate cyclase system and CGRP increases the accumulation of cAMP in the culture medium. Interestingly affinity purified antibodies against CGRP specifically inhibited growth of F9 cells by 50%. CGRP therefore stimulates F9 cell growth by an autocrine process, suggesting that CGRP may be a growth factor during early embryogenesis.

Animals↗

PubMed1991

A novel calcitonin carboxyl-terminal peptide produced in medullary thyroid carcinoma by alternative RNA processing of the calcitonin/calcitonin gene-related peptide gene.

The calcitonin/calcitonin gene-related peptide gene expresses two different mRNAs by tissue-specific alternative processing. The calcitonin mRNA is produced in thyroid C cells by splicing of the first three exons to the fourth polyadenylated exon. It encodes a protein precursor containing an amino-terminal peptide, calcitonin, and a carboxyl-terminal peptide (CCP I). Calcitonin gene-related peptide (CGRP) mRNA is produced in neuronal cells by splicing of the three common exons to the fifth exon and the polyadenylated sixth exon, leading to the production of a CGRP precursor. Our studies concerning the expression of the calcitonin/CGRP gene in human medullary thyroid carcinoma revealed the presence of a new RNA transcript. Amplification by polymerase chain reaction and direct sequencing showed that the novel transcript is composed of exons 1, 2, and 3, part of exon 4, exon 5, and a polyadenylated exon 6. This transcript contains an open reading frame coding for the known amino-terminal and calcitonin peptides, as well as for a novel calcitonin carboxyl-terminal peptide, CCP II. This third alternative pathway utilizes an internal donor site within the exon coding for calcitonin. The presence of CCP II was demonstrated in plasma and thyroidal tissues of medullary thyroid carcinoma patients, implying that this novel mRNA is actively translated in medullary thyroid carcinoma.

Amino Acid Sequence↗

PubMed1991

Calcitonin gene-related peptide immunoreactivity in a crustacean Nephrops norvegicus and correlation with calcitonin.

Immunoreactive calcitonin-gene-related peptide (ir-CGRP) was detected in the crustacean Nephrops norvegicus. High levels of ir-CGRP were present in the foregut and hepatopancreas (3 +/- 0.7 and 4.6 +/- 1.0 micrograms eq per 100 mg of fresh organ, respectively). Molecular sieving of acidic extracts of anterior gut of Nephrops norvegicus showed a high molecular weight immunoreactive peptide in the range 15,000 to 25,000 Da. Immunoreactivity related to salmon calcitonin was present in the high molecular weight fraction.

Animals↗

PubMed1991

Binding sites of calcitonin gene related peptide (CGRP) to trout tissues.

We localized specific binding sites for human calcitonin gene related peptide (hCGRP) in different organs of the trout using labelled human CGRP. Maximal binding was observed in gill and spleen membranes. The binding of 125I-hCGRP was time and temperature dependent. Scatchard analysis of binding data for the spleen and the gills disclosed two binding sites. The constants for the site of high affinity and low capacity (KAM-1 and Bmax (fmol/mg of proteins] were 2.9 x 10(9) for the spleen and 70 and 3.5 x 10(9) for the gill. Salmon calcitonin (sCT) inhibited the binding of 125I-hCGRP to spleen membranes with the same order of potency as hCGRP. In contrast sCT was less effective than hCGRP in suppressing the specific binding of 125I-hCGRP to gill membranes.

Animals↗

PubMed1991

Study of calcitonin and thyroglobulin gene expression in human mixed follicular and medullary thyroid carcinoma.

mRNAs were isolated from 2 patients suffering from a familial form of a rare variant of medullary carcinoma of the thyroid (MTC), called mixed follicular and medullary carcinoma. The presence of calcitonin (CT) and thyroglobulin (Tg) mRNAs was checked by northern and in situ hybridization and compared with immunohistochemical results. In each case, mRNAs hybridizing to probes specific for CT and Tg were detected. Both proteins were quantified by radioimmunoassay determination in tissue extracts. Patient 1 had 20 ng Tg and 68 ng CT per micrograms total protein, and patient 2 had 0.4 ng Tg and 1.7 ng CT per micrograms total protein. Northern analysis showed that mixed carcinoma expressed several species of both CT mRNAs and Tg mRNAs. The main Tg transcripts present in neoplastic cells (8.5 and 4.8 kb for patient 1 and patient 2) were identical to or smaller than those of normal thyroid tissue (8.5 kb). The tumor CT mRNA (1 kb) was identical to that of normal tissue. In situ hybridization confirmed the presence of CT and Tg mRNA in the great majority of tumor cells. Furthermore, the presence of small amounts of organified iodine was evidenced by analytical ion microscopy in 35% of these cells. This raises an important question regarding the histogenesis of this tumor.

Adenocarcinoma↗

PubMed1990

Expression of the calcitonin gene during migration of the Pacific salmon, Oncorhynchus gorbuscha.

Plasma calcitonin levels increase during reproduction in female salmon. Whether these changes in circulating levels of the hormone are due to increased secretion and or increased biosynthesis is not established. We measured total and poly A+ RNAs and calcitonin content of ultimobranchial bodies of male and female pink salmon during the different stages of the reproductive cycle. Calcitonin mRNAs were analysed by hybridization of northern and dot blots with a specific probe for salmon calcitonin. Dot blot autoradiography indicated that female animals had higher levels of CT mRNA than males and these values were correlated with plasma calcitonin levels. In conclusion, sexual hormones still to be identified, are probably implicated in the expression of the calcitonin gene in females during the reproductive cycle.

Animals↗

PubMed1990

PCR amplification of CGRP II mRNA. Variable expression in tumoral and non-tumoral human thyroid.

Two genes code for calcitonin gene-related peptides (CGRPs). One expresses by tissue-specific alternate splicing calcitonin and CGRP I mRNAs, the other CGRP II mRNA. Calcitonin is the marker of sporadic or hereditary human medullary thyroid carcinoma (MTC). CGRP II expression is not well established in normal or tumoral thyroid. After amplification by polymerase chain reaction, CGRP I and II mRNAs were detected in six cases of MTC associated with other endocrine neoplasia (MEN IIa) and in two cases of isolated MTC. CGRP I was detected in all non-C cell tumoral thyroids (6 samples), CGRP II was barely detectable in three out of six cases. CGRP II could be a specific tumoral marker of MTC.

Base Sequence↗

PubMed1990

Calcitonin gene-related peptide stimulates adenylate cyclase activity in trout gill cell membranes.

The physiological significance of calcitonin gene-related peptide (CGRP) was investigated by assessing the CGRP stimulated adenylate cyclase activity in various tissues of trout. The highest enzyme concentration was found in gill and stomach membranes. The maximal activity (190% of the basal value) was observed for a concentration of 53.3 nM CGRP I or II. In the presence of 58 nM sCT, the maximal enzyme activity represented 120% of the basal value. No additive effect was observed; this suggests that both CGRP and sCT activities are mediated through the same receptor. The present data are in favour of a role for this neuropeptide operating in branchial cell functions such as calcium transfer from the external to the internal milieu.

Adenylyl Cyclases↗

PubMed1990

Increase in calcitonin mRNA levels in rats at high risk of C cell tumours is genetically determined.

C-cells tumours are frequent (50%) in old WAG/Rij rats. In comparison to the original Wistar strain, three month old WAG/Rij rats are characterized by higher calcitonin synthesis and secretion, in addition to a genetically transmitted loss of calcitonin binding sites in the outer renal medulla. In order to determine if the increase of calcitonin gene expression is also of genetic origin, we quantified calcitonin and its specific messenger in the thyroid glands of second generation (Wistar x WAG/Rij) hybrids. These parameters ranged from low Wistar like to higher WAG/Rij like values. The amount of calcitonin messenger in the thyroid was highly correlated to the release of the hormone in plasma elicited by a calcium challenge and inversely correlated with the number of its binding sites in the kidney. Our results suggest that an enhanced expression of the calcitonin gene is genetically transmitted, probably as a consequence of the mutation involved in the loss of renal calcitonin binding sites. It may represent the first event leading to malignancy.

Animals↗

PubMed1990

Distribution of calcitonin gene-related peptide and calcitonin-like immunoreactivity in trout.

Radioimmunoassay and chromatography were used to study the occurrence of calcitonin gene-related peptide in various tissues of the rainbow trout, Salmo gairdnerii. The highest concentrations of the peptide were found in gill (1.68 +/- 0.09 ng/mg protein) and in intestine (1.06 +/- 0.4 ng/mg protein). Significant concentrations were also found in heart and stomach. The level in brain was very low. In trout, the plasma concentration accounted for 283 +/- 82 pg/ml. Chromatographic analysis of the calcitonin gene-related peptide (CGRP)-like immunoreactivity occurring in gills showed that two molecular forms cross-reacted with the anti-human CGRP antibody, one co-eluting with the synthetic human CGRP. In addition, calcitonin in fish is not confined to the ultimobranchial organ but is also present in organs as heart, intestine, kidney, spleen and stomach. The evidence of CGRP in fish emphasizes the role of this hormone in evolution and leads us to investigate its physiological role in this species.

Animals↗

PubMed1990

Calcitonin variations in male and female trout, Salmo gairdneri, during the annual cycle.

Calcitonin (CT) levels in the ultimobranchial body and in plasma were radioimmunoassayed in rainbow trout, Salmo gairdneri, as a function of the annual cycle. In male and female, there was an important increase in the CT levels in the ultimobranchial body and in plasma. These variations in CT levels in both male and female suggest that sexual maturity influences the synthesis and the secretion of calcitonin in fishes. A positive correlation was observed between plasma and ultimobranchial CT levels and the gonadosomatic index in both sexes, suggesting that CT has a role in the processes involved in gonadal development.

Animals↗

PubMed1990

Hypoprolactinemic effect of calcitonin gene-related peptide in the rat.

The effect of calcitonin gene-related peptide (CGRP) on prolactin (PRL) secretion was studied in female rats. Prepubertal female rats were submitted to the stressful stimuli of injection, blood puncture and thermal stress. Lactating rats were exposed to suckling stimulus. The effects of CGRP on PRL release were compared to those of calcitonin (CT). CGRP i.p. or s.c. prevents the increase in circulating PRL induced by stress. This effect was observed two hrs and even 24 hrs after CGRP administration. It was elicited at all doses tested, 2.5, 1.25 and 0.6 micrograms per animal. The time course of the actions of CGRP and CT are different. It is proposed that CGRP and CT act on different receptors.

Aging↗

PubMed1989

Loss of calcitonin receptors: a genetically transmitted defect in rats with high incidence of C-cell tumors.

C-cell tumors (medullary thyroid carcinoma) occur in humans and several other mammalian species. This tumor develops spontaneously with a high incidence (50%) in old Wag/Rij (Wistar-derived strain) rats. We have recently shown that calcitonin binding sites, which are present in the Wistar rats, are lost from renal medulla of the Wag/Rij rats before they reach the age of 1 month. In the present work, we investigated the distribution of calcitonin binding sites in the kidneys of first and second generation hybrids of Wistar x Wag/Rij rats. The absence of calcitonin binding sites from the renal medullas of 25% of F2 hybrids indicates that the deficiency is inherited in a Mendelian fashion and opens the way to establishing inbred strains lacking renal medullary calcitonin binding sites.

Aging↗

PubMed1989

Elevated levels of calcitonin mRNA: a marker for the spontaneous development of medullary thyroid carcinoma in rats.

The aging WAG/Rij rats (a Wistar derived strain) develop spontaneously medullary thyroid carcinoma with a high frequency (50%). We have studied calcitonin biosynthesis in Wistar and WAG/Rij rats strains in order to determine if early changes in this parameter occurred in the WAG/Rij strain. Thyroidal and plasma CT levels were measured in three months old WAG/Rij and Wistar rats before and after acute calcium challenge. Total RNA was extracted from thyroid glands and specific CT messenger RNA levels estimated by dot and Northern blot analysis with a 32P-labeled probe specific for CT mRNA. The capacity of mRNA to direct synthesis of CT precursor was also measured by translation in an in vitro system. Though mean basal circulating CT levels were equivalent in both strains, CT release after calcium stimulation was much increased in the WAG/Rij rat. CT content of the glands and CT mRNA levels were two fold higher in the WAG/Rij strain. Thus, in this strain, CT biosynthesis and secretion were increased long before the development of a C-cell carcinoma.

Animals↗

PubMed1989

[Detection and prognosis of medullary cancer of the thyroid gland. Influence of national multidisciplinary cooperation].

Medullary thyroid carcinoma is a rare and sometimes hereditary disease. The tumour can be diagnosed and followed up by measuring the amounts of calcitonin it secretes. The prognosis of this cancer largely depends on an early diagnosis and treatment. From the clinical and laboratory (calcitonin assays) data recorded in our department, we have endeavoured to determine the influence of a national multidisciplinary co-operative group (GETC: French medullary study group) on the diagnosis and prognosis of this malignancy. We are able to show that the number of medullary thyroid carcinomas detected (principally in their familial forms) has increased by 141 per cent after the GETC was created. Calcitonin levels at the time of diagnosis are significantly lower (P less than 0.05) in familial cases, which reflects an early detection. The same applies to post-operative calcitonin levels (P less than 0.005), so that in the long run a better prognosis can be expected. It seems therefore that together with a better knowledge of this cancer and its detection, the setting up of a national multidisciplinary co-operative group results in a better clinical and therapeutic approach of these patients, and particularly of the familial cases of medullary thyroid carcinoma.

Calcitonin↗

PubMed1989

Distribution of renal calcitonin binding sites in mammalian and nonmammalian vertebrates.

Calcitonin (CT), the hypocalcemic hypophosphatemic hormone, is present in many vertebrate species. The principal target organs for CT are, in mammals, kidney and bone, and in fish, bone and gill. We have investigated the presence of renal calcitonin binding sites in a fish (Salmo gairdneri), two amphibians (Bufo bufo and Rana esculenta), two reptiles (Pseudemys scripta and Gekko gecko), and two birds (Gallus domesticus and Cothurnix japonica). We compared their distribution to a mammal, the rat (Wistar strain), using quantitative autoradiography methods. Moreover, CT binding sites were also characterized in the chicken kidney by a membrane assay. No renal 125I-sCT binding sites were detected in the fish, the amphibians, and the reptiles studied. In the rat, binding sites were present in the outer medulla and in the cortex. In the chicken and in the quail, scattered binding sites were also observed in the medulla and in the cortex, whose pattern seemed to follow the distribution of the glomeruli and/or the collecting tubules. Chicken kidney membranes were also purified by sucrose gradient centrifugation. Scatchard analysis of the binding data revealed the presence of one type of 125I-sCT binding site with an apparent dissociation constant of 4.3 nM and a number of sites of 73 fmol/mg/protein. The present results suggest that calcitonin renal receptors appeared late in evolution and thus that a regulation of renal function by calcitonin was only effective in birds.

Animals↗